PMC:4576194 / 5156-18933 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4576194","sourcedb":"PMC","sourceid":"4576194","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4576194","text":"2. Results\n\n2.1. NS5A Interacts with eEF1A\nIn the yeast two-hybrid screen, only one protein, eEF1A, was identified to be a potential binding protein for NS5A (Figure 1A). To confirm the interaction between NS5A and eEF1A, glutathione S-transferase (GST) pulldown assay was performed with the GST-tagged NS5A protein expressed in Escherichia coli and the Myc-tagged eEF1A protein expressed in HEK293T cells. The results showed that GST-NS5A but not GST interacts with eEF1A (Figure 1B). To further verify the interaction between NS5A and eEF1A, coimmunoprecipitation (Co-IP) assay was performed with HEK293T cells overexpressing 3×Flag-tagged NS5A and Myc-tagged eEF1A. After incubation with anti-Flag monoclonal antibody (MAb) and Protein G-Agarose, the Myc-tagged eEF1A was found to coprecipitate with the 3×Flag-tagged NS5A (Figure 1C). Furthermore, the 3×Flag-tagged NS5A was shown to coprecipitate with Myc-eEF1A when the cell lysate was incubated with anti-Myc MAb and Protein G-Agarose (Figure 1D). Considering that both eEF1A and NS5A show a strong affinity for nucleic acids, we investigated whether the NS5A–eEF1A interaction might be mediated through a nonspecific RNA bridge. The results showed that the NS5A–eEF1A interaction was not influenced by RNase treatment, indicating that the interaction is not due to nonspecific RNA-mediated binding (Figure 1E). To examine the colocalization of NS5A protein with eEF1A, the subcellular localization of 3×Flag-NS5A and Myc-eEF1A was examined by confocal microscopy. Both 3×Flag-NS5A and Myc-eEF1A were colocalized in the cytoplasm (Figure 1F). Taken together, the results demonstrated that eEF1A is an interacting partner of the CSFV NS5A protein.\n\n2.2. The Domain I of eEF1A is Critical for the NS5A–eEF1A Interaction\nTo determine which domain(s) of eEF1A is responsible for the association with NS5A, a series of Myc-tagged deletion mutants of eEF1A were constructed (Figure 2A) and tested for the interaction with NS5A by Co-IP assay. The results showed that eEF1A(1-133) and eEF1A(1-237) were capable of binding NS5A (Figure 2B), indicating that the domain I is critical for the NS5A–eEF1A interaction.\nFigure 1 Interaction of NS5A and eEF1A. (A) Reactivity of NS5A with eEF1A in a yeast two-hybrid (Y2H) system. Yeast Y2HGold strain was cotransformed with pGBKT7-NS5A (BD-NS5A) as a bait and pGADT7-eEF1A (AD-eEF1A) as a prey. As a positive control, pGBKT7-p53 (BD-p53) and pGADT7-T (AD-T) were cotransformed into Y2HGold strain. Cotransformation of pGBKT7-Lamin C (BD-Lamin C) and AD-T was used as a negative control. All the yeast colonies cotransformed with the above plasmids were grown on synthetically defined (SD) medium lacking His and Leu (SD/-2), SD medium lacking His, Leu, Trp and Ade (SD/-4) and SD/-4 medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal) and aureobasidin A (Aba) (SD/-4/X-α-Gal/Aba); (B) Glutathione S-transferase (GST) pulldown assay. The GST or GST-NS5A fusion proteins expressed in Escherichia coli BL21(DE3) were purified with glutathione Sepharose 4B resin and incubated with the lysate of HEK293T cells overexpressing the Myc-tagged eEF1A. After washing with cold PBS, the bound proteins were subjected to sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and Western blotting using the anti-GST polyclonal antibody (PAb) (1:2000) and the anti-Myc monoclonal antibody (MAb) (1:1000); (C) Coimmunoprecipitation (Co-IP) analysis of Myc-tagged eEF1A and Flag-tagged NS5A. HEK293T cells were cotransfected with the indicated plasmids (+) or empty vectors (-) for 48 h. The transfected cells were lyzed and incubated with a mouse anti-Flag MAb, followed by incubation with the Protein G-Agarose for 6 h at 4 °C. The immunoprecipitate was analyzed by Western blotting using the anti-Flag MAb (1:1000) and a rabbit anti-Myc PAb (1:500); (D) Co-IP analysis of eEF1A and NS5A by the anti-Myc MAb. HEK293T cells were cotransfected with the indicated plasmids (+) or empty vectors (−), the cell lysate was collected at 48 h post-transfection (hpt), incubated with the anti-Myc MAb and Protein G-Agarose. The immunoprecipitate was examined by Western blotting using the anti-Flag MAb (1:1000) and the anti-Myc PAb (1:500); (E) Effects of RNase A/T1 treatment on the NS5A–eEF1A interaction. Extracts of HEK293T cells overexpressing different proteins were treated or untreated with RNase A/T1 for 1 h prior to Co-IP and immunoblotted with the indicated antibodies. The total RNA in the immunoprecipitate was visualized by ethidium bromide-staining; (F) Colocalization of NS5A protein with eEF1A. HEK293T cells were cotransfected with p3×Flag-NS5A and pMyc-eEF1A. Cells were fixed at 48 hpt and subjected to indirect immunofluorescence assay to detect 3×Flag-NS5A (green) and Myc-eEF1A (red) with mouse anti-Flag and rabbit anti-Myc antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. Samples were imaged and magnified 630 times on the Leica SP2 confocal system fitted with a 63× objective lens. Scale bar = 7.5 μm.\nFigure 2 The domain I of eEF1A is required for its association with NS5A. (A) Schematic representation of eEF1A protein domains and the individual eEF1A deletion mutants tested in this study; (B) Coimmunoprecipitation (Co-IP) analysis of the association of 3×Flag-NS5A with Myc-eEF1A mutants in cotransfected HEK293T cells.\n\n2.3. Overexpression of eEF1A Inhibits CSFV Replication\nTo investigate the effects of eEF1A on the CSFV replication, stable PK-15 cell line overexpressing EGFP-eEF1A (PK-EGFP-eEF1A) or EGFP (PK-EGFP) was generated and infected with the CSFV Shimen strain at a multiplicity of infection (MOI) of 0.1. The Npro expression in the CSFV-infected PK-EGFP-eEF1A cells was decreased at 48 and 72 h post-infection (hpi), compared with that in the PK-EGFP cells (Figure 3A,B). Real-time RT-PCR analysis revealed that the viral genome copies in the PK-EGFP-eEF1A cells were also reduced at 48 and 72 hpi, compared with the PK-EGFP cells (Figure 3C). Additionally, yields of infectious virus in culture supernatant of PK-EGFP-eEF1A cells were decreased at 48 and 72 hpi (Figure 3D). The results indicated the eEF1A negatively modulates the CSFV replication.\n\n2.4. Knockdown of eEF1A by Lentivirus-Mediated shRNAs Enhances CSFV Replication\nTo further determine the effects of eEF1A on CSFV replication, recombinant lentiviruses expressing short hairpin RNA (shRNA) against eEF1A (sheEF1A) or non-targeting negative control shRNA (shNC) were generated and transduced into PK-15 cells, resulting in efficient knockdown of the eEF1A expression (Figure 4A). Twenty-four hours after lentivirus-mediated shRNAs transduction, PK-15 cells were infected with the CSFV Shimen strain at an MOI of 0.1. The viral replication was analyzed by real-time RT-PCR, virus titration and Western blotting. In comparison with the shNC control, the viral genome copies in the eEF1A-knocked down cells were increased at 48 and 72 hpi (Figure 4A). Similarly, the CSFV titers were significantly increased after eEF1A knockdown in PK-15 cells (Figure 4B), and the Npro protein expression in the sheEF1A-transduced cells was increased at 48 and 72 hpi (Figure 4C). The results indicate that the eEF1A negatively modulates the CSFV replication.\nFigure 3 Overexpression of eEF1A suppresses the CSFV growth. (A and B) Western blotting analysis of overexpressed EGFP-eEF1A and CSFV Npro. PK-15 cells were transduced with the lentivirus expressing EGFP-eEF1A or EGFP, followed by infection with CSFV at a multiplicity of infection (MOI) of 0.1. Protein expression was examined by Western blotting at 48 (A) and 72 h (B) post-infection (hpi) using a rabbit anti-eEF1A monoclonal antibody (MAb) (1:2000) and a mouse anti-Npro polyclonal antibody (PAb) (1:500) (produced in-house). β-Tubulin served as an internal control; (C) Real-time RT-PCR analysis of CSFV genomic copy numbers. The culture supernatant of the CSFV-infected cells at 48 and 72 hpi were harvested and evaluated for the viral genomic copy numbers by real-time RT-PCR as described previously [25]. (D) CSFV titers in eEF1A-overexpressed cells. Virus titers in the supernatant were determined at 48 and 72 hpi and expressed as 50% tissue culture infective doses (TCID50)/mL. Error bars represent the standard deviations of the means from three independent experiments. p-values were indicated above the bars.\nFigure 4 Knockdown of eEF1A increases CSFV growth. (A) CSFV genome copies in eEF1A knockdown cells. PK-15 cells transduced with the lenti-sheEF1A or lenti-NC for 24 h were infected with the CSFV Shimen strain at a multiplicity of infection (MOI) of 0.1 for 48 h or 72 h. The CSFV genome copy numbers were assessed using a real-time RT-PCR assay as described previously [25]. (B) CSFV titers in eEF1A-knocked down cells. Virus titers in the supernatant were determined at 48 and 72 h post-infection (hpi) and expressed as 50% tissue culture infective doses (TCID50)/mL. Error bars represent the standard deviations of the means from three independent experiments. p-values were indicated above the bars. (C) Knockdown of eEF1A by lentivirus-mediated shRNAs increased expression of the CSFV Npro protein. The endogenous eEF1A and the CSFV Npro protein were detected by Western blotting using an anti-eEF1A monoclonal antibody (MAb) and an anti-Npro polyclonal antibody (PAb), respectively.\n\n2.5. eEF1A Reduces the Translation Efficiency of CSFV IRES \nA previous study showed that NS5A decreases the CSFV IRES-mediated translation in a dose-dependent manner [13]. To examine the effects of eEF1A on the NS5A-mediated inhibition of the CSFV IRES activity, the luciferase reporter assay was used in this study. The results showed that eEF1A (Figure 5A), as well as NS5A (Figure 5B), inhibited the CSFV IRES activity in a dose-dependent manner. To further investigate the inhibitory effects of eEF1A and NS5A coexpression, eEF1A- and NS5A-expressing plasmids were cotransfected with the luciferase reporter plasmids into HEK293T cells. The results indicated that the eEF1A did not antagonize the inhibition of the IRES activity by NS5A when NS5A and eEF1A were coexpressed (Figure 5C).\nFigure 5 Overexpressed eEF1A or/and NS5A inhibits the CSFV internal ribosome entry site (IRES) activity. (A) eEF1A inhibitory effect on the CSFV IRES activity in a dose-dependent manner. The plasmids pMyc-eEF1A (0, 0.25, 0.5 or 1 μg), pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells. The reporter gene activity was detected and expressed as fold induction. Protein expression of Myc-tagged eEF1A was examined by Western blotting using a rabbit anti-Myc polyclonal antibody (PAb) (1:500); (B) NS5A inhibitory effect on the CSFV IRES activity in a dose-dependent manner. The plasmids p3×Flag-NS5A (0, 0.25, 0.5 or 1 μg), pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells. The reporter gene activity was detected and presented as fold induction. Western blotting was performed using a mouse anti-Flag monoclonal antibody (MAb) (1:1000) to verify the expression of 3×Flag-NS5A; (C) Effects of eEF1A on the NS5A-mediated inhibition of the CSFV IRES activity. Examination of the CSFV IRES activity using a luciferase reporter assay. Luciferase reporter plasmids pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells with or without p3×Flag-NS5A (0.5 μg) and pMyc-eEF1A (0.5 μg). The reporter gene activity was detected and presented as fold induction. To prove the expression of 3×Flag-NS5A and Myc-eEF1A, Western blotting was performed using anti-Flag (1:1000) and anti-Myc (1:500) antibodies. GAPDH was included as an internal control. Rluc level represented the CSFV IRES activity. The Fluc gene under the control of the T7 promoter was used as an internal control. p-values were indicated above the bars. The data were averaged from six replicates of two independent experiments.\nFigure 6 eEF1A binds to the CSFV internal ribosome entry site (IRES). (A) eEF1A as well as NS5A interacts with the CSFV IRES. HEK293T cells were transfected with pMyc-eEF1A or pMyc-NS5A. The cell lysate was incubated with the biotinylated CSFV IRES or porcine reproductive and respiratory syndrome virus (PRRSV) 3ʹ-UTR, followed by incubation with streptavidin beads. The bound proteins were analyzed by immunoblotting using the anti-Myc monoclonal antibody (MAb) (1:1000); (B) eEF1A interacts with the CSFV IRES in a dose-dependent manner. HEK293T cells grown in the 6-well plate were transfected with an increased amount of pMyc-eEF1A (0, 1, 2 and 4 μg), followed by streptavidin pulldown assay as described above; (C) Competitive RNA pulldown assay. HEK293T cells grown in the 6-well plate were transfected with the indicated pMyc-eEF1A (4 μg). The cell lysate was incubated with an increased amount of the IRES RNA (0, 0.75, 1.5 and 3.0 μg) for 1 h at 4 °C and incubated with the biotinylated IRES RNA and streptavidin beads. The bound proteins were subjected to immunoblotting using the anti-Myc MAb.\n\n2.6. eEF1A Interacts with the CSFV IRES\nThe observation that overexpression of eEF1A resulted in the inhibition of the CSFV IRES activity prompted an examination of the interaction between eEF1A and IRES of CSFV. Streptavidin pulldown assay showed that the biotinylated CSFV IRES, but not the biotinylated porcine reproductive and respiratory syndrome virus (PRRSV) 3ʹ-UTR, could bind to eEF1A in a dose-dependent manner (Figure 6A,B). Competitive RNA pulldown assay indicated that the association of eEF1A with the CSFV IRES could be inhibited by the unlabeled CSFV IRES (Figure 6C). The results demonstrated that eEF1A binds to the CSFV IRES and inhibits its activity.\n","divisions":[{"label":"Title","span":{"begin":0,"end":10}},{"label":"Section","span":{"begin":12,"end":1703}},{"label":"Title","span":{"begin":12,"end":42}},{"label":"Section","span":{"begin":1705,"end":5407}},{"label":"Title","span":{"begin":1705,"end":1774}},{"label":"Figure caption","span":{"begin":2163,"end":5081}},{"label":"Figure caption","span":{"begin":5080,"end":5406}},{"label":"Section","span":{"begin":5406,"end":6250}},{"label":"Title","span":{"begin":5406,"end":5460}},{"label":"Section","span":{"begin":6252,"end":9423}},{"label":"Title","span":{"begin":6252,"end":6331}},{"label":"Figure caption","span":{"begin":7308,"end":8433}},{"label":"Figure caption","span":{"begin":8432,"end":9422}},{"label":"Section","span":{"begin":9422,"end":13107}},{"label":"Title","span":{"begin":9422,"end":9481}},{"label":"Figure caption","span":{"begin":10213,"end":11999}},{"label":"Figure caption","span":{"begin":11998,"end":13106}},{"label":"Section","span":{"begin":13106,"end":13776}},{"label":"Title","span":{"begin":13106,"end":13145}}],"tracks":[{"project":"2_test","denotations":[{"id":"26266418-17658704-144197538","span":{"begin":8117,"end":8119},"obj":"17658704"},{"id":"26266418-17658704-144197539","span":{"begin":8803,"end":8805},"obj":"17658704"},{"id":"26266418-19264615-144197540","span":{"begin":9589,"end":9591},"obj":"19264615"}],"attributes":[{"subj":"26266418-17658704-144197538","pred":"source","obj":"2_test"},{"subj":"26266418-17658704-144197539","pred":"source","obj":"2_test"},{"subj":"26266418-19264615-144197540","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#bd93ec","default":true}]}]}}