PMC:4576194 / 23104-34743 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":"4. Materials and Methods\n\n4.1. Cells, Viruses and Virus Titration Assay\nPK-15 or SK6 cells (porcine kidney cell lines) and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) confirmed to be free of BVDV and anti-BVDV antibodies. The CSFV Shimen strain was propagated in PK-15 or SK6 cells. Viral titers in the culture supernatant of CSFV-infected cells were determined as described previously [38].\nviruses-07-02833-t001_Table 1 Table 1 Primers used in this study.\n\n4.2. Construction of Expression Vectors\nThe swine eEF1A gene (GenBank accession no. NM_001097418.2) and its various domains were amplified by PCR and cloned into the pMyc vector (Clontech, Palo Alto, CA, USA) to generate pMyc-eEF1A, pMyc-eEF1A(1-333), pMyc-eEF1A(1-237), pMyc-eEF1A(238-462) and pCMV-Myc-eEF1A(201-333), respectively. The CSFV NS5A gene (GenBank accession no. EU497410.1) was amplified by PCR and cloned into the p3×Flag-CMV-10 vector (Sigma-Aldrich, ST. Louis, MO, USA) or pCMV-Myc to generate p3×Flag-NS5A or pMyc-NS5A. The primers for amplifying eEF1A and NS5A genes are listed in Table 1.\n\n4.3. Yeast Two-Hybrid Screen\nA Matchmaker two-hybrid system (catalog no. 630489; Clontech) was used to screen host proteins that interact with NS5A. Briefly, the bait construct pGBKT7-NS5A (BD-NS5A) was transformed into the yeast strain Y2HGold and hybridized with a cDNA library derived from porcine macrophages [39]. Transformants were selected for growth on synthetically defined (SD) medium lacking His, Leu, Trp and Ade (SD/-4) for 3 to 6 days at 30 °C. The clones were then transferred to 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). Blue colonies were selected and inoculated with 5 mL of SD/-4 medium for shaking culture at 30 °C for 1 to 3 days. Yeast plasmids were extracted using a yeast plasmid kit (catalog no. D3376; Omega, Guangzhou, China) according to the manufacturer’s instructions, and the target inserts were verified by sequencing using the Gal4 AD and 3ʹAD primers. To validate the interaction between NS5A and the cellular proteins, the bait and prey plasmids were cotransformed into the Y2HGold yeast strain using a yeast transformation system 2 (catalog no. 630439; Clontech). The interaction between murine p53 and SV40 large T-antigen was used as a positive control, and the human lamin C protein, which does not interact with the SV40 large T-antigen, was included as a negative control.\n\n4.4. Plasmid Transfection\nHEK293T cells grown in 6-well plates (Nunc, Waltham, MA, USA) were cotransfected with 5 µg of pMyc-eEF1A and 3 µg of p3×Flag-NS5A using an X-tremeGENE HP DNA transfection reagent (catalog no. 06366236001; Roche, Penzberg, Germany) according to the manufacturer’s instructions. Briefly, the cells were transfected with 400 µL of serum-free DMEM containing 8 µg of plasmids and 12 µL of X-tremeGENE HP DNA transfection reagent. At 6 h post-transfection (hpt), the transfection mixture was replaced with DMEM supplemented with 10% FBS and incubated for an additional 48 h before being assayed.\n\n4.5. GST Pulldown Assay\nFor expression of the GST-tagged NS5A protein, the NS5A gene was subcloned into the pGEX-6p-1 vector (catalog no. 28-9546-48; GE Healthcare, Piscataway, NJ, USA), creating pGEX-NS5A. The GST or GST-NS5A fusion protein expressed in E. coli BL21(DE3) were purified with the glutathione-Sepharose 4B resin (catalog no. 10049253; GE Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. Briefly, the expression of GST or GST-NS5A protein was induced by addition of 1 mM isopropylthiogalactoside (IPTG) (catalog no. ST098; Beyotime, Shanghai, China).The bacterial cells were harvested and resuspended in cold phosphate-buffered saline (PBS) containing 1 mg/mL protease inhibitor cocktail (catalog no. 11873580001; Roche, Penzberg, Germany), followed by mild sonication. Insoluble components were removed by centrifugation at 10,000 ×g for 20 min. Subsequently, the soluble GST or GST-NS5A protein was incubated with the glutathione-Sepharose 4B resin for 4 h at 4 °C. The resins were washed four times with cold PBS and incubated with 300 μL of the lysate of the HEK293T cells transfected with pMyc-eEF1A for 2 h at 4 °C. After an extensive wash with PBS, the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting using the anti-Myc MAb (1:1000) (catalog no. M4439; Sigma-Aldrich) and an anti-GST polyclonal antibody (PAb) (1:2000) (catalog no. AB101; Tiangen, Beijing, China).\n\n4.6. Coimmunoprecipitation Assay\nFor Co-IP assay, the p3×Flag-NS5A and pMyc-eEF1A were cotransfected into HEK293T cells as described above. The cells were collected at 48 hpt, washed two times with cold PBS, and lyzed with the CHAPS lysis buffer (1% CHAPS, 150 mM NaCl, 5 mM MgCl2 and 10 mM Tris-HCl [pH 7.5]) containing 1 mM PMSF (catalog no. ST506; Beyotime) and 1 mg/mL protease inhibitor cocktail at 4 °C for 1 h. The cell lysate was centrifuged at 13,000 ×g for 20 min at 4 °C. The clarified lysate was treated with a mixture of RNase A (25 U/mL) (catalog no. 2158; TaKaRa, Dalian, China) and RNase T1 (1 U/µL) (catalog no. PF0059; Fermentas, Burlington, Canada) for 1 h at room temperature prior to Co-IP analysis. The lysate was precleared with the Protein G-Agarose (catalog no. 11243233001; Roche) at 4 °C for 4 h followed by incubation with the indicated antibodies at 4 °C for 4 h, and the Protein G-Agarose was then added and incubated at 4 °C for 2 h. The Protein G-Agarose was washed three times with PBS, and the bound proteins were separated by SDS-PAGE followed by Western blotting using the indicated antibodies.\n\n4.7. Confocal Microscopy\nHEK293T cells were cotransfected with pMyc-eEF1A (3 μg) and p3×Flag-NS5A (2 μg). After 48-h incubation, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were then incubated with an anti-Myc PAb (1:100) (catalog no. C3956; Sigma-Aldrich) or anti-Flag MAb (1:100) (catalog no. F1804; Sigma-Aldrich) for 2 h. The cells were incubated with goat anti-mouse IgG (whole molecule)-fluorescein isothiocyanate (FITC) antibody (1:100) (catalog no. F2012; Sigma-Aldrich) and goat anti-rabbit IgG (whole molecule)-tetramethyl rhodamine isocyanate (TRITC) antibody (1:100) (catalog no. T6778; Sigma-Aldrich). Subsequently, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:1000) (catalog no. D8417; Sigma-Aldrich) for 15 min and examined using a Leica SP2 confocal system (Leica Microsystems, GmbH, Wetzlar, Germany).\n\n4.8. Construction of a Stable Cell Line Overexpressing eEF1A\nTo construct a stable cell line overexpressing eEF1A, the swine eEF1A gene was subcloned into the lentivirus vector pFUGW (Addgene, Cambridge, MA, USA). HEK293T cells were cotransfected with the recombinant plasmid pFUGW-eEF1A or the empty vector pFUGW, and the packaging plasmids psPAX2 (Addgene) and pMD2.G (Addgene). At 6 hpt, the medium was replaced with DMEM containing 5% FBS. After 48-h incubation, the supernatant of the cell culture was harvested and ultra-centrifuged to concentrate the recombinant lentiviruses. Subsequently, PK-15 cells grown on 6-well plates were transduced with the lentiviruses of 10 transduction units (TU) per cell. The expression of EGFP-eEF1A in the transduced PK-15 cells was examined by Western blotting using a rabbit anti-eEF1A MAb (1:2000) (catalog no. ab157455; Abcam, Cambridge, UK).\n\n4.9. Gene Knockdown by shRNAs\nTo knock down the expression of eEF1A in PK-15 cells, a lentivirus vector-mediated shRNA targeting eEF1A was constructed. Briefly, the upper and lower sequences of shRNAs for eEF1A and non-targeting negative control (NC) are listed in Table 1. The oligonucleotides were annealed and cloned into the lentivirus vector pLVX-shRNA2 (Clontech). The resulting recombinant plasmids pLVX-eEF1A-shRNA1, pLVX-eEF1A-shRNA2 or pLVX-NC-shRNA were cotransfected with the packaging plasmids psPAX2 and pMD2.G into HEK293T cells in 10-cm cell dishes. At 48 hpt, the lentiviruses in the culture supernatant were concentrated at 6000 ×g for 20 min at 4 °C using an Amicon Ultra-15 centrifugal filter unit with Ultracel-100 membrane (catalog no. UFC910096; Millipore, Billerica, MA, USA). The lentiviuses were titrated and transduced into PK-15 cells as described above. Knockdown of eEF1A was validated by Western blotting using the anti-eEF1A MAb (1:2000).\n\n4.10. Real-Time RT-PCR\nTotal RNA was extracted from the culture supernatant of CSFV-infected PK-15 cells using a TRIzol reagent (catalog no. 15596026; Invitrogen, Waltham, MA, USA). The isolated RNA was then reverse transcribed to cDNA with reverse transcriptase XL (AMV) (catalog no. 2621; TaKaRa, Dalian, China) according to the manufacturer’s instructions. Genomic copies of CSFV were quantified by a real-time RT-PCR assay as described previously [25]. The experiment was carried out in triplicates.\n\n4.11. Luciferase Reporter Assay\nIn luciferase reporter assay, the reporter plasmid pFluc/IRES/Rluc [40], a gift from Prof. Belsham, was used, which contains the firefly luciferase (Fluc) gene under the control of the T7 promoter and the renilla luciferase (Rluc) gene under the control of the CSFV IRES. The plasmid pLXSN-T7 expressing the T7 RNA polymerase [41] was also used in the assay. For the luciferase reporter assay, HEK293T cells grown on 24-well plates were cotransfected with 750 ng of pFluc/IRES/Rluc, 300 ng of pLXSN-T7 and different amounts of p3×Flag-NS5A or pMyc-eEF1A. After 48-h incubation, the reporter gene activity was analyzed with a dual-luciferase reporter assay system (catalog no. E1910; Promega, Madison, WI, USA) and measured with the TD-20/20 Luminometer (Turner Designs, Morgan Hill, CA, USA) according to the manufacturer’s instructions. The data represent the Rluc activity normalized to the Fluc activity. Three independent experiments were carried out in duplicates.\n\n4.12. Streptavidin Pulldown Assay\nThe CSFV IRES or PRRSV 3ʹ-UTR fragment was amplified and cloned into the pcDNA3.1(+) vector to generate pcDNA-IRES or pcDNA-PRRSV-3ʹ-UTR. Subsequently, the plasmids were linearized by EcoRV and transcribed in vitro using a RiboMAX™ large scale RNA production system (catalog no. P1300; Promega) according to the manufacturer’s instructions. The resulting RNA was labeled with photobiotin (catalog no. A14216; Baoman, Shanghai, China) using a mercury vapor lamp for 30 min. HEK293T cells grown on 6-well plates were transfected with the indicated plasmids (pMyc-NS5A and pMyc-eEF1A) and lyzed with the lysis buffer. The lysate collected from two wells of the 6-well plate (lyzed with 150 μL of lysis buffer per well) was incubated with the biotinylated IRES RNA (2 μg) for 4 h at 4 °C, and then incubated with streptavidin beads (catalog no. 11205D; Invitrogen) for another 30 min at room temperature. The beads were washed three times with lysis buffer and analyzed by immunoblotting with the anti-Myc MAb (1:1000). For competitive RNA pulldown, the cell lysate was incubated with an increased amount of unlabeled IRES RNA (0, 0.75, 1.5 and 3 μg) for 1 h at 4 °C and then incubated with the biotinylated IRES and streptavidin beads. The experiments were carried out in triplicates.\n\n4.13. Statistical Analysis \nStatistical analysis was performed using the SPSS 17.0 software. Student’s t-test or one-way analysis of variance was used to compare the CSFV genomic copies. A p-value of \u003c0.05 was considered significant. 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