PMC:7464876 / 8981-22096 JSONTXT

{"project":"2_test","denotations":[{"id":"32806600-2153228-20296188","span":{"begin":284,"end":286},"obj":"2153228"},{"id":"32806600-10792995-20296189","span":{"begin":443,"end":445},"obj":"10792995"},{"id":"32806600-10792995-20296190","span":{"begin":549,"end":551},"obj":"10792995"},{"id":"32806600-2153228-20296191","span":{"begin":890,"end":892},"obj":"2153228"},{"id":"32806600-21129143-20296192","span":{"begin":1047,"end":1049},"obj":"21129143"},{"id":"32806600-24899172-20296193","span":{"begin":1308,"end":1310},"obj":"24899172"},{"id":"32806600-8392600-20296194","span":{"begin":1586,"end":1588},"obj":"8392600"},{"id":"32806600-7835336-20296195","span":{"begin":1771,"end":1773},"obj":"7835336"},{"id":"32806600-24614091-20296196","span":{"begin":1977,"end":1979},"obj":"24614091"},{"id":"32806600-24614091-20296197","span":{"begin":4430,"end":4432},"obj":"24614091"},{"id":"32806600-32017353-20296198","span":{"begin":4433,"end":4435},"obj":"32017353"},{"id":"32806600-20406418-20296199","span":{"begin":4715,"end":4717},"obj":"20406418"},{"id":"32806600-24237698-20296200","span":{"begin":4718,"end":4720},"obj":"24237698"},{"id":"32806600-21129143-20296201","span":{"begin":5610,"end":5612},"obj":"21129143"},{"id":"32806600-10792995-20296202","span":{"begin":6337,"end":6339},"obj":"10792995"},{"id":"32806600-32017353-20296203","span":{"begin":7966,"end":7968},"obj":"32017353"},{"id":"32806600-32017353-20296204","span":{"begin":8092,"end":8094},"obj":"32017353"},{"id":"32806600-24614091-20296205","span":{"begin":8598,"end":8600},"obj":"24614091"},{"id":"32806600-24614091-20296206","span":{"begin":9354,"end":9356},"obj":"24614091"},{"id":"32806600-29367244-20296207","span":{"begin":11415,"end":11417},"obj":"29367244"}],"text":"2. Materials and Methods\n\n2.1. Expression Constructs\nThe plasmid pHBV carries a 1.1 mer of the HBV genome (nt 2922 to 84, derived from genotype D, GenBank™ accession number V01460) under the transcriptional control of the human metallothionein (hMT) IIA promoter in a pUC19 backbone [32]. To prevent expression of the L, M and S envelope proteins (HBV.Envminus), their translational start codons were inactivated by mutagenesis, as described [33]. In the HBV.Lminus construct, only the L-specific start codon was substituted by a threonine residue [33]. To ablate the core expression from the HBV replicon (HBV.Cminus), a stop codon at triplet 38 of the core gene was created [34]. The mutant HBV.Polminus replicon carries an aspartic acid-to-histidine substitution at amino acid (aa) position 540 of the HBV polymerase and has been shown to be defective in reverse transcriptase activity [32]. For trans-complementation studies of the HBV.Cminus replicon, the vector pNI2.C containing the HBV core gene preceded by the hMT IIa promoter was used [35]. The core mutant C.K96A, defective in capsid envelopment, has been described [34]. The plasmid pT7HB2.7, which directs the synthesis of all three HBV envelope proteins under their authentic promoters, was kindly provided by C. Sureau (INTS, Paris, France) [13] and used for trans-complementation of the HBV.Envminus replicon. To inactivate the N-glycan acceptor site within the S domain of the three envelope proteins, the asparagine residue at aa position 146 (N146) was replaced by a glutamic acid residue (Env.N146D), as described [36]. For the sole expression of the S protein under the control of the hMT IIa promoter, plasmid pNI2.S.HA carrying the S gene with a C-terminal-fused haemagglutinin (HA) tag was used [10]. Where indicated, HBV replication was studied in cells transfected with pCEP4∆CMV∆SV40/1.1 × HBV (termed pHBV* herein), which carries the 1.1 mer of the HBV genome in a modified pCEP4 plasmid backbone [22]. The plasmid pEYFP-ER encodes an enhanced yellow fluorescent protein (EYFP) with an N-terminal-fused ER-targeting sequence of calreticulin plus a C-terminal-fused ER retrieval sequence and was purchased from Clontech (Clontech, Mountain View, CA, USA). The vector pGRASP65-GFP (green fluorescent protein) was kindly provided by E. Hildt (PEI, Langen, Germany) and encodes a fusion protein of GRASP65, a peripheral membrane protein that is localized to the cis-Golgi, and of the green fluorescent protein (GFP). The plasmid pDsRed-Golgi carries the DsRed-coding sequence fused to the N-terminal 81 aa of human β 1,4-galactosyltransferase, which targets the fusion protein to the trans-medial region of the Golgi (Clontech, Mountain View, CA, USA). The expression vector encoding human Vps4A with a C-terminal EGFP tag was generously provided by W. Sundquist (University of Utah, Salt Lake Cit, UT, USA). The vector pcDNA3.1(+)-ERGIC-53 carrying the human ERGIC-53 gene (NM_005570.3) under the control of the CMV promoter was purchased from GenScript (GenScript Biotech, Leiden, Netherlands). For tagging with the Myc epitope, the aa sequence 28–37 of ERGIC-53 was mutated to EQKLISEEDL by Q5® Site-Directed Mutagenesis (New England Biolabs, Ipswich, MA, USA) with the oligonucleotides 5′- CCGAGGAGGACCTCCTGGTGCAGAGCGACGG-3′ and 5′- AGATTAATTTTTGCTCGAAACGGCGATGTGGC-3′.\n\n2.2. SiRNAs, Cell Culture and Transfection\nFor transient expression analyses, the human hepatocellular carcinoma cell line HuH-7 was used that was obtained by the European Collection of Authenticated Cell Cultures (http://cellbank.nibiohn.go.jp/english/). This cell line is not susceptible to HBV infection, because it expresses very low levels of the NTCP receptor and is therefore a useful model to study the production and release of the virus rather than infection [7]. Cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 5-µg/mL ciprofloxacin (Fresenius Kabi, Bad Homburg, Germany). Transfections of cells with plasmid DNAs were performed with Lipofectamine™ Plus (Thermo Fisher Scientific, Waltham, MA, USA). For depletion of EAP20 (NM_032353.3), Sar1A (NM_01142648), Sar1B (NM_016103.3), Sec23A (NM_006364.4), Sec23B (NM_006363), Sec24A (NM_021982) or Sec24B (NM_006323), single siRNA duplexes or siGENOME SMARTpool RNAs (Dharmacon, Lafayette, CO, USA) were used as described [22,29]. To silence the expression of ERGIC-53 (NM_005570), a siRNA (5′-GGACAGAAUCGUAUUCAUC-3′) targeting nucleotide positions 1009–1027 was obtained from Dharmacon (Dharmacon, Lafayette, CO, USA). The specificity and efficacy of this siRNA has been approved in independent studies [37,38,39]. A control siRNA with no known homology to mammalian genes was purchased from Qiagen (Qiagen, Hilden, Germany). For combined transfection, HuH-7 cells were first transfected with siRNAs by using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). In a typical experiment, 3 × 105 cells per well of a 12-well plate were transfected with a final concentration of 20-nM siRNA, according to the protocol of the supplier. After 48 to 72 h, cells were retransfected with 2-µg plasmid DNA, and transfected cells were harvested after an additional 48 to 72 h, as indicated in the text. For drug treatment, cells were incubated with 1-µM brefeldin A (BFA; Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C.\n\n2.3. Antibodies\nPolyclonal antisera against recombinant native (K45) or denatured (K46) HBV core particles were raised in rabbits, as described [35]. In addition, commercially available polyclonal rabbit (B0586; Dako, Carpinteria, CA, USA) or monoclonal mouse (3HB17; HyTest, Turku, Finland) antisera against the core antigen or capsid particle, respectively, were used. For immunodetection of the HBV L protein, a rabbit antibody raised against a recombinant peptide encoding aa 1–42 of L fused to glutathione S-transferase (K1350; Eurogentec, Liège, Belgium) or the mouse antibodies HepB preS1 (sc-57761; Santa Cruz, CA, USA) and MA18/7 (D. Glebe, University of Giessen, Giessen, Germany), recognizing L-specific epitopes, were employed. For immunoprecipitation (IP) of all three HBV envelope proteins, a rabbit polyclonal antiserum (K38) against the S domain was used [33]. Antibodies obtained from Covance (Covance, HISS Diagnostics, Freiburg im Breisgau, Germany) were mouse anti-HA (16B12) and anti-Myc (9E10). The mouse anti-EAP20 (VPS25: sc-271648) antibody was purchased from Santa Cruz (Santa Cruz, CA, USA), while the mouse anti-ERGIC-53 (OTI1A8) antibody was from Enzo (Enzo Life Sciences, Farmingdale, NY, USA). Antibodies obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) were rabbit anti-ERGIC-53 (E1031), rabbit anti-Sec24A (HPA056825), anti-β-actin (AC15) and anti-α-tubulin (B-5-1-2). Peroxidase-labeled secondary antibodies were obtained from Dianova (Hamburg, Germany), and fluorophore-labeled antibodies were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA).\n\n2.4. Quantitative Reverse Transcription PCR (qRT-PCR) Analysis\nTotal mRNAs were isolated from cells using the peqGold TriFast (Peqlab Biotechnologie, Darmstadt, Germany) and the Direct-zol™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). The mRNA was digested with 5 U RNase-free, recombinant DNase I (Roche Diagnostics, Rotkreuz, Switzerland), and cDNA synthesis was performed by using the qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA). qRT-PCR reactions were conducted as described [40]. For data analysis, the comparative cycle threshold method (CT) was used, and data were reported as the fold change normalized to an endogenous reference gene (β-actin). To quantify the ERGIC-53 transcript levels, the primers 5′- CAGATCAAATTCGAGTAGCACCA-3′ and 5′-AATATTCCAACACCATTCCACAGA-3′ were used. All other primer pairs have been described previously [29].\n\n2.5. Subviral Particle Analysis\nTo probe for subviral particle production, an SVP release assay was used, as described [29]. Briefly, cells were lysed for 20 min on ice, and lysates were centrifuged for 5 min at 15,000× g and 4 °C. To analyze the secretion of SVPs from transfected cells, clarified culture medium was concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion (4 h at 100,000× g and 4 °C), and samples were subjected to Western blot (WB) analysis.\n\n2.6. Viral Particle Analysis\nThe production and release of HBV particles were determined by a TaqMan® chemistry-based real-time PCR, as described [22]. Briefly, cell lysates and supernatants were harvested, and intracellular nucleocapsids and extracellular virions were isolated by immunomagnetic separation using PureProteome™ Protein G Magnetic Beads (Millipore, Billerica, MA, USA) coated with capsid (K45)- and envelope-specific (K38 and K1350) antibodies, respectively. After isolation of the viral DNA, PCR analyses were performed with a 7500 Real-Time PCR System and Sequence Detection Software 4.0 (Applied Biosystems, Foster, CA, USA). Dependent on the transfected HBV replicon constructs, simplex or multiplex PCR analysis were conducted, with the latter using two primer/probe sets targeting either the HBV genome or the hygromycin resistance gene of the pCEP plasmid backbone, as described [22]. Where indicated, cells were lysed by osmotic shock in a hypotonic lysis buffer (10-mM Tris-HCl, pH 7.5, 10-mM NaCl and 1.5-mM MgCl2) for 15 min on ice and three subsequent freeze-thaw cycles (using liquid nitrogen to freeze and a 37 °C water bath to thaw). Lysates were supplemented with NaCl to a final concentration of 150 mM and centrifuged for 30 min at 15,000× g and 4 °C. Intracellular enveloped viral particles were immunoisolated by an envelope-specific IP in the absence of detergents and assayed by PCR.\n\n2.7. Endoglycosidase H (EndoH) Treatment\nFor EndoH digestion, cell supernatants were concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion (4 h at 100,000× g and 4 °C), and pellets were suspended in Tris-buffered saline TBS buffer (TBS; 50-mM Tris-HCl, pH 7.5, and 150-mM NaCl). Cells were lysed in TBS supplemented with 0.5% Nonidet P-40 (NP-40), and lysates were centrifuged for 5 min at 15,000× g and 4 °C. After the addition of 10 × glycoprotein denaturing buffer (New England Biolabs, Ipswich, MA, USA), samples were heated at 100 °C for 10 min. Next, samples were adjusted to 10 × GlycoBuffer 3 (New England Biolabs, Ipswich, MA, USA), divided in two portions and incubated for 1 h at 37 °C with or without 2-µL EndoH (New England Biolabs, Ipswich, MA, USA).\n\n2.8. Coimmunoprecipitation (CO-IP) Assay\nTo probe for intracellular protein interactions, cells were either lysed with a 2% CHAPS solution dissolved in HBS (50-mM Hepes-KCl, pH 7.5, 200-mM NaCl and 20-mM N-ethylmaleimide) or with Triton buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 1-mM CaCl2 and 1% Triton X-100) in the presence of 1 × protease inhibitor mixture for 20 min on ice. After centrifugation for 20 min at 15,000× g and 4 °C, lysates were immediately subjected to immunoprecipitation using tosyl-activated, superparamagnetic polystyrene beads (Dynabeads Sheep anti-rabbit or Dynabeads Sheep anti-mouse IgGs; Thermo Fisher Scientific, Waltham, MA, USA) that had been precoated with the anti-core (K45) or anti-L (57761) antibodies, as described [41]. After incubation for 3 h at 4 °C with agitation, the immune complexes were washed three times with either 0.5% CHAPS/HBS (CHAPS-lysed cells) or with 50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 1-mM CaCl2 and 0.1% Triton X-100 (Triton-lysed cells).\n\n2.9. Fluorescence Microscopy\nFor immunofluorescence (IF), cells grown on coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.2% Triton X-100 for 10 min. Cells were blocked in phosphate-buffered saline (PBS) containing 1% BSA, incubated with the indicated primary antibodies for 1 h at 37 °C, rinsed with PBS and then incubated with Alexa Fluor-tagged secondary antibodies for 1 h at 37 °C. Cell nuclei (DNA) were stained with Hoechst 33,342 (Sigma-Aldrich, St. Louis, MO, USA). Images were acquired separately for each channel using a Zeiss Axiovert 200 M microscope equipped with a Plan-Apochromat 100× (1.4 NA) and a Zeiss Axiocam digital camera (Zeiss, Oberkochen, Germany). AxioVision software 4.7.1 (Zeiss, Oberkochen, Germany) was used for merging pictures, and Tiffs were assembled into figures using Adobe Photoshop CS6. For quantitative colocalization analysis (QCA), digital photographs were quantitated using the AxioVision colocalization software (4.7.1; Zeiss, Oberkochen, Germany) by calculating the pixel intensity-based Pearson’s correlation coefficients (PCC) of randomly selected cells (25 cells per coverslip).\n\n2.10. Statistics\nStatistical differences between groups and statistical graphs were assessed with a two-tailed, unpaired t-test using Microsoft Office Excel 2016. Differences between groups were considered significant when the p-value was * p \u003c 0.05 or ** p \u003c 0.01."}