PMC:4576194 / 11408-14579
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4576194","sourcedb":"PMC","sourceid":"4576194","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4576194","text":"2.4. 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","divisions":[{"label":"Title","span":{"begin":0,"end":79}},{"label":"Figure caption","span":{"begin":1056,"end":2181}},{"label":"Figure caption","span":{"begin":2180,"end":3170}}],"tracks":[{"project":"2_test","denotations":[{"id":"26266418-17658704-144197538","span":{"begin":1865,"end":1867},"obj":"17658704"},{"id":"26266418-17658704-144197539","span":{"begin":2551,"end":2553},"obj":"17658704"}],"attributes":[{"subj":"26266418-17658704-144197538","pred":"source","obj":"2_test"},{"subj":"26266418-17658704-144197539","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93bfec","default":true}]}]}}