PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T89	0-94	Sentence	denotes	Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65
T1	0-94	Sentence	denotes	Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65
T2	97-105	Sentence	denotes	Abstract
T90	106-248	Sentence	denotes	Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection.
T3	106-248	Sentence	denotes	Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection.
T91	249-347	Sentence	denotes	Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS.
T4	249-347	Sentence	denotes	Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS.
T92	348-568	Sentence	denotes	We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex.
T5	348-568	Sentence	denotes	We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex.
T93	569-696	Sentence	denotes	Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity.
T6	569-696	Sentence	denotes	Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity.
T94	697-841	Sentence	denotes	The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity.
T7	697-841	Sentence	denotes	The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity.
T95	842-1148	Sentence	denotes	Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2.
T8	842-1148	Sentence	denotes	Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2.
T96	1149-1358	Sentence	denotes	Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.
T9	1149-1358	Sentence	denotes	Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.
T10	1360-1372	Sentence	denotes	INTRODUCTION
T1429	1373-1516	Sentence	denotes	Nuclear factor (NF)-κB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1).
T11	1373-1516	Sentence	denotes	Nuclear factor (NF)-κB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1).
T1430	1517-1698	Sentence	denotes	The NF-κB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding.
T12	1517-1698	Sentence	denotes	The NF-κB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding.
T1431	1699-1891	Sentence	denotes	The transcriptional activity of the NF-κB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).
T13	1699-1891	Sentence	denotes	The transcriptional activity of the NF-κB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).
T1432	1892-1993	Sentence	denotes	Inhibitors of NF-κB (IκB) associate with the NF-κB complex and interfere with its binding to DNA (3).
T14	1892-1993	Sentence	denotes	Inhibitors of NF-κB (IκB) associate with the NF-κB complex and interfere with its binding to DNA (3).
T1433	1994-2248	Sentence	denotes	In the canonical pathway of NF-κB activation, the activated IκB kinase (IKK) phosphorylates IκB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-κB complex in the nucleus.
T15	1994-2248	Sentence	denotes	In the canonical pathway of NF-κB activation, the activated IκB kinase (IKK) phosphorylates IκB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-κB complex in the nucleus.
T1434	2249-2438	Sentence	denotes	IκB-α, the most abundant inhibitor of NF-κB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6).
T16	2249-2438	Sentence	denotes	IκB-α, the most abundant inhibitor of NF-κB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6).
T1435	2439-2575	Sentence	denotes	The NF-κB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCζ (9) and Ser536 by IKKα (10,11).
T17	2439-2575	Sentence	denotes	The NF-κB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCζ (9) and Ser536 by IKKα (10,11).
T1436	2576-2758	Sentence	denotes	Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IκB-α, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13).
T18	2576-2758	Sentence	denotes	Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IκB-α, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13).
T1437	2759-2951	Sentence	denotes	Post-activation turn off of NF-κB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-κB, such as IκB-α and ubiquitin-editing protein A20 (14–20).
T19	2759-2951	Sentence	denotes	Post-activation turn off of NF-κB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-κB, such as IκB-α and ubiquitin-editing protein A20 (14–20).
T1438	2952-3095	Sentence	denotes	Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-κB activity (12,21,22).
T20	2952-3095	Sentence	denotes	Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-κB activity (12,21,22).
T1439	3096-3363	Sentence	denotes	Persistent activation of NF-κB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-κB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23–25).
T21	3096-3363	Sentence	denotes	Persistent activation of NF-κB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-κB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23–25).
T1440	3364-3451	Sentence	denotes	Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27).
T22	3364-3451	Sentence	denotes	Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27).
T1441	3452-3670	Sentence	denotes	Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29).
T23	3452-3670	Sentence	denotes	Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29).
T1442	3671-3789	Sentence	denotes	Thus, understanding the mechanisms of NF-κB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.
T24	3671-3789	Sentence	denotes	Thus, understanding the mechanisms of NF-κB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.
T1443	3790-3928	Sentence	denotes	In HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-κB activity by activation of IKK (30) and procaspase 8 (31).
T25	3790-3928	Sentence	denotes	In HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-κB activity by activation of IKK (30) and procaspase 8 (31).
T1444	3929-4078	Sentence	denotes	Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication.
T26	3929-4078	Sentence	denotes	Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication.
T1445	4079-4328	Sentence	denotes	Tat binds to RNA stem-loop structures generated by the 5′ end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor β (TNFβ) (33) and interleukin-6 (IL-6) (34) to activate gene transcription.
T27	4079-4328	Sentence	denotes	Tat binds to RNA stem-loop structures generated by the 5′ end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor β (TNFβ) (33) and interleukin-6 (IL-6) (34) to activate gene transcription.
T1446	4329-4591	Sentence	denotes	Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPβ (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41–43).
T28	4329-4591	Sentence	denotes	Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPβ (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41–43).
T1447	4592-4774	Sentence	denotes	When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).
T29	4592-4774	Sentence	denotes	When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).
T1448	4775-4886	Sentence	denotes	We first reported that NF-κB was constitutively active in Jurkat cells that stably expressed the Tat gene (47).
T30	4775-4886	Sentence	denotes	We first reported that NF-κB was constitutively active in Jurkat cells that stably expressed the Tat gene (47).
T1449	4887-5126	Sentence	denotes	Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IκB-α (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49).
T31	4887-5126	Sentence	denotes	Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IκB-α (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49).
T1450	5127-5358	Sentence	denotes	These findings suggested that Tat modulates crucial enzymes involved in NF-κB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-κB, which is mainly dependent on de novo synthesis of IκB-α (15,17).
T32	5127-5358	Sentence	denotes	These findings suggested that Tat modulates crucial enzymes involved in NF-κB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-κB, which is mainly dependent on de novo synthesis of IκB-α (15,17).
T1451	5359-5471	Sentence	denotes	We previously found that IκB-α binds to Tat and promotes the nuclear export of the viral transactivator (50,51).
T33	5359-5471	Sentence	denotes	We previously found that IκB-α binds to Tat and promotes the nuclear export of the viral transactivator (50,51).
T1452	5472-5684	Sentence	denotes	In this study, we report that Tat counteracts the post-activation turn off of NF-κB through direct interaction with IκB-α and p65, which enhances the DNA binding and transcriptional activity of the NF-κB complex.
T34	5472-5684	Sentence	denotes	In this study, we report that Tat counteracts the post-activation turn off of NF-κB through direct interaction with IκB-α and p65, which enhances the DNA binding and transcriptional activity of the NF-κB complex.
T1453	5685-5823	Sentence	denotes	The new mechanism of NF-κB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.
T35	5685-5823	Sentence	denotes	The new mechanism of NF-κB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.
T36	5825-5846	Sentence	denotes	MATERIALS AND METHODS
T4409	5848-5856	Sentence	denotes	Plasmids
T37	5848-5856	Sentence	denotes	Plasmids
T4410	5857-6081	Sentence	denotes	The plasmids pcDNA-3xHA-IκB-α, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50).
T38	5857-6081	Sentence	denotes	The plasmids pcDNA-3xHA-IκB-α, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50).
T4411	6082-6294	Sentence	denotes	The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pκBluc and pSV-β-Gal were purchased from Promega (Madison, WI, USA).
T39	6082-6294	Sentence	denotes	The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pκBluc and pSV-β-Gal were purchased from Promega (Madison, WI, USA).
T4412	6295-6560	Sentence	denotes	The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1–318), pRc/CMV-3xHA-p65ΔN(122–551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.
T40	6295-6560	Sentence	denotes	The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1–318), pRc/CMV-3xHA-p65ΔN(122–551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.
T4906	6562-6614	Sentence	denotes	Cells, transfection, treatments and luciferase assay
T41	6562-6614	Sentence	denotes	Cells, transfection, treatments and luciferase assay
T4907	6615-6841	Sentence	denotes	HeLa, p50−/−p65−/− mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640.
T42	6615-6841	Sentence	denotes	HeLa, p50−/−p65−/− mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640.
T4908	6842-6891	Sentence	denotes	PBMCs were isolated as previously described (53).
T43	6842-6891	Sentence	denotes	PBMCs were isolated as previously described (53).
T4909	6892-7008	Sentence	denotes	Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany).
T44	6892-7008	Sentence	denotes	Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany).
T4910	7009-7278	Sentence	denotes	HeLa, p50−/−p65−/− MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA).
T45	7009-7278	Sentence	denotes	HeLa, p50−/−p65−/− MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA).
T4911	7279-7567	Sentence	denotes	For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-α (TNF-α; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture.
T46	7279-7567	Sentence	denotes	For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-α (TNF-α; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture.
T4912	7568-7671	Sentence	denotes	For luciferase assays, pSV-β-Gal was co-transfected with pκBluc to monitor the transfection efficiency.
T47	7568-7671	Sentence	denotes	For luciferase assays, pSV-β-Gal was co-transfected with pκBluc to monitor the transfection efficiency.
T4913	7672-7971	Sentence	denotes	Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and β-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA).
T48	7672-7971	Sentence	denotes	Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and β-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA).
T4914	7972-8079	Sentence	denotes	The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units.
T49	7972-8079	Sentence	denotes	The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units.
T5677	8081-8097	Sentence	denotes	RNA interference
T50	8081-8097	Sentence	denotes	RNA interference
T5678	8098-8223	Sentence	denotes	Jurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA).
T51	8098-8223	Sentence	denotes	Jurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA).
T5679	8224-8506	Sentence	denotes	Briefly, aliquots (5 × 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 µF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium.
T52	8224-8506	Sentence	denotes	Briefly, aliquots (5 × 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 µF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium.
T5680	8507-8726	Sentence	denotes	RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU.
T53	8507-8726	Sentence	denotes	RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU.
T5681	8727-8841	Sentence	denotes	RNA interference of p65 and IκB-α was performed with SMART pool siRNA p65 and IκB-α (Dharmacon, Chicago, IL, USA).
T54	8727-8841	Sentence	denotes	RNA interference of p65 and IκB-α was performed with SMART pool siRNA p65 and IκB-α (Dharmacon, Chicago, IL, USA).
T6045	8843-8889	Sentence	denotes	Pseudotyped virions and single round infection
T55	8843-8889	Sentence	denotes	Pseudotyped virions and single round infection
T6046	8890-9072	Sentence	denotes	293T cells (1 × 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 µg) together with pHXB2 Env (10 µg), and 48-h post-transfection cell supernatant was collected.
T56	8890-9072	Sentence	denotes	293T cells (1 × 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 µg) together with pHXB2 Env (10 µg), and 48-h post-transfection cell supernatant was collected.
T6047	9073-9169	Sentence	denotes	Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration.
T57	9073-9169	Sentence	denotes	Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration.
T6048	9170-9319	Sentence	denotes	PBMCs, Jurkat or U937 cells (5 × 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).
T58	9170-9319	Sentence	denotes	PBMCs, Jurkat or U937 cells (5 × 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).
T6288	9321-9388	Sentence	denotes	Cell extracts, western blotting, IKK activity and NF-κB DNA binding
T59	9321-9388	Sentence	denotes	Cell extracts, western blotting, IKK activity and NF-κB DNA binding
T60	9389-9515	Sentence	denotes	Total, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data.
T6289	9389-10064	Sentence	denotes	Total, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data. Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris–HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% β-mercaptoethanol, 25% glycerol), resolved on 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories).
T61	9516-10064	Sentence	denotes	Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris–HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% β-mercaptoethanol, 25% glycerol), resolved on 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories).
T6290	10065-10155	Sentence	denotes	Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham).
T62	10065-10155	Sentence	denotes	Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham).
T6291	10156-10195	Sentence	denotes	Primary antibodies were purchased from:
T63	10156-10195	Sentence	denotes	Primary antibodies were purchased from:
T6292	10196-10396	Sentence	denotes	Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IκB-α C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-γ-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65).
T64	10196-10396	Sentence	denotes	Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IκB-α C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-γ-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65).
T6293	10397-10477	Sentence	denotes	Densitometry of single bands was analysed by ImageJ software package (NIH, USA).
T65	10397-10477	Sentence	denotes	Densitometry of single bands was analysed by ImageJ software package (NIH, USA).
T6294	10478-10611	Sentence	denotes	IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA).
T66	10478-10611	Sentence	denotes	IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA).
T6295	10612-10798	Sentence	denotes	Binding of p65, p50 and FLAG-Tat to the double-stranded NF-κB oligonucleotide was measured using NF-κB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
T67	10612-10798	Sentence	denotes	Binding of p65, p50 and FLAG-Tat to the double-stranded NF-κB oligonucleotide was measured using NF-κB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
T6296	10799-10931	Sentence	denotes	Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.
T68	10799-10931	Sentence	denotes	Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.
T6715	10933-10953	Sentence	denotes	In vitro translation
T69	10933-10953	Sentence	denotes	In vitro translation
T6716	10954-11137	Sentence	denotes	HA-IκB-α, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50).
T70	10954-11137	Sentence	denotes	HA-IκB-α, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50).
T6717	11138-11182	Sentence	denotes	Details are described in Supplementary Data.
T71	11138-11182	Sentence	denotes	Details are described in Supplementary Data.
T6879	11184-11227	Sentence	denotes	Immunoprecipitation assay and GST-pull down
T72	11184-11227	Sentence	denotes	Immunoprecipitation assay and GST-pull down
T6880	11228-11370	Sentence	denotes	Immunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50).
T73	11228-11370	Sentence	denotes	Immunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50).
T6881	11371-11415	Sentence	denotes	Details are described in Supplementary Data.
T74	11371-11415	Sentence	denotes	Details are described in Supplementary Data.
T7009	11417-11430	Sentence	denotes	Real-time PCR
T75	11417-11430	Sentence	denotes	Real-time PCR
T7010	11431-11681	Sentence	denotes	Total RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.
T76	11431-11681	Sentence	denotes	Total RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.
T7011	11682-11878	Sentence	denotes	Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions:
T77	11682-11878	Sentence	denotes	Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions:
T7012	11879-11921	Sentence	denotes	95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.
T78	11879-11921	Sentence	denotes	95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.
T79	11922-11982	Sentence	denotes	Primers for Tat and MIP-1α are listed in Supplementary Data.
T7013	11922-12144	Sentence	denotes	Primers for Tat and MIP-1α are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-κB signaling pathway (QIAGEN Sciences, MD, USA).
T80	11983-12144	Sentence	denotes	Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-κB signaling pathway (QIAGEN Sciences, MD, USA).
T7014	12145-12282	Sentence	denotes	Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control.
T81	12145-12282	Sentence	denotes	Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control.
T7015	12283-12363	Sentence	denotes	Relative expression was calculated as 2(Ct gene under investigation − Ct GAPDH).
T82	12283-12363	Sentence	denotes	Relative expression was calculated as 2(Ct gene under investigation − Ct GAPDH).
T7515	12365-12400	Sentence	denotes	Chromatin immunoprecipitation assay
T83	12365-12400	Sentence	denotes	Chromatin immunoprecipitation assay
T7516	12401-12490	Sentence	denotes	Cells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%.
T84	12401-12490	Sentence	denotes	Cells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%.
T7517	12491-12627	Sentence	denotes	After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped.
T85	12491-12627	Sentence	denotes	After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped.
T7518	12628-12806	Sentence	denotes	After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1× Complete Protease Inhibitor (Roche Diagnostic GmbH).
T86	12628-12806	Sentence	denotes	After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1× Complete Protease Inhibitor (Roche Diagnostic GmbH).
T7519	12807-12929	Sentence	denotes	Nuclei were pelletted (1000 × g, 5 min), and resuspended in sonication buffer (50 mM Tris–HCl pH 8.0, 1% SDS, 10 mM EDTA).
T87	12807-12929	Sentence	denotes	Nuclei were pelletted (1000 × g, 5 min), and resuspended in sonication buffer (50 mM Tris–HCl pH 8.0, 1% SDS, 10 mM EDTA).
T7520	12930-13194	Sentence	denotes	Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 × g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris–HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA).
T88	12930-13194	Sentence	denotes	Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 × g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris–HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA).
T7521	13195-13345	Sentence	denotes	Samples were pre-cleared by 3-h incubation with 20 µl of protein G agarose beads followed by incubation with antibodies against the analysed proteins.
T89	13195-13345	Sentence	denotes	Samples were pre-cleared by 3-h incubation with 20 µl of protein G agarose beads followed by incubation with antibodies against the analysed proteins.
T90	13346-13497	Sentence	denotes	Primary antibodies were: anti-p65 (sc-372), anti-IκBα (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma–Aldrich.
T7522	13346-13944	Sentence	denotes	Primary antibodies were: anti-p65 (sc-372), anti-IκBα (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma–Aldrich. Immunoprecipitations were carried out at 4°C overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS.
T91	13498-13944	Sentence	denotes	Immunoprecipitations were carried out at 4°C overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS.
T7523	13945-14012	Sentence	denotes	Protein–DNA cross-links were reverted by heating at 65°C overnight.
T92	13945-14012	Sentence	denotes	Protein–DNA cross-links were reverted by heating at 65°C overnight.
T7524	14013-14124	Sentence	denotes	DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 µl sterile distilled water.
T93	14013-14124	Sentence	denotes	DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 µl sterile distilled water.
T7525	14125-14294	Sentence	denotes	Specific enrichment in NF-κB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen).
T94	14125-14294	Sentence	denotes	Specific enrichment in NF-κB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen).
T7526	14295-14423	Sentence	denotes	Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions:
T95	14295-14423	Sentence	denotes	Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions:
T7527	14424-14466	Sentence	denotes	95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.
T96	14424-14466	Sentence	denotes	95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.
T97	14467-14540	Sentence	denotes	Primers used for MIP-1α, GAPDH and ACTB are listed in Supplementary Data.
T7528	14467-14637	Sentence	denotes	Primers used for MIP-1α, GAPDH and ACTB are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN).
T98	14541-14637	Sentence	denotes	Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN).
T7529	14638-14746	Sentence	denotes	For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.
T99	14638-14746	Sentence	denotes	For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.
T8155	14748-14768	Sentence	denotes	Statistical analysis
T100	14748-14768	Sentence	denotes	Statistical analysis
T8156	14769-14842	Sentence	denotes	Statistical analysis was performed by two-tail unpaired Student's t-test.
T101	14769-14842	Sentence	denotes	Statistical analysis was performed by two-tail unpaired Student's t-test.
T8157	14843-14876	Sentence	denotes	Data were reported as means ± SE.
T102	14843-14876	Sentence	denotes	Data were reported as means ± SE.
T8158	14877-14980	Sentence	denotes	Differences between the means were considered as statistically significant at the 95% level (P < 0.05).
T103	14877-14980	Sentence	denotes	Differences between the means were considered as statistically significant at the 95% level (P < 0.05).
T104	14982-14989	Sentence	denotes	RESULTS
T8532	14991-15094	Sentence	denotes	Tat enhances the NF-κB activity by hijacking IκB-α and inhibiting the post-activation turn off of NF-κB
T105	14991-15094	Sentence	denotes	Tat enhances the NF-κB activity by hijacking IκB-α and inhibiting the post-activation turn off of NF-κB
T8533	15095-15229	Sentence	denotes	We analysed the kinetic of NF-κB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference.
T106	15095-15229	Sentence	denotes	We analysed the kinetic of NF-κB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference.
T8534	15230-15398	Sentence	denotes	Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R−E− virions.
T107	15230-15398	Sentence	denotes	Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R−E− virions.
T8535	15399-15533	Sentence	denotes	By cytofluorimetric analysis, ∼40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1).
T108	15399-15533	Sentence	denotes	By cytofluorimetric analysis, ∼40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1).
T8536	15534-15756	Sentence	denotes	The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A).
T109	15534-15756	Sentence	denotes	The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A).
T8537	15757-16051	Sentence	denotes	The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).
T110	15757-16051	Sentence	denotes	The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).
T111	16052-16061	Sentence	denotes	Figure 1.
T23911	16062-16148	Sentence	denotes	Tat counteracts the post-activation turn off of NF-κB in single round HIV-1-infection.
T112	16062-16148	Sentence	denotes	Tat counteracts the post-activation turn off of NF-κB in single round HIV-1-infection.
T23912	16149-16861	Sentence	denotes	Jurkat cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 µg) were analysed for the p65 and p50 binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IκB-α was analysed by 12% SDS–PAGE and western blotting of nuclear or cytosolic extracts (20 µg) using anti-p65 and anti-IκB-α antibodies.
T113	16149-16861	Sentence	denotes	Jurkat cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 µg) were analysed for the p65 and p50 binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IκB-α was analysed by 12% SDS–PAGE and western blotting of nuclear or cytosolic extracts (20 µg) using anti-p65 and anti-IκB-α antibodies.
T23913	16862-16989	Sentence	denotes	Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively.
T114	16862-16989	Sentence	denotes	Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively.
T23914	16990-17207	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK Kinase Assay (Cell Signaling Technology).
T115	16990-17207	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK Kinase Assay (Cell Signaling Technology).
T23915	17208-17244	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T116	17208-17244	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T8538	17245-17738	Sentence	denotes	The progressive increase in NF-κB activity, as measured by p65–p50 binding to an NF-κB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C).
T117	17245-17738	Sentence	denotes	The progressive increase in NF-κB activity, as measured by p65–p50 binding to an NF-κB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C).
T8539	17739-17942	Sentence	denotes	IκB-α was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol).
T118	17739-17942	Sentence	denotes	IκB-α was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol).
T8540	17943-18094	Sentence	denotes	Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D).
T119	17943-18094	Sentence	denotes	Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D).
T8541	18095-18360	Sentence	denotes	Similar kinetics of NF-κB activity, IKK activation and IκB-α degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2).
T120	18095-18360	Sentence	denotes	Similar kinetics of NF-κB activity, IKK activation and IκB-α degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2).
T8542	18361-18659	Sentence	denotes	These results indicated that in single-round HIV-1 infection the NF-κB activation initially correlated with the IKK activation and IκB-α degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IκB-α.
T121	18361-18659	Sentence	denotes	These results indicated that in single-round HIV-1 infection the NF-κB activation initially correlated with the IKK activation and IκB-α degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IκB-α.
T8543	18660-18749	Sentence	denotes	Next, we tested the single action of Tat on the kinetic of NF-κB activity induced by PMA.
T122	18660-18749	Sentence	denotes	Next, we tested the single action of Tat on the kinetic of NF-κB activity induced by PMA.
T8544	18750-18942	Sentence	denotes	To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-κB activation (17,18).
T123	18750-18942	Sentence	denotes	To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-κB activation (17,18).
T8545	18943-19143	Sentence	denotes	In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IκB-α content was not significantly affected (Figure 2B, cytosol).
T124	18943-19143	Sentence	denotes	In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IκB-α content was not significantly affected (Figure 2B, cytosol).
T8546	19144-19491	Sentence	denotes	Transient stimulation with PMA increased the NF-κB DNA binding activity and nuclear p65 at 5 min peaking at 60–120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B).
T125	19144-19491	Sentence	denotes	Transient stimulation with PMA increased the NF-κB DNA binding activity and nuclear p65 at 5 min peaking at 60–120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B).
T8547	19492-19716	Sentence	denotes	In addition, Tat bound to the NF-κB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-κB complex bound to DNA.
T126	19492-19716	Sentence	denotes	In addition, Tat bound to the NF-κB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-κB complex bound to DNA.
T8548	19717-19913	Sentence	denotes	Degradation of IκB-α was progressively induced at 5–30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol).
T127	19717-19913	Sentence	denotes	Degradation of IκB-α was progressively induced at 5–30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol).
T8549	19914-20039	Sentence	denotes	Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C).
T128	19914-20039	Sentence	denotes	Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C).
T8550	20040-20196	Sentence	denotes	These results indicated that Tat enhanced the NF-κB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IκB-α content.
T129	20040-20196	Sentence	denotes	These results indicated that Tat enhanced the NF-κB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IκB-α content.
T130	20197-20206	Sentence	denotes	Figure 2.
T24351	20207-20330	Sentence	denotes	Tat enhances the NF-κB activity following PMA stimulation by associating with IκB-α and counteracting the NF-κB repression.
T131	20207-20330	Sentence	denotes	Tat enhances the NF-κB activity following PMA stimulation by associating with IκB-α and counteracting the NF-κB repression.
T24352	20331-20558	Sentence	denotes	HeLa cells (5 × 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 µg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times.
T132	20331-20558	Sentence	denotes	HeLa cells (5 × 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 µg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times.
T24353	20559-21007	Sentence	denotes	Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 µg) were separated by 12% SDS–PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IκB-α and anti-Hexokinase-II antibodies.
T133	20559-21007	Sentence	denotes	Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 µg) were separated by 12% SDS–PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IκB-α and anti-Hexokinase-II antibodies.
T24354	21008-21441	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 × 106) were transfected with or without p3xFLAG-Tat (5 µg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min.
T134	21008-21441	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 × 106) were transfected with or without p3xFLAG-Tat (5 µg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min.
T24355	21442-21546	Sentence	denotes	Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IκB-α antibody.
T135	21442-21546	Sentence	denotes	Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IκB-α antibody.
T24356	21547-21678	Sentence	denotes	Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies.
T136	21547-21678	Sentence	denotes	Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies.
T24357	21679-21775	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1).
T137	21679-21775	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1).
T24358	21776-21812	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T138	21776-21812	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T8551	21813-21967	Sentence	denotes	As we previously found that Tat binds to the sixth ankyrin of IκB-α (50,51), we tested whether Tat counteracted the generation of the IκB-α/NF-κB complex.
T139	21813-21967	Sentence	denotes	As we previously found that Tat binds to the sixth ankyrin of IκB-α (50,51), we tested whether Tat counteracted the generation of the IκB-α/NF-κB complex.
T8552	21968-22195	Sentence	denotes	To this end, the association of IκB-α with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IκB-α, respectively.
T140	21968-22195	Sentence	denotes	To this end, the association of IκB-α with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IκB-α, respectively.
T8553	22196-22494	Sentence	denotes	Coimmunoprecipitation of IκB-α with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IκB-α (Figure 2D, lanes 2 and 4), indicating that Tat competed the IκB-α binding to p65.
T141	22196-22494	Sentence	denotes	Coimmunoprecipitation of IκB-α with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IκB-α (Figure 2D, lanes 2 and 4), indicating that Tat competed the IκB-α binding to p65.
T11602	22496-22574	Sentence	denotes	Tat counteracts the IκB-α inhibition of p65 by competing the repressor binding
T142	22496-22574	Sentence	denotes	Tat counteracts the IκB-α inhibition of p65 by competing the repressor binding
T11603	22575-22709	Sentence	denotes	We further investigated the physical interaction of Tat with IκB-α using in vitro translated proteins in coimmunoprecipitation assays.
T143	22575-22709	Sentence	denotes	We further investigated the physical interaction of Tat with IκB-α using in vitro translated proteins in coimmunoprecipitation assays.
T11604	22710-22781	Sentence	denotes	For mapping the interaction domains, we used the following Tat mutants:
T144	22710-22781	Sentence	denotes	For mapping the interaction domains, we used the following Tat mutants:
T11605	22782-22861	Sentence	denotes	Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49–57)A (Figure 3A).
T145	22782-22861	Sentence	denotes	Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49–57)A (Figure 3A).
T11606	22862-22945	Sentence	denotes	IκB-α coimmunoprecipitated with all Tat proteins, except Tat R(49–57)A (Figure 3B).
T146	22862-22945	Sentence	denotes	IκB-α coimmunoprecipitated with all Tat proteins, except Tat R(49–57)A (Figure 3B).
T11607	22946-23101	Sentence	denotes	These results indicated that the arginine-rich domain of Tat was involved in the binding to IκB-α, which was consistent with previous observations (50,51).
T147	22946-23101	Sentence	denotes	These results indicated that the arginine-rich domain of Tat was involved in the binding to IκB-α, which was consistent with previous observations (50,51).
T11608	23102-23389	Sentence	denotes	Moreover, Tat associating with IκB-α competed the binding of IκB-α to p65 in a dose-dependent manner (Figure 3C, lanes 3–5), while Tat R(49–57)A, which lacked the binding site for IκB-α, did not associate with IκB-α and did not compete the binding of IκB-α to p65 (Figure 3C, lanes 6–8).
T148	23102-23389	Sentence	denotes	Moreover, Tat associating with IκB-α competed the binding of IκB-α to p65 in a dose-dependent manner (Figure 3C, lanes 3–5), while Tat R(49–57)A, which lacked the binding site for IκB-α, did not associate with IκB-α and did not compete the binding of IκB-α to p65 (Figure 3C, lanes 6–8).
T149	23390-23399	Sentence	denotes	Figure 3.
T25204	23400-23710	Sentence	denotes	Tat relieves p65 from the IκB-α inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IκB-α (5 µl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49–57)A or FLAG-Tat C(22,25,27)A (10 µl) using in vitro translated proteins.
T150	23400-23710	Sentence	denotes	Tat relieves p65 from the IκB-α inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IκB-α (5 µl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49–57)A or FLAG-Tat C(22,25,27)A (10 µl) using in vitro translated proteins.
T25205	23711-24080	Sentence	denotes	Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 µl) was incubated with in vitro translated HA-IκB-α (5 µl) in presence or absence of FLAG-Tat or FLAG-Tat R(49–57)A (5, 10 or 20 µl).
T151	23711-24080	Sentence	denotes	Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 µl) was incubated with in vitro translated HA-IκB-α (5 µl) in presence or absence of FLAG-Tat or FLAG-Tat R(49–57)A (5, 10 or 20 µl).
T25206	24081-24288	Sentence	denotes	HA-IκB-α was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65).
T152	24081-24288	Sentence	denotes	HA-IκB-α was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65).
T25207	24289-24475	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins.
T153	24289-24475	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins.
T25208	24476-24777	Sentence	denotes	32P-labeled-NF-κB double-stranded oligonucleotide was incubated with p65 (0.5 µl) in presence or absence of HA-IκB-α (1 µl), FLAG-Tat or FLAG-Tat R(49–57)A (5 and 10 µl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-κB double-stranded oligonucleotide.
T154	24476-24777	Sentence	denotes	32P-labeled-NF-κB double-stranded oligonucleotide was incubated with p65 (0.5 µl) in presence or absence of HA-IκB-α (1 µl), FLAG-Tat or FLAG-Tat R(49–57)A (5 and 10 µl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-κB double-stranded oligonucleotide.
T25209	24778-25073	Sentence	denotes	DNA/protein complexes were run on 6% PAGE–TBE and analysed by autoradiography. (E) p50−/−p65−/−MEFs (3 × 105 cells) were transfected with pκBLuc (0.5 µg) and pSV-β-Gal (0.1 µg) with or without pRc/CMV-p65 (0.5 µg), pCMV4-HA-IκB-α (0.5 µg), p3xFLAG-Tat or p3xFLAG-Tat R(49–57)A (0.5, 1 and 2 µg).
T155	24778-25073	Sentence	denotes	DNA/protein complexes were run on 6% PAGE–TBE and analysed by autoradiography. (E) p50−/−p65−/−MEFs (3 × 105 cells) were transfected with pκBLuc (0.5 µg) and pSV-β-Gal (0.1 µg) with or without pRc/CMV-p65 (0.5 µg), pCMV4-HA-IκB-α (0.5 µg), p3xFLAG-Tat or p3xFLAG-Tat R(49–57)A (0.5, 1 and 2 µg).
T25210	25074-25194	Sentence	denotes	The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to β-galactosidase activity.
T156	25074-25194	Sentence	denotes	The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to β-galactosidase activity.
T25211	25195-25279	Sentence	denotes	Fold activation was calculated relative to transfection of the pκBLuc plasmid alone.
T157	25195-25279	Sentence	denotes	Fold activation was calculated relative to transfection of the pκBLuc plasmid alone.
T25212	25280-25316	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T158	25280-25316	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T25213	25317-25483	Sentence	denotes	As control of protein expression, aliquots of cell extracts (20 µg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-γ-tubulin antibodies.
T159	25317-25483	Sentence	denotes	As control of protein expression, aliquots of cell extracts (20 µg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-γ-tubulin antibodies.
T11609	25484-25705	Sentence	denotes	Next, we evaluated whether Tat counteracted the IκB-α-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IκB-α proteins with the NF-κB probe in presence or absence of Tat followed by EMSA.
T160	25484-25705	Sentence	denotes	Next, we evaluated whether Tat counteracted the IκB-α-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IκB-α proteins with the NF-κB probe in presence or absence of Tat followed by EMSA.
T11610	25706-25852	Sentence	denotes	The p65 DNA binding activity was inhibited by IκB-α and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57) (Figure 3D).
T161	25706-25852	Sentence	denotes	The p65 DNA binding activity was inhibited by IκB-α and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57) (Figure 3D).
T162	25853-26087	Sentence	denotes	Further, we analysed the Tat effect on the IκB-α repression of p65 transcriptional activity by transfecting p50−/−p65−/−MEFs with the NF-κB-Luc reporter together with expression vectors of p65 and IκB-α, in presence or absence of Tat.
T11611	25853-26258	Sentence	denotes	Further, we analysed the Tat effect on the IκB-α repression of p65 transcriptional activity by transfecting p50−/−p65−/−MEFs with the NF-κB-Luc reporter together with expression vectors of p65 and IκB-α, in presence or absence of Tat. IκB-α inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57)A (Figure 3E).
T163	26088-26258	Sentence	denotes	IκB-α inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57)A (Figure 3E).
T11612	26259-26457	Sentence	denotes	Altogether these results indicated that Tat counteracts the IκB-α repression of the p65 DNA-binding and transcriptional activity by associating with IκB-α and competing the repressor binding to p65.
T164	26259-26457	Sentence	denotes	Altogether these results indicated that Tat counteracts the IκB-α repression of the p65 DNA-binding and transcriptional activity by associating with IκB-α and competing the repressor binding to p65.
T13324	26459-26533	Sentence	denotes	Tat increases the p65 affinity binding to DNA through association with p65
T165	26459-26533	Sentence	denotes	Tat increases the p65 affinity binding to DNA through association with p65
T13325	26534-26587	Sentence	denotes	We tested whether Tat physically interacted with p65.
T166	26534-26587	Sentence	denotes	We tested whether Tat physically interacted with p65.
T13326	26588-26729	Sentence	denotes	HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts.
T167	26588-26729	Sentence	denotes	HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts.
T13327	26730-26973	Sentence	denotes	The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A).
T168	26730-26973	Sentence	denotes	The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A).
T13328	26974-27202	Sentence	denotes	Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B).
T169	26974-27202	Sentence	denotes	Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B).
T13329	27203-27368	Sentence	denotes	These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.
T170	27203-27368	Sentence	denotes	These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.
T171	27369-27378	Sentence	denotes	Figure 4.
T26499	27379-27646	Sentence	denotes	Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg).
T172	27379-27646	Sentence	denotes	Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg).
T173	27647-28028	Sentence	denotes	FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose.
T26500	27647-28416	Sentence	denotes	FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg).
T174	28029-28416	Sentence	denotes	Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg).
T26501	28417-28947	Sentence	denotes	Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide.
T175	28417-28947	Sentence	denotes	Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide.
T26502	28948-29092	Sentence	denotes	DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.
T176	28948-29092	Sentence	denotes	DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.
T13330	29093-29328	Sentence	denotes	To identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C).
T177	29093-29328	Sentence	denotes	To identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C).
T13331	29329-29497	Sentence	denotes	Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.
T178	29329-29497	Sentence	denotes	Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.
T13332	29498-29764	Sentence	denotes	To address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA.
T179	29498-29764	Sentence	denotes	To address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA.
T13333	29765-29929	Sentence	denotes	Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E).
T180	29765-29929	Sentence	denotes	Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E).
T13334	29930-30143	Sentence	denotes	The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).
T181	29930-30143	Sentence	denotes	The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).
T15219	30145-30300	Sentence	denotes	Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement
T182	30145-30300	Sentence	denotes	Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement
T15220	30301-30383	Sentence	denotes	We analysed the action of Tat on the expression of NF-κB-responsive genes in vivo.
T183	30301-30383	Sentence	denotes	We analysed the action of Tat on the expression of NF-κB-responsive genes in vivo.
T15221	30384-30582	Sentence	denotes	To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR.
T184	30384-30582	Sentence	denotes	To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR.
T15222	30583-30749	Sentence	denotes	Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E).
T185	30583-30749	Sentence	denotes	Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E).
T15223	30750-30956	Sentence	denotes	The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E).
T186	30750-30956	Sentence	denotes	The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E).
T15224	30957-31093	Sentence	denotes	Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G).
T187	30957-31093	Sentence	denotes	Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G).
T15225	31094-31383	Sentence	denotes	These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.
T188	31094-31383	Sentence	denotes	These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.
T189	31384-31393	Sentence	denotes	Figure 5.
T27258	31394-31554	Sentence	denotes	Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement.
T190	31394-31554	Sentence	denotes	Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement.
T27259	31555-31808	Sentence	denotes	HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol).
T191	31555-31808	Sentence	denotes	HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol).
T27260	31809-32150	Sentence	denotes	Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody.
T192	31809-32150	Sentence	denotes	Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody.
T27261	32151-32435	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody.
T193	32151-32435	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody.
T27262	32436-32715	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody.
T194	32436-32715	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody.
T27263	32716-32794	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters.
T195	32716-32794	Sentence	denotes	Real-time PCR was performed with primers specific for the indicated promoters.
T27264	32795-32831	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T196	32795-32831	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T27265	32832-32977	Sentence	denotes	The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).
T197	32832-32977	Sentence	denotes	The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).
T15226	32978-33115	Sentence	denotes	MIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56).
T198	32978-33115	Sentence	denotes	MIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56).
T15227	33116-33331	Sentence	denotes	Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62).
T199	33116-33331	Sentence	denotes	Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62).
T15228	33332-33403	Sentence	denotes	The transcriptional regulation of MIP-1α has been poorly characterized.
T200	33332-33403	Sentence	denotes	The transcriptional regulation of MIP-1α has been poorly characterized.
T15229	33404-33669	Sentence	denotes	Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A).
T201	33404-33669	Sentence	denotes	Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A).
T15230	33670-33917	Sentence	denotes	To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP.
T202	33670-33917	Sentence	denotes	To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP.
T15231	33918-34127	Sentence	denotes	TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C).
T203	33918-34127	Sentence	denotes	TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C).
T15232	34128-34287	Sentence	denotes	Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D).
T204	34128-34287	Sentence	denotes	Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D).
T15233	34288-34434	Sentence	denotes	Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.
T205	34288-34434	Sentence	denotes	Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.
T15234	34435-34619	Sentence	denotes	Similarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H).
T206	34435-34619	Sentence	denotes	Similarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H).
T15235	34620-34839	Sentence	denotes	By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction.
T207	34620-34839	Sentence	denotes	By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction.
T15236	34840-35098	Sentence	denotes	As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters.
T208	34840-35098	Sentence	denotes	As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters.
T17783	35100-35247	Sentence	denotes	In HIV-1-infected monocytes Tat sustains the NF-κB activity and promotes the transcriptional activation of MIP-1α by interacting with IκB-α and p65
T209	35100-35247	Sentence	denotes	In HIV-1-infected monocytes Tat sustains the NF-κB activity and promotes the transcriptional activation of MIP-1α by interacting with IκB-α and p65
T17784	35248-35367	Sentence	denotes	We next analysed whether Tat affected the NF-κB activity and the expression of MIP-1α in the course of HIV-1 infection.
T210	35248-35367	Sentence	denotes	We next analysed whether Tat affected the NF-κB activity and the expression of MIP-1α in the course of HIV-1 infection.
T17785	35368-35536	Sentence	denotes	To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R−E− virions, which were used in single-round infection of U937, a human monocytic cell line.
T211	35368-35536	Sentence	denotes	To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R−E− virions, which were used in single-round infection of U937, a human monocytic cell line.
T17786	35537-35724	Sentence	denotes	Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-κB activation following viral infection.
T212	35537-35724	Sentence	denotes	Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-κB activation following viral infection.
T17787	35725-35870	Sentence	denotes	The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).
T213	35725-35870	Sentence	denotes	The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).
T214	35871-35880	Sentence	denotes	Figure 6.
T28289	35881-36002	Sentence	denotes	In HIV-1 infection Tat sustains the NF-κB activity and enhances the MIP-1α expression via interaction with IκB-α and p65.
T215	35881-36002	Sentence	denotes	In HIV-1 infection Tat sustains the NF-κB activity and enhances the MIP-1α expression via interaction with IκB-α and p65.
T28290	36003-36655	Sentence	denotes	U937 cells (5 × 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts (10 µg) using the NF-κB Transcription Factor ELISA assay kit (Cayman). (C) The IκB-α content was analysed by 12% SDS–PAGE and western blotting of cytosolic extracts (20 µg) using anti-IκB-α antibody.
T216	36003-36655	Sentence	denotes	U937 cells (5 × 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts (10 µg) using the NF-κB Transcription Factor ELISA assay kit (Cayman). (C) The IκB-α content was analysed by 12% SDS–PAGE and western blotting of cytosolic extracts (20 µg) using anti-IκB-α antibody.
T28291	36656-36985	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 µg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 × 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T217	36656-36985	Sentence	denotes	Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 µg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 × 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T28292	36986-37116	Sentence	denotes	Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody.
T218	36986-37116	Sentence	denotes	Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody.
T28293	37117-37467	Sentence	denotes	Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies. (F) U937 cells (5 × 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T219	37117-37467	Sentence	denotes	Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies. (F) U937 cells (5 × 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T28294	37468-37779	Sentence	denotes	Twenty-four-hour post-infection, total RNA was analysed for MIP-1α expression by real-time PCR. (G and H) U937 cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T220	37468-37779	Sentence	denotes	Twenty-four-hour post-infection, total RNA was analysed for MIP-1α expression by real-time PCR. (G and H) U937 cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.
T28295	37780-37867	Sentence	denotes	Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H).
T221	37780-37867	Sentence	denotes	Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H).
T28296	37868-37949	Sentence	denotes	Real-time PCR was performed with primers specific for MIP-1α and GAPDH promoters.
T222	37868-37949	Sentence	denotes	Real-time PCR was performed with primers specific for MIP-1α and GAPDH promoters.
T28297	37950-37986	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T223	37950-37986	Sentence	denotes	Values (mean ± SE, n = 3) are shown.
T28298	37987-38125	Sentence	denotes	The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).
T224	37987-38125	Sentence	denotes	The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).
T17788	38126-38359	Sentence	denotes	The NF-κB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B).
T225	38126-38359	Sentence	denotes	The NF-κB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B).
T17789	38360-38518	Sentence	denotes	Degradation of IκB-α was observed at 3-h post-infection, and was followed by de novo synthesis of IκB-α at 12 h independently of the Tat presence (Figure 6C).
T226	38360-38518	Sentence	denotes	Degradation of IκB-α was observed at 3-h post-infection, and was followed by de novo synthesis of IκB-α at 12 h independently of the Tat presence (Figure 6C).
T17790	38519-38653	Sentence	denotes	Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D).
T227	38519-38653	Sentence	denotes	Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D).
T17791	38654-38882	Sentence	denotes	These results agreed with the kinetic of NF-κB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-κB.
T228	38654-38882	Sentence	denotes	These results agreed with the kinetic of NF-κB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-κB.
T17792	38883-39136	Sentence	denotes	In HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IκB-α and p65 (Figure 6E), and activated the MIP-1α expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F).
T229	38883-39136	Sentence	denotes	In HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IκB-α and p65 (Figure 6E), and activated the MIP-1α expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F).
T17793	39137-39281	Sentence	denotes	Moreover, Tat and p65 were both recruited to the NF-κB enhancer of MIP-1α, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H).
T230	39137-39281	Sentence	denotes	Moreover, Tat and p65 were both recruited to the NF-κB enhancer of MIP-1α, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H).
T17794	39282-39525	Sentence	denotes	Altogether these results indicated that in single round HIV-1 infection Tat associated with IκB-α and p65, and induced the p65-dependent activation of MIP-1α expression through occupancy of the MIP-1α promoter and increased recruitment of p65.
T231	39282-39525	Sentence	denotes	Altogether these results indicated that in single round HIV-1 infection Tat associated with IκB-α and p65, and induced the p65-dependent activation of MIP-1α expression through occupancy of the MIP-1α promoter and increased recruitment of p65.
T232	39527-39537	Sentence	denotes	DISCUSSION
T19880	39538-39627	Sentence	denotes	This study reports a novel mechanism of NF-κB activation by the HIV-1 Tat transactivator.
T233	39538-39627	Sentence	denotes	This study reports a novel mechanism of NF-κB activation by the HIV-1 Tat transactivator.
T19881	39628-39901	Sentence	denotes	Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-κB (49,63,64), and physically interacted with the IκB-α repressor (50,51), we investigated the possibility that Tat could activate NF-κB via direct interaction with IκB-α and p65.
T234	39628-39901	Sentence	denotes	Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-κB (49,63,64), and physically interacted with the IκB-α repressor (50,51), we investigated the possibility that Tat could activate NF-κB via direct interaction with IκB-α and p65.
T19882	39902-40033	Sentence	denotes	To this end, the NF-κB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression.
T235	39902-40033	Sentence	denotes	To this end, the NF-κB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression.
T19883	40034-40186	Sentence	denotes	By this approach, we avoided the perpetuation of NF-κB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31).
T236	40034-40186	Sentence	denotes	By this approach, we avoided the perpetuation of NF-κB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31).
T237	40187-40323	Sentence	denotes	Upon HIV-1 infection, the early NF-κB activation occurred concomitantly with IKK activation and IκB-α degradation in the absence of Tat.
T19884	40187-40512	Sentence	denotes	Upon HIV-1 infection, the early NF-κB activation occurred concomitantly with IKK activation and IκB-α degradation in the absence of Tat. Soon after the shut off of IKK activity and new synthesis of IκB-α, the NF-κB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene.
T238	40324-40512	Sentence	denotes	Soon after the shut off of IKK activity and new synthesis of IκB-α, the NF-κB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene.
T19885	40513-40669	Sentence	denotes	These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-κB activity without affecting the IKK activity and the half-life of IκB-α.
T239	40513-40669	Sentence	denotes	These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-κB activity without affecting the IKK activity and the half-life of IκB-α.
T19886	40670-40867	Sentence	denotes	Similar kinetic of NF-κB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-κB in presence of newly synthesized IκB-α.
T240	40670-40867	Sentence	denotes	Similar kinetic of NF-κB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-κB in presence of newly synthesized IκB-α.
T19887	40868-41033	Sentence	denotes	The Tat-dependent activation of NF-κB correlated with the association of the viral protein with IκB-α, which interfered with the generation of the IκB-α/p65 complex.
T241	40868-41033	Sentence	denotes	The Tat-dependent activation of NF-κB correlated with the association of the viral protein with IκB-α, which interfered with the generation of the IκB-α/p65 complex.
T19888	41034-41153	Sentence	denotes	This evidence demonstrated that Tat competed for the binding of IκB-α to p65 and prevented the IκB-α repression of p65.
T242	41034-41153	Sentence	denotes	This evidence demonstrated that Tat competed for the binding of IκB-α to p65 and prevented the IκB-α repression of p65.
T19889	41154-41490	Sentence	denotes	As additional mechanism of NF-κB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-κB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65.
T243	41154-41490	Sentence	denotes	As additional mechanism of NF-κB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-κB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65.
T19890	41491-41627	Sentence	denotes	We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-κB activation.
T244	41491-41627	Sentence	denotes	We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-κB activation.
T19891	41628-41809	Sentence	denotes	Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IκB-α (50,51), and for releasing p65 from the IκB-α inhibition.
T245	41628-41809	Sentence	denotes	Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IκB-α (50,51), and for releasing p65 from the IκB-α inhibition.
T19892	41810-42018	Sentence	denotes	Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-κB by hijacking the IκB-α repressor, shielding p65 from the IκB-α embrace, and enforcing the p65 binding to DNA.
T246	41810-42018	Sentence	denotes	Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-κB by hijacking the IκB-α repressor, shielding p65 from the IκB-α embrace, and enforcing the p65 binding to DNA.
T19893	42019-42282	Sentence	denotes	Further evidence of Tat activation of NF-κB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-κB-responsive genes, including MIP-1α, CSF3, LTA, NFKBIA and TLR2.
T247	42019-42282	Sentence	denotes	Further evidence of Tat activation of NF-κB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-κB-responsive genes, including MIP-1α, CSF3, LTA, NFKBIA and TLR2.
T19894	42283-42438	Sentence	denotes	By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-κB enhancers of the activated genes, and was recovered at the same sites.
T248	42283-42438	Sentence	denotes	By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-κB enhancers of the activated genes, and was recovered at the same sites.
T19895	42439-42585	Sentence	denotes	RNA interference of p65 abolished the Tat occupancy of the NF-κB enhancers indicating that the Tat binding to the NF-κB sites was mediated by p65.
T249	42439-42585	Sentence	denotes	RNA interference of p65 abolished the Tat occupancy of the NF-κB enhancers indicating that the Tat binding to the NF-κB sites was mediated by p65.
T19896	42586-42785	Sentence	denotes	These results were consistent with the evidence that Tat displaced p65 from the binding to IκB-α (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F).
T250	42586-42785	Sentence	denotes	These results were consistent with the evidence that Tat displaced p65 from the binding to IκB-α (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F).
T19897	42786-42962	Sentence	denotes	Thus, Tat likely increased the recruitment of p65 to the NF-κB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IκB-α.
T251	42786-42962	Sentence	denotes	Thus, Tat likely increased the recruitment of p65 to the NF-κB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IκB-α.
T252	42963-43087	Sentence	denotes	Of interest, IκB-α was found associated to the NF-κB enhancers in absence of Tat, where it was displaced in presence of Tat.
T19898	42963-43205	Sentence	denotes	Of interest, IκB-α was found associated to the NF-κB enhancers in absence of Tat, where it was displaced in presence of Tat. These results suggest a negative regulatory role of IκB-α in gene regulation through occupancy of specific promoters.
T253	43088-43205	Sentence	denotes	These results suggest a negative regulatory role of IκB-α in gene regulation through occupancy of specific promoters.
T19899	43206-43412	Sentence	denotes	Indeed, IκB-α was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-α stimulation causing histone acetylation and hes1 transcriptional activation (65).
T254	43206-43412	Sentence	denotes	Indeed, IκB-α was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-α stimulation causing histone acetylation and hes1 transcriptional activation (65).
T19900	43413-43621	Sentence	denotes	A similar mechanism of gene regulation could apply to the NF-κB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IκB-α and promoting the p65 loading (Figure 7).
T255	43413-43621	Sentence	denotes	A similar mechanism of gene regulation could apply to the NF-κB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IκB-α and promoting the p65 loading (Figure 7).
T256	43622-43631	Sentence	denotes	Figure 7.
T29103	43632-44044	Sentence	denotes	Model of MIP-1α transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IκB-α occupies the NF-κB enhancer of the MIP-1α promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1α expression by removing the IκB-α repressor from the NF-κB enhancer, and by increasing the binding of p65 NF-κB complex to the NF-κB enhancer.
T257	43632-44044	Sentence	denotes	Model of MIP-1α transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IκB-α occupies the NF-κB enhancer of the MIP-1α promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1α expression by removing the IκB-α repressor from the NF-κB enhancer, and by increasing the binding of p65 NF-κB complex to the NF-κB enhancer.
T19901	44045-44541	Sentence	denotes	The physiological relevance of Tat cross talk with NF-κB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-κB, as shown by: (i) sustained NF-κB activity by Tat in presence of newly synthesized IκB-α; (ii) coimmunoprecipitation of Tat with IκB-α and p65; (iii) induction of MIP-1α expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1α NF-κB enhancer associated with increased recruitment of p65.
T258	44045-44541	Sentence	denotes	The physiological relevance of Tat cross talk with NF-κB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-κB, as shown by: (i) sustained NF-κB activity by Tat in presence of newly synthesized IκB-α; (ii) coimmunoprecipitation of Tat with IκB-α and p65; (iii) induction of MIP-1α expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1α NF-κB enhancer associated with increased recruitment of p65.
T19902	44542-44645	Sentence	denotes	This study supports the pro-inflammatory action of Tat through physical interaction with p65 and IκB-α.
T259	44542-44645	Sentence	denotes	This study supports the pro-inflammatory action of Tat through physical interaction with p65 and IκB-α.
T19903	44646-44832	Sentence	denotes	Several inflammatory cytokines under the transcriptional control of NF-κB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-α (68), and MIP-1α (57–62,69–71).
T260	44646-44832	Sentence	denotes	Several inflammatory cytokines under the transcriptional control of NF-κB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-α (68), and MIP-1α (57–62,69–71).

T89

0-94

Sentence

denotes

Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65

T1

0-94

Sentence

denotes

Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65

T2

97-105

Sentence

denotes

Abstract

T90

106-248

Sentence

denotes

Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection.

T3

106-248

Sentence

denotes

Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection.

T91

249-347

Sentence

denotes

Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS.

T4

249-347

Sentence

denotes

Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS.

T92

348-568

Sentence

denotes

We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex.

T5

348-568

Sentence

denotes

We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex.

T93

569-696

Sentence

denotes

Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity.

T6

569-696

Sentence

denotes

Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity.

T94

697-841

Sentence

denotes

The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity.

T7

697-841

Sentence

denotes

The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity.

T95

842-1148

Sentence

denotes

Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2.

T8

842-1148

Sentence

denotes

Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2.

T96

1149-1358

Sentence

denotes

Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.

T9

1149-1358

Sentence

denotes

Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.

T10

1360-1372

Sentence

denotes

INTRODUCTION

T1429

1373-1516

Sentence

denotes

Nuclear factor (NF)-κB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1).

T11

1373-1516

Sentence

denotes

Nuclear factor (NF)-κB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1).

T1430

1517-1698

Sentence

denotes

The NF-κB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding.

T12

1517-1698

Sentence

denotes

The NF-κB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding.

T1431

1699-1891

Sentence

denotes

The transcriptional activity of the NF-κB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).

T13

1699-1891

Sentence

denotes

The transcriptional activity of the NF-κB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).

T1432

1892-1993

Sentence

denotes

Inhibitors of NF-κB (IκB) associate with the NF-κB complex and interfere with its binding to DNA (3).

T14

1892-1993

Sentence

denotes

Inhibitors of NF-κB (IκB) associate with the NF-κB complex and interfere with its binding to DNA (3).

T1433

1994-2248

Sentence

denotes

In the canonical pathway of NF-κB activation, the activated IκB kinase (IKK) phosphorylates IκB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-κB complex in the nucleus.

T15

1994-2248

Sentence

denotes

In the canonical pathway of NF-κB activation, the activated IκB kinase (IKK) phosphorylates IκB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-κB complex in the nucleus.

T1434

2249-2438

Sentence

denotes

IκB-α, the most abundant inhibitor of NF-κB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6).

T16

2249-2438

Sentence

denotes

IκB-α, the most abundant inhibitor of NF-κB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6).

T1435

2439-2575

Sentence

denotes

The NF-κB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCζ (9) and Ser536 by IKKα (10,11).

T17

2439-2575

Sentence

denotes

The NF-κB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCζ (9) and Ser536 by IKKα (10,11).

T1436

2576-2758

Sentence

denotes

Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IκB-α, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13).

T18

2576-2758

Sentence

denotes

Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IκB-α, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13).

T1437

2759-2951

Sentence

denotes

Post-activation turn off of NF-κB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-κB, such as IκB-α and ubiquitin-editing protein A20 (14–20).

T19

2759-2951

Sentence

denotes

Post-activation turn off of NF-κB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-κB, such as IκB-α and ubiquitin-editing protein A20 (14–20).

T1438

2952-3095

Sentence

denotes

Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-κB activity (12,21,22).

T20

2952-3095

Sentence

denotes

Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-κB activity (12,21,22).

T1439

3096-3363

Sentence

denotes

Persistent activation of NF-κB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-κB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23–25).

T21

3096-3363

Sentence

denotes

Persistent activation of NF-κB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-κB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23–25).

T1440

3364-3451

Sentence

denotes

Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27).

T22

3364-3451

Sentence

denotes

Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27).

T1441

3452-3670

Sentence

denotes

Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29).

T23

3452-3670

Sentence

denotes

Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29).

T1442

3671-3789

Sentence

denotes

Thus, understanding the mechanisms of NF-κB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.

T24

3671-3789

Sentence

denotes

Thus, understanding the mechanisms of NF-κB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.

T1443

3790-3928

Sentence

denotes

In HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-κB activity by activation of IKK (30) and procaspase 8 (31).

T25

3790-3928

Sentence

denotes

In HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-κB activity by activation of IKK (30) and procaspase 8 (31).

T1444

3929-4078

Sentence

denotes

Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication.

T26

3929-4078

Sentence

denotes

Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication.

T1445

4079-4328

Sentence

denotes

Tat binds to RNA stem-loop structures generated by the 5′ end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor β (TNFβ) (33) and interleukin-6 (IL-6) (34) to activate gene transcription.

T27

4079-4328

Sentence

denotes

Tat binds to RNA stem-loop structures generated by the 5′ end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor β (TNFβ) (33) and interleukin-6 (IL-6) (34) to activate gene transcription.

T1446

4329-4591

Sentence

denotes

Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPβ (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41–43).

T28

4329-4591

Sentence

denotes

Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPβ (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41–43).

T1447

4592-4774

Sentence

denotes

When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).

T29

4592-4774

Sentence

denotes

When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).

T1448

4775-4886

Sentence

denotes

We first reported that NF-κB was constitutively active in Jurkat cells that stably expressed the Tat gene (47).

T30

4775-4886

Sentence

denotes

We first reported that NF-κB was constitutively active in Jurkat cells that stably expressed the Tat gene (47).

T1449

4887-5126

Sentence

denotes

Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IκB-α (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49).

T31

4887-5126

Sentence

denotes

Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IκB-α (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49).

T1450

5127-5358

Sentence

denotes

These findings suggested that Tat modulates crucial enzymes involved in NF-κB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-κB, which is mainly dependent on de novo synthesis of IκB-α (15,17).

T32

5127-5358

Sentence

denotes

These findings suggested that Tat modulates crucial enzymes involved in NF-κB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-κB, which is mainly dependent on de novo synthesis of IκB-α (15,17).

T1451

5359-5471

Sentence

denotes

We previously found that IκB-α binds to Tat and promotes the nuclear export of the viral transactivator (50,51).

T33

5359-5471

Sentence

denotes

We previously found that IκB-α binds to Tat and promotes the nuclear export of the viral transactivator (50,51).

T1452

5472-5684

Sentence

denotes

In this study, we report that Tat counteracts the post-activation turn off of NF-κB through direct interaction with IκB-α and p65, which enhances the DNA binding and transcriptional activity of the NF-κB complex.

T34

5472-5684

Sentence

denotes

In this study, we report that Tat counteracts the post-activation turn off of NF-κB through direct interaction with IκB-α and p65, which enhances the DNA binding and transcriptional activity of the NF-κB complex.

T1453

5685-5823

Sentence

denotes

The new mechanism of NF-κB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.

T35

5685-5823

Sentence

denotes

The new mechanism of NF-κB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.

T36

5825-5846

Sentence

denotes

MATERIALS AND METHODS

T4409

5848-5856

Sentence

denotes

Plasmids

T37

5848-5856

Sentence

denotes

Plasmids

T4410

5857-6081

Sentence

denotes

The plasmids pcDNA-3xHA-IκB-α, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50).

T38

5857-6081

Sentence

denotes

The plasmids pcDNA-3xHA-IκB-α, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50).

T4411

6082-6294

Sentence

denotes

The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pκBluc and pSV-β-Gal were purchased from Promega (Madison, WI, USA).

T39

6082-6294

Sentence

denotes

The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pκBluc and pSV-β-Gal were purchased from Promega (Madison, WI, USA).

T4412

6295-6560

Sentence

denotes

The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1–318), pRc/CMV-3xHA-p65ΔN(122–551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.

T40

6295-6560

Sentence

denotes

The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1–318), pRc/CMV-3xHA-p65ΔN(122–551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.

T4906

6562-6614

Sentence

denotes

Cells, transfection, treatments and luciferase assay

T41

6562-6614

Sentence

denotes

Cells, transfection, treatments and luciferase assay

T4907

6615-6841

Sentence

denotes

HeLa, p50−/−p65−/− mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640.

T42

6615-6841

Sentence

denotes

HeLa, p50−/−p65−/− mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640.

T4908

6842-6891

Sentence

denotes

PBMCs were isolated as previously described (53).

T43

6842-6891

Sentence

denotes

PBMCs were isolated as previously described (53).

T4909

6892-7008

Sentence

denotes

Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany).

T44

6892-7008

Sentence

denotes

Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany).

T4910

7009-7278

Sentence

denotes

HeLa, p50−/−p65−/− MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA).

T45

7009-7278

Sentence

denotes

HeLa, p50−/−p65−/− MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA).

T4911

7279-7567

Sentence

denotes

For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-α (TNF-α; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture.

T46

7279-7567

Sentence

denotes

For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-α (TNF-α; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture.

T4912

7568-7671

Sentence

denotes

For luciferase assays, pSV-β-Gal was co-transfected with pκBluc to monitor the transfection efficiency.

T47

7568-7671

Sentence

denotes

For luciferase assays, pSV-β-Gal was co-transfected with pκBluc to monitor the transfection efficiency.

T4913

7672-7971

Sentence

denotes

Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and β-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA).

T48

7672-7971

Sentence

denotes

Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and β-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA).

T4914

7972-8079

Sentence

denotes

The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units.

T49

7972-8079

Sentence

denotes

The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units.

T5677

8081-8097

Sentence

denotes

RNA interference

T50

8081-8097

Sentence

denotes

RNA interference

T5678

8098-8223

Sentence

denotes

Jurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA).

T51

8098-8223

Sentence

denotes

Jurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA).

T5679

8224-8506

Sentence

denotes

Briefly, aliquots (5 × 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 µF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium.

T52

8224-8506

Sentence

denotes

Briefly, aliquots (5 × 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 µF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium.

T5680

8507-8726

Sentence

denotes

RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU.

T53

8507-8726

Sentence

denotes

RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU.

T5681

8727-8841

Sentence

denotes

RNA interference of p65 and IκB-α was performed with SMART pool siRNA p65 and IκB-α (Dharmacon, Chicago, IL, USA).

T54

8727-8841

Sentence

denotes

RNA interference of p65 and IκB-α was performed with SMART pool siRNA p65 and IκB-α (Dharmacon, Chicago, IL, USA).

T6045

8843-8889

Sentence

denotes

Pseudotyped virions and single round infection

T55

8843-8889

Sentence

denotes

Pseudotyped virions and single round infection

T6046

8890-9072

Sentence

denotes

293T cells (1 × 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 µg) together with pHXB2 Env (10 µg), and 48-h post-transfection cell supernatant was collected.

T56

8890-9072

Sentence

denotes

293T cells (1 × 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 µg) together with pHXB2 Env (10 µg), and 48-h post-transfection cell supernatant was collected.

T6047

9073-9169

Sentence

denotes

Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration.

T57

9073-9169

Sentence

denotes

Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration.

T6048

9170-9319

Sentence

denotes

PBMCs, Jurkat or U937 cells (5 × 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).

T58

9170-9319

Sentence

denotes

PBMCs, Jurkat or U937 cells (5 × 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).

T6288

9321-9388

Sentence

denotes

Cell extracts, western blotting, IKK activity and NF-κB DNA binding

T59

9321-9388

Sentence

denotes

Cell extracts, western blotting, IKK activity and NF-κB DNA binding

T60

9389-9515

Sentence

denotes

Total, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data.

T6289

9389-10064

Sentence

denotes

Total, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data. Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris–HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% β-mercaptoethanol, 25% glycerol), resolved on 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories).

T61

9516-10064

Sentence

denotes

Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris–HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% β-mercaptoethanol, 25% glycerol), resolved on 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories).

T6290

10065-10155

Sentence

denotes

Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham).

T62

10065-10155

Sentence

denotes

Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham).

T6291

10156-10195

Sentence

denotes

Primary antibodies were purchased from:

T63

10156-10195

Sentence

denotes

Primary antibodies were purchased from:

T6292

10196-10396

Sentence

denotes

Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IκB-α C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-γ-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65).

T64

10196-10396

Sentence

denotes

Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IκB-α C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-γ-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65).

T6293

10397-10477

Sentence

denotes

Densitometry of single bands was analysed by ImageJ software package (NIH, USA).

T65

10397-10477

Sentence

denotes

Densitometry of single bands was analysed by ImageJ software package (NIH, USA).

T6294

10478-10611

Sentence

denotes

IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA).

T66

10478-10611

Sentence

denotes

IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA).

T6295

10612-10798

Sentence

denotes

Binding of p65, p50 and FLAG-Tat to the double-stranded NF-κB oligonucleotide was measured using NF-κB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).

T67

10612-10798

Sentence

denotes

Binding of p65, p50 and FLAG-Tat to the double-stranded NF-κB oligonucleotide was measured using NF-κB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).

T6296

10799-10931

Sentence

denotes

Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.

T68

10799-10931

Sentence

denotes

Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.

T6715

10933-10953

Sentence

denotes

In vitro translation

T69

10933-10953

Sentence

denotes

In vitro translation

T6716

10954-11137

Sentence

denotes

HA-IκB-α, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50).

T70

10954-11137

Sentence

denotes

HA-IκB-α, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50).

T6717

11138-11182

Sentence

denotes

Details are described in Supplementary Data.

T71

11138-11182

Sentence

denotes

Details are described in Supplementary Data.

T6879

11184-11227

Sentence

denotes

Immunoprecipitation assay and GST-pull down

T72

11184-11227

Sentence

denotes

Immunoprecipitation assay and GST-pull down

T6880

11228-11370

Sentence

denotes

Immunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50).

T73

11228-11370

Sentence

denotes

Immunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50).

T6881

11371-11415

Sentence

denotes

Details are described in Supplementary Data.

T74

11371-11415

Sentence

denotes

Details are described in Supplementary Data.

T7009

11417-11430

Sentence

denotes

Real-time PCR

T75

11417-11430

Sentence

denotes

Real-time PCR

T7010

11431-11681

Sentence

denotes

Total RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.

T76

11431-11681

Sentence

denotes

Total RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.

T7011

11682-11878

Sentence

denotes

Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions:

T77

11682-11878

Sentence

denotes

Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions:

T7012

11879-11921

Sentence

denotes

95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.

T78

11879-11921

Sentence

denotes

95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.

T79

11922-11982

Sentence

denotes

Primers for Tat and MIP-1α are listed in Supplementary Data.

T7013

11922-12144

Sentence

denotes

Primers for Tat and MIP-1α are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-κB signaling pathway (QIAGEN Sciences, MD, USA).

T80

11983-12144

Sentence

denotes

Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-κB signaling pathway (QIAGEN Sciences, MD, USA).

T7014

12145-12282

Sentence

denotes

Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control.

T81

12145-12282

Sentence

denotes

Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control.

T7015

12283-12363

Sentence

denotes

Relative expression was calculated as 2(Ct gene under investigation − Ct GAPDH).

T82

12283-12363

Sentence

denotes

Relative expression was calculated as 2(Ct gene under investigation − Ct GAPDH).

T7515

12365-12400

Sentence

denotes

Chromatin immunoprecipitation assay

T83

12365-12400

Sentence

denotes

Chromatin immunoprecipitation assay

T7516

12401-12490

Sentence

denotes

Cells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%.

T84

12401-12490

Sentence

denotes

Cells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%.

T7517

12491-12627

Sentence

denotes

After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped.

T85

12491-12627

Sentence

denotes

After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped.

T7518

12628-12806

Sentence

denotes

After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1× Complete Protease Inhibitor (Roche Diagnostic GmbH).

T86

12628-12806

Sentence

denotes

After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1× Complete Protease Inhibitor (Roche Diagnostic GmbH).

T7519

12807-12929

Sentence

denotes

Nuclei were pelletted (1000 × g, 5 min), and resuspended in sonication buffer (50 mM Tris–HCl pH 8.0, 1% SDS, 10 mM EDTA).

T87

12807-12929

Sentence

denotes

Nuclei were pelletted (1000 × g, 5 min), and resuspended in sonication buffer (50 mM Tris–HCl pH 8.0, 1% SDS, 10 mM EDTA).

T7520

12930-13194

Sentence

denotes

Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 × g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris–HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA).

T88

12930-13194

Sentence

denotes

Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 × g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris–HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA).

T7521

13195-13345

Sentence

denotes

Samples were pre-cleared by 3-h incubation with 20 µl of protein G agarose beads followed by incubation with antibodies against the analysed proteins.

T89

13195-13345

Sentence

denotes

Samples were pre-cleared by 3-h incubation with 20 µl of protein G agarose beads followed by incubation with antibodies against the analysed proteins.

T90

13346-13497

Sentence

denotes

Primary antibodies were: anti-p65 (sc-372), anti-IκBα (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma–Aldrich.

T7522

13346-13944

Sentence

denotes

Primary antibodies were: anti-p65 (sc-372), anti-IκBα (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma–Aldrich. Immunoprecipitations were carried out at 4°C overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS.

T91

13498-13944

Sentence

denotes

Immunoprecipitations were carried out at 4°C overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris–HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS.

T7523

13945-14012

Sentence

denotes

Protein–DNA cross-links were reverted by heating at 65°C overnight.

T92

13945-14012

Sentence

denotes

Protein–DNA cross-links were reverted by heating at 65°C overnight.

T7524

14013-14124

Sentence

denotes

DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 µl sterile distilled water.

T93

14013-14124

Sentence

denotes

DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 µl sterile distilled water.

T7525

14125-14294

Sentence

denotes

Specific enrichment in NF-κB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen).

T94

14125-14294

Sentence

denotes

Specific enrichment in NF-κB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen).

T7526

14295-14423

Sentence

denotes

Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions:

T95

14295-14423

Sentence

denotes

Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions:

T7527

14424-14466

Sentence

denotes

95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.

T96

14424-14466

Sentence

denotes

95°C, 1 min; (94°C, 10 s; 60°C, 30 s) ×40.

T97

14467-14540

Sentence

denotes

Primers used for MIP-1α, GAPDH and ACTB are listed in Supplementary Data.

T7528

14467-14637

Sentence

denotes

Primers used for MIP-1α, GAPDH and ACTB are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN).

T98

14541-14637

Sentence

denotes

Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN).

T7529

14638-14746

Sentence

denotes

For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.

T99

14638-14746

Sentence

denotes

For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.

T8155

14748-14768

Sentence

denotes

Statistical analysis

T100

14748-14768

Sentence

denotes

Statistical analysis

T8156

14769-14842

Sentence

denotes

Statistical analysis was performed by two-tail unpaired Student's t-test.

T101

14769-14842

Sentence

denotes

Statistical analysis was performed by two-tail unpaired Student's t-test.

T8157

14843-14876

Sentence

denotes

Data were reported as means ± SE.

T102

14843-14876

Sentence

denotes

Data were reported as means ± SE.

T8158

14877-14980

Sentence

denotes

Differences between the means were considered as statistically significant at the 95% level (P < 0.05).

T103

14877-14980

Sentence

denotes

Differences between the means were considered as statistically significant at the 95% level (P < 0.05).

T104

14982-14989

Sentence

denotes

RESULTS

T8532

14991-15094

Sentence

denotes

Tat enhances the NF-κB activity by hijacking IκB-α and inhibiting the post-activation turn off of NF-κB

T105

14991-15094

Sentence

denotes

Tat enhances the NF-κB activity by hijacking IκB-α and inhibiting the post-activation turn off of NF-κB

T8533

15095-15229

Sentence

denotes

We analysed the kinetic of NF-κB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference.

T106

15095-15229

Sentence

denotes

We analysed the kinetic of NF-κB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference.

T8534

15230-15398

Sentence

denotes

Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R−E− virions.

T107

15230-15398

Sentence

denotes

Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R−E− virions.

T8535

15399-15533

Sentence

denotes

By cytofluorimetric analysis, ∼40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1).

T108

15399-15533

Sentence

denotes

By cytofluorimetric analysis, ∼40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1).

T8536

15534-15756

Sentence

denotes

The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A).

T109

15534-15756

Sentence

denotes

The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A).

T8537

15757-16051

Sentence

denotes

The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).

T110

15757-16051

Sentence

denotes

The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).

T111

16052-16061

Sentence

denotes

Figure 1.

T23911

16062-16148

Sentence

denotes

Tat counteracts the post-activation turn off of NF-κB in single round HIV-1-infection.

T112

16062-16148

Sentence

denotes

Tat counteracts the post-activation turn off of NF-κB in single round HIV-1-infection.

T23912

16149-16861

Sentence

denotes

Jurkat cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 µg) were analysed for the p65 and p50 binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IκB-α was analysed by 12% SDS–PAGE and western blotting of nuclear or cytosolic extracts (20 µg) using anti-p65 and anti-IκB-α antibodies.

T113

16149-16861

Sentence

denotes

Jurkat cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 µg) were analysed for the p65 and p50 binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IκB-α was analysed by 12% SDS–PAGE and western blotting of nuclear or cytosolic extracts (20 µg) using anti-p65 and anti-IκB-α antibodies.

T23913

16862-16989

Sentence

denotes

Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively.

T114

16862-16989

Sentence

denotes

Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively.

T23914

16990-17207

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK Kinase Assay (Cell Signaling Technology).

T115

16990-17207

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK Kinase Assay (Cell Signaling Technology).

T23915

17208-17244

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T116

17208-17244

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T8538

17245-17738

Sentence

denotes

The progressive increase in NF-κB activity, as measured by p65–p50 binding to an NF-κB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C).

T117

17245-17738

Sentence

denotes

The progressive increase in NF-κB activity, as measured by p65–p50 binding to an NF-κB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C).

T8539

17739-17942

Sentence

denotes

IκB-α was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol).

T118

17739-17942

Sentence

denotes

IκB-α was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol).

T8540

17943-18094

Sentence

denotes

Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D).

T119

17943-18094

Sentence

denotes

Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D).

T8541

18095-18360

Sentence

denotes

Similar kinetics of NF-κB activity, IKK activation and IκB-α degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2).

T120

18095-18360

Sentence

denotes

Similar kinetics of NF-κB activity, IKK activation and IκB-α degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2).

T8542

18361-18659

Sentence

denotes

These results indicated that in single-round HIV-1 infection the NF-κB activation initially correlated with the IKK activation and IκB-α degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IκB-α.

T121

18361-18659

Sentence

denotes

These results indicated that in single-round HIV-1 infection the NF-κB activation initially correlated with the IKK activation and IκB-α degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IκB-α.

T8543

18660-18749

Sentence

denotes

Next, we tested the single action of Tat on the kinetic of NF-κB activity induced by PMA.

T122

18660-18749

Sentence

denotes

Next, we tested the single action of Tat on the kinetic of NF-κB activity induced by PMA.

T8544

18750-18942

Sentence

denotes

To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-κB activation (17,18).

T123

18750-18942

Sentence

denotes

To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-κB activation (17,18).

T8545

18943-19143

Sentence

denotes

In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IκB-α content was not significantly affected (Figure 2B, cytosol).

T124

18943-19143

Sentence

denotes

In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IκB-α content was not significantly affected (Figure 2B, cytosol).

T8546

19144-19491

Sentence

denotes

Transient stimulation with PMA increased the NF-κB DNA binding activity and nuclear p65 at 5 min peaking at 60–120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B).

T125

19144-19491

Sentence

denotes

Transient stimulation with PMA increased the NF-κB DNA binding activity and nuclear p65 at 5 min peaking at 60–120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B).

T8547

19492-19716

Sentence

denotes

In addition, Tat bound to the NF-κB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-κB complex bound to DNA.

T126

19492-19716

Sentence

denotes

In addition, Tat bound to the NF-κB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-κB complex bound to DNA.

T8548

19717-19913

Sentence

denotes

Degradation of IκB-α was progressively induced at 5–30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol).

T127

19717-19913

Sentence

denotes

Degradation of IκB-α was progressively induced at 5–30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol).

T8549

19914-20039

Sentence

denotes

Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C).

T128

19914-20039

Sentence

denotes

Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C).

T8550

20040-20196

Sentence

denotes

These results indicated that Tat enhanced the NF-κB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IκB-α content.

T129

20040-20196

Sentence

denotes

These results indicated that Tat enhanced the NF-κB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IκB-α content.

T130

20197-20206

Sentence

denotes

Figure 2.

T24351

20207-20330

Sentence

denotes

Tat enhances the NF-κB activity following PMA stimulation by associating with IκB-α and counteracting the NF-κB repression.

T131

20207-20330

Sentence

denotes

Tat enhances the NF-κB activity following PMA stimulation by associating with IκB-α and counteracting the NF-κB repression.

T24352

20331-20558

Sentence

denotes

HeLa cells (5 × 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 µg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times.

T132

20331-20558

Sentence

denotes

HeLa cells (5 × 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 µg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times.

T24353

20559-21007

Sentence

denotes

Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 µg) were separated by 12% SDS–PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IκB-α and anti-Hexokinase-II antibodies.

T133

20559-21007

Sentence

denotes

Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-κB double-stranded oligonucleotide using the NF-κB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 µg) were separated by 12% SDS–PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IκB-α and anti-Hexokinase-II antibodies.

T24354

21008-21441

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 × 106) were transfected with or without p3xFLAG-Tat (5 µg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min.

T134

21008-21441

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 µg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 × 106) were transfected with or without p3xFLAG-Tat (5 µg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min.

T24355

21442-21546

Sentence

denotes

Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IκB-α antibody.

T135

21442-21546

Sentence

denotes

Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IκB-α antibody.

T24356

21547-21678

Sentence

denotes

Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies.

T136

21547-21678

Sentence

denotes

Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies.

T24357

21679-21775

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1).

T137

21679-21775

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1).

T24358

21776-21812

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T138

21776-21812

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T8551

21813-21967

Sentence

denotes

As we previously found that Tat binds to the sixth ankyrin of IκB-α (50,51), we tested whether Tat counteracted the generation of the IκB-α/NF-κB complex.

T139

21813-21967

Sentence

denotes

As we previously found that Tat binds to the sixth ankyrin of IκB-α (50,51), we tested whether Tat counteracted the generation of the IκB-α/NF-κB complex.

T8552

21968-22195

Sentence

denotes

To this end, the association of IκB-α with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IκB-α, respectively.

T140

21968-22195

Sentence

denotes

To this end, the association of IκB-α with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IκB-α, respectively.

T8553

22196-22494

Sentence

denotes

Coimmunoprecipitation of IκB-α with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IκB-α (Figure 2D, lanes 2 and 4), indicating that Tat competed the IκB-α binding to p65.

T141

22196-22494

Sentence

denotes

Coimmunoprecipitation of IκB-α with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IκB-α (Figure 2D, lanes 2 and 4), indicating that Tat competed the IκB-α binding to p65.

T11602

22496-22574

Sentence

denotes

Tat counteracts the IκB-α inhibition of p65 by competing the repressor binding

T142

22496-22574

Sentence

denotes

Tat counteracts the IκB-α inhibition of p65 by competing the repressor binding

T11603

22575-22709

Sentence

denotes

We further investigated the physical interaction of Tat with IκB-α using in vitro translated proteins in coimmunoprecipitation assays.

T143

22575-22709

Sentence

denotes

We further investigated the physical interaction of Tat with IκB-α using in vitro translated proteins in coimmunoprecipitation assays.

T11604

22710-22781

Sentence

denotes

For mapping the interaction domains, we used the following Tat mutants:

T144

22710-22781

Sentence

denotes

For mapping the interaction domains, we used the following Tat mutants:

T11605

22782-22861

Sentence

denotes

Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49–57)A (Figure 3A).

T145

22782-22861

Sentence

denotes

Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49–57)A (Figure 3A).

T11606

22862-22945

Sentence

denotes

IκB-α coimmunoprecipitated with all Tat proteins, except Tat R(49–57)A (Figure 3B).

T146

22862-22945

Sentence

denotes

IκB-α coimmunoprecipitated with all Tat proteins, except Tat R(49–57)A (Figure 3B).

T11607

22946-23101

Sentence

denotes

These results indicated that the arginine-rich domain of Tat was involved in the binding to IκB-α, which was consistent with previous observations (50,51).

T147

22946-23101

Sentence

denotes

These results indicated that the arginine-rich domain of Tat was involved in the binding to IκB-α, which was consistent with previous observations (50,51).

T11608

23102-23389

Sentence

denotes

Moreover, Tat associating with IκB-α competed the binding of IκB-α to p65 in a dose-dependent manner (Figure 3C, lanes 3–5), while Tat R(49–57)A, which lacked the binding site for IκB-α, did not associate with IκB-α and did not compete the binding of IκB-α to p65 (Figure 3C, lanes 6–8).

T148

23102-23389

Sentence

denotes

Moreover, Tat associating with IκB-α competed the binding of IκB-α to p65 in a dose-dependent manner (Figure 3C, lanes 3–5), while Tat R(49–57)A, which lacked the binding site for IκB-α, did not associate with IκB-α and did not compete the binding of IκB-α to p65 (Figure 3C, lanes 6–8).

T149

23390-23399

Sentence

denotes

Figure 3.

T25204

23400-23710

Sentence

denotes

Tat relieves p65 from the IκB-α inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IκB-α (5 µl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49–57)A or FLAG-Tat C(22,25,27)A (10 µl) using in vitro translated proteins.

T150

23400-23710

Sentence

denotes

Tat relieves p65 from the IκB-α inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IκB-α (5 µl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49–57)A or FLAG-Tat C(22,25,27)A (10 µl) using in vitro translated proteins.

T25205

23711-24080

Sentence

denotes

Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 µl) was incubated with in vitro translated HA-IκB-α (5 µl) in presence or absence of FLAG-Tat or FLAG-Tat R(49–57)A (5, 10 or 20 µl).

T151

23711-24080

Sentence

denotes

Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 µl) was incubated with in vitro translated HA-IκB-α (5 µl) in presence or absence of FLAG-Tat or FLAG-Tat R(49–57)A (5, 10 or 20 µl).

T25206

24081-24288

Sentence

denotes

HA-IκB-α was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65).

T152

24081-24288

Sentence

denotes

HA-IκB-α was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65).

T25207

24289-24475

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins.

T153

24289-24475

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins.

T25208

24476-24777

Sentence

denotes

32P-labeled-NF-κB double-stranded oligonucleotide was incubated with p65 (0.5 µl) in presence or absence of HA-IκB-α (1 µl), FLAG-Tat or FLAG-Tat R(49–57)A (5 and 10 µl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-κB double-stranded oligonucleotide.

T154

24476-24777

Sentence

denotes

32P-labeled-NF-κB double-stranded oligonucleotide was incubated with p65 (0.5 µl) in presence or absence of HA-IκB-α (1 µl), FLAG-Tat or FLAG-Tat R(49–57)A (5 and 10 µl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-κB double-stranded oligonucleotide.

T25209

24778-25073

Sentence

denotes

DNA/protein complexes were run on 6% PAGE–TBE and analysed by autoradiography. (E) p50−/−p65−/−MEFs (3 × 105 cells) were transfected with pκBLuc (0.5 µg) and pSV-β-Gal (0.1 µg) with or without pRc/CMV-p65 (0.5 µg), pCMV4-HA-IκB-α (0.5 µg), p3xFLAG-Tat or p3xFLAG-Tat R(49–57)A (0.5, 1 and 2 µg).

T155

24778-25073

Sentence

denotes

DNA/protein complexes were run on 6% PAGE–TBE and analysed by autoradiography. (E) p50−/−p65−/−MEFs (3 × 105 cells) were transfected with pκBLuc (0.5 µg) and pSV-β-Gal (0.1 µg) with or without pRc/CMV-p65 (0.5 µg), pCMV4-HA-IκB-α (0.5 µg), p3xFLAG-Tat or p3xFLAG-Tat R(49–57)A (0.5, 1 and 2 µg).

T25210

25074-25194

Sentence

denotes

The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to β-galactosidase activity.

T156

25074-25194

Sentence

denotes

The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to β-galactosidase activity.

T25211

25195-25279

Sentence

denotes

Fold activation was calculated relative to transfection of the pκBLuc plasmid alone.

T157

25195-25279

Sentence

denotes

Fold activation was calculated relative to transfection of the pκBLuc plasmid alone.

T25212

25280-25316

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T158

25280-25316

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T25213

25317-25483

Sentence

denotes

As control of protein expression, aliquots of cell extracts (20 µg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-γ-tubulin antibodies.

T159

25317-25483

Sentence

denotes

As control of protein expression, aliquots of cell extracts (20 µg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-γ-tubulin antibodies.

T11609

25484-25705

Sentence

denotes

Next, we evaluated whether Tat counteracted the IκB-α-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IκB-α proteins with the NF-κB probe in presence or absence of Tat followed by EMSA.

T160

25484-25705

Sentence

denotes

Next, we evaluated whether Tat counteracted the IκB-α-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IκB-α proteins with the NF-κB probe in presence or absence of Tat followed by EMSA.

T11610

25706-25852

Sentence

denotes

The p65 DNA binding activity was inhibited by IκB-α and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57) (Figure 3D).

T161

25706-25852

Sentence

denotes

The p65 DNA binding activity was inhibited by IκB-α and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57) (Figure 3D).

T162

25853-26087

Sentence

denotes

Further, we analysed the Tat effect on the IκB-α repression of p65 transcriptional activity by transfecting p50−/−p65−/−MEFs with the NF-κB-Luc reporter together with expression vectors of p65 and IκB-α, in presence or absence of Tat.

T11611

25853-26258

Sentence

denotes

Further, we analysed the Tat effect on the IκB-α repression of p65 transcriptional activity by transfecting p50−/−p65−/−MEFs with the NF-κB-Luc reporter together with expression vectors of p65 and IκB-α, in presence or absence of Tat. IκB-α inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57)A (Figure 3E).

T163

26088-26258

Sentence

denotes

IκB-α inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49–57)A (Figure 3E).

T11612

26259-26457

Sentence

denotes

Altogether these results indicated that Tat counteracts the IκB-α repression of the p65 DNA-binding and transcriptional activity by associating with IκB-α and competing the repressor binding to p65.

T164

26259-26457

Sentence

denotes

Altogether these results indicated that Tat counteracts the IκB-α repression of the p65 DNA-binding and transcriptional activity by associating with IκB-α and competing the repressor binding to p65.

T13324

26459-26533

Sentence

denotes

Tat increases the p65 affinity binding to DNA through association with p65

T165

26459-26533

Sentence

denotes

Tat increases the p65 affinity binding to DNA through association with p65

T13325

26534-26587

Sentence

denotes

We tested whether Tat physically interacted with p65.

T166

26534-26587

Sentence

denotes

We tested whether Tat physically interacted with p65.

T13326

26588-26729

Sentence

denotes

HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts.

T167

26588-26729

Sentence

denotes

HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts.

T13327

26730-26973

Sentence

denotes

The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A).

T168

26730-26973

Sentence

denotes

The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A).

T13328

26974-27202

Sentence

denotes

Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B).

T169

26974-27202

Sentence

denotes

Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B).

T13329

27203-27368

Sentence

denotes

These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.

T170

27203-27368

Sentence

denotes

These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.

T171

27369-27378

Sentence

denotes

Figure 4.

T26499

27379-27646

Sentence

denotes

Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg).

T172

27379-27646

Sentence

denotes

Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg).

T173

27647-28028

Sentence

denotes

FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose.

T26500

27647-28416

Sentence

denotes

FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg).

T174

28029-28416

Sentence

denotes

Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg).

T26501

28417-28947

Sentence

denotes

Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide.

T175

28417-28947

Sentence

denotes

Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide.

T26502

28948-29092

Sentence

denotes

DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.

T176

28948-29092

Sentence

denotes

DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.

T13330

29093-29328

Sentence

denotes

To identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C).

T177

29093-29328

Sentence

denotes

To identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C).

T13331

29329-29497

Sentence

denotes

Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.

T178

29329-29497

Sentence

denotes

Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.

T13332

29498-29764

Sentence

denotes

To address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA.

T179

29498-29764

Sentence

denotes

To address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA.

T13333

29765-29929

Sentence

denotes

Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E).

T180

29765-29929

Sentence

denotes

Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E).

T13334

29930-30143

Sentence

denotes

The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).

T181

29930-30143

Sentence

denotes

The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).

T15219

30145-30300

Sentence

denotes

Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement

T182

30145-30300

Sentence

denotes

Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement

T15220

30301-30383

Sentence

denotes

We analysed the action of Tat on the expression of NF-κB-responsive genes in vivo.

T183

30301-30383

Sentence

denotes

We analysed the action of Tat on the expression of NF-κB-responsive genes in vivo.

T15221

30384-30582

Sentence

denotes

To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR.

T184

30384-30582

Sentence

denotes

To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR.

T15222

30583-30749

Sentence

denotes

Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E).

T185

30583-30749

Sentence

denotes

Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E).

T15223

30750-30956

Sentence

denotes

The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E).

T186

30750-30956

Sentence

denotes

The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E).

T15224

30957-31093

Sentence

denotes

Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G).

T187

30957-31093

Sentence

denotes

Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G).

T15225

31094-31383

Sentence

denotes

These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.

T188

31094-31383

Sentence

denotes

These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.

T189

31384-31393

Sentence

denotes

Figure 5.

T27258

31394-31554

Sentence

denotes

Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement.

T190

31394-31554

Sentence

denotes

Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement.

T27259

31555-31808

Sentence

denotes

HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol).

T191

31555-31808

Sentence

denotes

HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol).

T27260

31809-32150

Sentence

denotes

Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody.

T192

31809-32150

Sentence

denotes

Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody.

T27261

32151-32435

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody.

T193

32151-32435

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody.

T27262

32436-32715

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody.

T194

32436-32715

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody.

T27263

32716-32794

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters.

T195

32716-32794

Sentence

denotes

Real-time PCR was performed with primers specific for the indicated promoters.

T27264

32795-32831

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T196

32795-32831

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T27265

32832-32977

Sentence

denotes

The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).

T197

32832-32977

Sentence

denotes

The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).

T15226

32978-33115

Sentence

denotes

MIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56).

T198

32978-33115

Sentence

denotes

MIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56).

T15227

33116-33331

Sentence

denotes

Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62).

T199

33116-33331

Sentence

denotes

Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62).

T15228

33332-33403

Sentence

denotes

The transcriptional regulation of MIP-1α has been poorly characterized.

T200

33332-33403

Sentence

denotes

The transcriptional regulation of MIP-1α has been poorly characterized.

T15229

33404-33669

Sentence

denotes

Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A).

T201

33404-33669

Sentence

denotes

Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A).

T15230

33670-33917

Sentence

denotes

To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP.

T202

33670-33917

Sentence

denotes

To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP.

T15231

33918-34127

Sentence

denotes

TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C).

T203

33918-34127

Sentence

denotes

TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C).

T15232

34128-34287

Sentence

denotes

Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D).

T204

34128-34287

Sentence

denotes

Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D).

T15233

34288-34434

Sentence

denotes

Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.

T205

34288-34434

Sentence

denotes

Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.

T15234

34435-34619

Sentence

denotes

Similarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H).

T206

34435-34619

Sentence

denotes

Similarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H).

T15235

34620-34839

Sentence

denotes

By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction.

T207

34620-34839

Sentence

denotes

By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction.

T15236

34840-35098

Sentence

denotes

As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters.

T208

34840-35098

Sentence

denotes

As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters.

T17783

35100-35247

Sentence

denotes

In HIV-1-infected monocytes Tat sustains the NF-κB activity and promotes the transcriptional activation of MIP-1α by interacting with IκB-α and p65

T209

35100-35247

Sentence

denotes

In HIV-1-infected monocytes Tat sustains the NF-κB activity and promotes the transcriptional activation of MIP-1α by interacting with IκB-α and p65

T17784

35248-35367

Sentence

denotes

We next analysed whether Tat affected the NF-κB activity and the expression of MIP-1α in the course of HIV-1 infection.

T210

35248-35367

Sentence

denotes

We next analysed whether Tat affected the NF-κB activity and the expression of MIP-1α in the course of HIV-1 infection.

T17785

35368-35536

Sentence

denotes

To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R−E− virions, which were used in single-round infection of U937, a human monocytic cell line.

T211

35368-35536

Sentence

denotes

To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R−E− virions, which were used in single-round infection of U937, a human monocytic cell line.

T17786

35537-35724

Sentence

denotes

Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-κB activation following viral infection.

T212

35537-35724

Sentence

denotes

Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-κB activation following viral infection.

T17787

35725-35870

Sentence

denotes

The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).

T213

35725-35870

Sentence

denotes

The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).

T214

35871-35880

Sentence

denotes

Figure 6.

T28289

35881-36002

Sentence

denotes

In HIV-1 infection Tat sustains the NF-κB activity and enhances the MIP-1α expression via interaction with IκB-α and p65.

T215

35881-36002

Sentence

denotes

In HIV-1 infection Tat sustains the NF-κB activity and enhances the MIP-1α expression via interaction with IκB-α and p65.

T28290

36003-36655

Sentence

denotes

U937 cells (5 × 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts (10 µg) using the NF-κB Transcription Factor ELISA assay kit (Cayman). (C) The IκB-α content was analysed by 12% SDS–PAGE and western blotting of cytosolic extracts (20 µg) using anti-IκB-α antibody.

T216

36003-36655

Sentence

denotes

U937 cells (5 × 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts (10 µg) using the NF-κB Transcription Factor ELISA assay kit (Cayman). (C) The IκB-α content was analysed by 12% SDS–PAGE and western blotting of cytosolic extracts (20 µg) using anti-IκB-α antibody.

T28291

36656-36985

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 µg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 × 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T217

36656-36985

Sentence

denotes

Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 µg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 × 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T28292

36986-37116

Sentence

denotes

Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody.

T218

36986-37116

Sentence

denotes

Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody.

T28293

37117-37467

Sentence

denotes

Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies. (F) U937 cells (5 × 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T219

37117-37467

Sentence

denotes

Immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IκB-α antibodies. (F) U937 cells (5 × 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T28294

37468-37779

Sentence

denotes

Twenty-four-hour post-infection, total RNA was analysed for MIP-1α expression by real-time PCR. (G and H) U937 cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T220

37468-37779

Sentence

denotes

Twenty-four-hour post-infection, total RNA was analysed for MIP-1α expression by real-time PCR. (G and H) U937 cells (5 × 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R−E− virions, or left uninfected.

T28295

37780-37867

Sentence

denotes

Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H).

T221

37780-37867

Sentence

denotes

Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H).

T28296

37868-37949

Sentence

denotes

Real-time PCR was performed with primers specific for MIP-1α and GAPDH promoters.

T222

37868-37949

Sentence

denotes

Real-time PCR was performed with primers specific for MIP-1α and GAPDH promoters.

T28297

37950-37986

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T223

37950-37986

Sentence

denotes

Values (mean ± SE, n = 3) are shown.

T28298

37987-38125

Sentence

denotes

The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).

T224

37987-38125

Sentence

denotes

The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).

T17788

38126-38359

Sentence

denotes

The NF-κB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B).

T225

38126-38359

Sentence

denotes

The NF-κB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B).

T17789

38360-38518

Sentence

denotes

Degradation of IκB-α was observed at 3-h post-infection, and was followed by de novo synthesis of IκB-α at 12 h independently of the Tat presence (Figure 6C).

T226

38360-38518

Sentence

denotes

Degradation of IκB-α was observed at 3-h post-infection, and was followed by de novo synthesis of IκB-α at 12 h independently of the Tat presence (Figure 6C).

T17790

38519-38653

Sentence

denotes

Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D).

T227

38519-38653

Sentence

denotes

Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D).

T17791

38654-38882

Sentence

denotes

These results agreed with the kinetic of NF-κB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-κB.

T228

38654-38882

Sentence

denotes

These results agreed with the kinetic of NF-κB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-κB.

T17792

38883-39136

Sentence

denotes

In HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IκB-α and p65 (Figure 6E), and activated the MIP-1α expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F).

T229

38883-39136

Sentence

denotes

In HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IκB-α and p65 (Figure 6E), and activated the MIP-1α expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F).

T17793

39137-39281

Sentence

denotes

Moreover, Tat and p65 were both recruited to the NF-κB enhancer of MIP-1α, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H).

T230

39137-39281

Sentence

denotes

Moreover, Tat and p65 were both recruited to the NF-κB enhancer of MIP-1α, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H).

T17794

39282-39525

Sentence

denotes

Altogether these results indicated that in single round HIV-1 infection Tat associated with IκB-α and p65, and induced the p65-dependent activation of MIP-1α expression through occupancy of the MIP-1α promoter and increased recruitment of p65.

T231

39282-39525

Sentence

denotes

Altogether these results indicated that in single round HIV-1 infection Tat associated with IκB-α and p65, and induced the p65-dependent activation of MIP-1α expression through occupancy of the MIP-1α promoter and increased recruitment of p65.

T232

39527-39537

Sentence

denotes

DISCUSSION

T19880

39538-39627

Sentence

denotes

This study reports a novel mechanism of NF-κB activation by the HIV-1 Tat transactivator.

T233

39538-39627

Sentence

denotes

This study reports a novel mechanism of NF-κB activation by the HIV-1 Tat transactivator.

T19881

39628-39901

Sentence

denotes

Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-κB (49,63,64), and physically interacted with the IκB-α repressor (50,51), we investigated the possibility that Tat could activate NF-κB via direct interaction with IκB-α and p65.

T234

39628-39901

Sentence

denotes

Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-κB (49,63,64), and physically interacted with the IκB-α repressor (50,51), we investigated the possibility that Tat could activate NF-κB via direct interaction with IκB-α and p65.

T19882

39902-40033

Sentence

denotes

To this end, the NF-κB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression.

T235

39902-40033

Sentence

denotes

To this end, the NF-κB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression.

T19883

40034-40186

Sentence

denotes

By this approach, we avoided the perpetuation of NF-κB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31).

T236

40034-40186

Sentence

denotes

By this approach, we avoided the perpetuation of NF-κB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31).

T237

40187-40323

Sentence

denotes

Upon HIV-1 infection, the early NF-κB activation occurred concomitantly with IKK activation and IκB-α degradation in the absence of Tat.

T19884

40187-40512

Sentence

denotes

Upon HIV-1 infection, the early NF-κB activation occurred concomitantly with IKK activation and IκB-α degradation in the absence of Tat. Soon after the shut off of IKK activity and new synthesis of IκB-α, the NF-κB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene.

T238

40324-40512

Sentence

denotes

Soon after the shut off of IKK activity and new synthesis of IκB-α, the NF-κB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene.

T19885

40513-40669

Sentence

denotes

These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-κB activity without affecting the IKK activity and the half-life of IκB-α.

T239

40513-40669

Sentence

denotes

These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-κB activity without affecting the IKK activity and the half-life of IκB-α.

T19886

40670-40867

Sentence

denotes

Similar kinetic of NF-κB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-κB in presence of newly synthesized IκB-α.

T240

40670-40867

Sentence

denotes

Similar kinetic of NF-κB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-κB in presence of newly synthesized IκB-α.

T19887

40868-41033

Sentence

denotes

The Tat-dependent activation of NF-κB correlated with the association of the viral protein with IκB-α, which interfered with the generation of the IκB-α/p65 complex.

T241

40868-41033

Sentence

denotes

The Tat-dependent activation of NF-κB correlated with the association of the viral protein with IκB-α, which interfered with the generation of the IκB-α/p65 complex.

T19888

41034-41153

Sentence

denotes

This evidence demonstrated that Tat competed for the binding of IκB-α to p65 and prevented the IκB-α repression of p65.

T242

41034-41153

Sentence

denotes

This evidence demonstrated that Tat competed for the binding of IκB-α to p65 and prevented the IκB-α repression of p65.

T19889

41154-41490

Sentence

denotes

As additional mechanism of NF-κB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-κB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65.

T243

41154-41490

Sentence

denotes

As additional mechanism of NF-κB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-κB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65.

T19890

41491-41627

Sentence

denotes

We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-κB activation.

T244

41491-41627

Sentence

denotes

We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-κB activation.

T19891

41628-41809

Sentence

denotes

Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IκB-α (50,51), and for releasing p65 from the IκB-α inhibition.

T245

41628-41809

Sentence

denotes

Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IκB-α (50,51), and for releasing p65 from the IκB-α inhibition.

T19892

41810-42018

Sentence

denotes

Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-κB by hijacking the IκB-α repressor, shielding p65 from the IκB-α embrace, and enforcing the p65 binding to DNA.

T246

41810-42018

Sentence

denotes

Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-κB by hijacking the IκB-α repressor, shielding p65 from the IκB-α embrace, and enforcing the p65 binding to DNA.

T19893

42019-42282

Sentence

denotes

Further evidence of Tat activation of NF-κB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-κB-responsive genes, including MIP-1α, CSF3, LTA, NFKBIA and TLR2.

T247

42019-42282

Sentence

denotes

Further evidence of Tat activation of NF-κB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-κB-responsive genes, including MIP-1α, CSF3, LTA, NFKBIA and TLR2.

T19894

42283-42438

Sentence

denotes

By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-κB enhancers of the activated genes, and was recovered at the same sites.

T248

42283-42438

Sentence

denotes

By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-κB enhancers of the activated genes, and was recovered at the same sites.

T19895

42439-42585

Sentence

denotes

RNA interference of p65 abolished the Tat occupancy of the NF-κB enhancers indicating that the Tat binding to the NF-κB sites was mediated by p65.

T249

42439-42585

Sentence

denotes

RNA interference of p65 abolished the Tat occupancy of the NF-κB enhancers indicating that the Tat binding to the NF-κB sites was mediated by p65.

T19896

42586-42785

Sentence

denotes

These results were consistent with the evidence that Tat displaced p65 from the binding to IκB-α (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F).

T250

42586-42785

Sentence

denotes

These results were consistent with the evidence that Tat displaced p65 from the binding to IκB-α (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F).

T19897

42786-42962

Sentence

denotes

Thus, Tat likely increased the recruitment of p65 to the NF-κB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IκB-α.

T251

42786-42962

Sentence

denotes

Thus, Tat likely increased the recruitment of p65 to the NF-κB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IκB-α.

T252

42963-43087

Sentence

denotes

Of interest, IκB-α was found associated to the NF-κB enhancers in absence of Tat, where it was displaced in presence of Tat.

T19898

42963-43205

Sentence

denotes

Of interest, IκB-α was found associated to the NF-κB enhancers in absence of Tat, where it was displaced in presence of Tat. These results suggest a negative regulatory role of IκB-α in gene regulation through occupancy of specific promoters.

T253

43088-43205

Sentence

denotes

These results suggest a negative regulatory role of IκB-α in gene regulation through occupancy of specific promoters.

T19899

43206-43412

Sentence

denotes

Indeed, IκB-α was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-α stimulation causing histone acetylation and hes1 transcriptional activation (65).

T254

43206-43412

Sentence

denotes

Indeed, IκB-α was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-α stimulation causing histone acetylation and hes1 transcriptional activation (65).

T19900

43413-43621

Sentence

denotes

A similar mechanism of gene regulation could apply to the NF-κB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IκB-α and promoting the p65 loading (Figure 7).

T255

43413-43621

Sentence

denotes

A similar mechanism of gene regulation could apply to the NF-κB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IκB-α and promoting the p65 loading (Figure 7).

T256

43622-43631

Sentence

denotes

Figure 7.

T29103

43632-44044

Sentence

denotes

Model of MIP-1α transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IκB-α occupies the NF-κB enhancer of the MIP-1α promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1α expression by removing the IκB-α repressor from the NF-κB enhancer, and by increasing the binding of p65 NF-κB complex to the NF-κB enhancer.

T257

43632-44044

Sentence

denotes

Model of MIP-1α transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IκB-α occupies the NF-κB enhancer of the MIP-1α promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1α expression by removing the IκB-α repressor from the NF-κB enhancer, and by increasing the binding of p65 NF-κB complex to the NF-κB enhancer.

T19901

44045-44541

Sentence

denotes

The physiological relevance of Tat cross talk with NF-κB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-κB, as shown by: (i) sustained NF-κB activity by Tat in presence of newly synthesized IκB-α; (ii) coimmunoprecipitation of Tat with IκB-α and p65; (iii) induction of MIP-1α expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1α NF-κB enhancer associated with increased recruitment of p65.

T258

44045-44541

Sentence

denotes

The physiological relevance of Tat cross talk with NF-κB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-κB, as shown by: (i) sustained NF-κB activity by Tat in presence of newly synthesized IκB-α; (ii) coimmunoprecipitation of Tat with IκB-α and p65; (iii) induction of MIP-1α expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1α NF-κB enhancer associated with increased recruitment of p65.

T19902

44542-44645

Sentence

denotes

This study supports the pro-inflammatory action of Tat through physical interaction with p65 and IκB-α.

T259

44542-44645

Sentence

denotes

This study supports the pro-inflammatory action of Tat through physical interaction with p65 and IκB-α.

T19903

44646-44832

Sentence

denotes

Several inflammatory cytokines under the transcriptional control of NF-κB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-α (68), and MIP-1α (57–62,69–71).

T260

44646-44832

Sentence

denotes

Several inflammatory cytokines under the transcriptional control of NF-κB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-α (68), and MIP-1α (57–62,69–71).

T19904

44833-45025

Sentence

denotes

In the case of TNF-α and IL-6, Tat activated the cytokine expression by binding to the TAR-like stem loop of the 5′ transcript, thus likely promoting the transcriptional elongation (33,37,47).

T261

44833-45025

Sentence

denotes

In the case of TNF-α and IL-6, Tat activated the cytokine expression by binding to the TAR-like stem loop of the 5′ transcript, thus likely promoting the transcriptional elongation (33,37,47).

T19905

45026-45245

Sentence

denotes

Here, we have shown that the Tat-dependent transcriptional activation of a number of pro-inflammatory genes required the assembly of Tat with p65 at the NF-κB enhancer together with the displacement of IκB-α (Figure 5).

T262

45026-45245

Sentence

denotes

Here, we have shown that the Tat-dependent transcriptional activation of a number of pro-inflammatory genes required the assembly of Tat with p65 at the NF-κB enhancer together with the displacement of IκB-α (Figure 5).

T19906

45246-45467

Sentence

denotes

Thus, in the set of distinct NF-κB-responsive genes, Tat acted as a component of the transcriptional initiation complex, which is consistent with previous reports on Tat promoting the transcription via DNA motifs (72,73).

T263

45246-45467

Sentence

denotes

Thus, in the set of distinct NF-κB-responsive genes, Tat acted as a component of the transcriptional initiation complex, which is consistent with previous reports on Tat promoting the transcription via DNA motifs (72,73).

T19907

45468-45612

Sentence

denotes

Overproduction of pro-inflammatory cytokines is considered a major mechanism of immune deregulation (26) and neuron dysfunction in AIDS (74,75).

T264

45468-45612

Sentence

denotes

Overproduction of pro-inflammatory cytokines is considered a major mechanism of immune deregulation (26) and neuron dysfunction in AIDS (74,75).

T19908

45613-45803

Sentence

denotes

In this scenario, inhibition of NF-κB activity could lead to a therapeutic strategy for counteracting the abnormal production of pro-inflammatory cytokines and chemokines in HIV-1 infection.

T265

45613-45803

Sentence

denotes

In this scenario, inhibition of NF-κB activity could lead to a therapeutic strategy for counteracting the abnormal production of pro-inflammatory cytokines and chemokines in HIV-1 infection.

T19909

45804-46069

Sentence

denotes

NF-κB inhibitors, such as anti-oxidants, proteasome and IKK inhibitors, significantly reduced the HIV-1 replication and the associated inflammatory response (76,77); however, these NF-κB inhibitors acted indiscriminately in both HIV-1-infected and uninfected cells.

T266

45804-46069

Sentence

denotes

NF-κB inhibitors, such as anti-oxidants, proteasome and IKK inhibitors, significantly reduced the HIV-1 replication and the associated inflammatory response (76,77); however, these NF-κB inhibitors acted indiscriminately in both HIV-1-infected and uninfected cells.

T19910

46070-46232

Sentence

denotes

The evidence that Tat activates NF-κB through direct interaction with IκB-α and p65 may lead to specific inhibitors to counteract the Tat pro-inflammatory action.

T267

46070-46232

Sentence

denotes

The evidence that Tat activates NF-κB through direct interaction with IκB-α and p65 may lead to specific inhibitors to counteract the Tat pro-inflammatory action.

T268

46234-46252

Sentence

denotes

SUPPLEMENTARY DATA

T269

46253-46300

Sentence

denotes

Supplementary Data are available at NAR Online:

T270

46301-46367

Sentence

denotes

Supplementary Figures 1–3 and Supplementary Materials and Methods.

T271

46369-46376

Sentence

denotes

FUNDING

T272

46377-46654

Sentence

denotes

Istituto Superiore di Sanità-National Research Program on AIDS (Grant number 40C.77 to I.Q.); Ministero dell'Istruzione, dell'Università e della Ricerca (Grant number FIRB RBLA033WJX_006 to I.Q.); EUROPRISE (Grant number LSHP-CT-2006-037611 to G.S.); FIRC fellowship (to A.R.).

T273

46655-46732

Sentence

denotes

Funding for open access charge: EUROPRISE (grant number LSHP-CT-2006-037611).

T274

46733-46764

Sentence

denotes

Conflict of interest statement.

T275

46765-46779

Sentence

denotes

None declared.

T276

46781-46803

Sentence

denotes

Supplementary Material

T277

46804-46822

Sentence

denotes

Supplementary Data

PMC:3333881 JSON TXT 24 Projects

Annnotations TAB TSV DIC JSON TextAE

PMC:3333881 JSONTXT 24 Projects

Annnotations TAB TSV DIC JSON TextAE

PMC:3333881 JSON TXT 24 Projects