Inhibition of Porcine Rotavirus in vitro Replication by Guanylate-binding Proteins GBP1 and GBP2
The aim of this study was to investigate the effects of the full length,fragmentation and GTPase activity of guanylate-binding protein-1(GBP1)and guanylate-binding protein-2(GBP2)on the replication of porcine rotavirus(PoRV).The full-length and truncations(G,M,E,ME regions)of GBP1 and GBP 2 genes were amplified by PCR using porcine kidney cell cDNA and GBP1 and GBP2 plasmids as templates and cloned into pCDNA3.1(+)eukaryotic expression vector.Overexpression or silencing of GBP1 and GBP 2 genes on rhesus monkey cells(MA104)cells was performed to explore the effect on PoRV replication.Different time variables were set to screen the time points at which GBP1 and GBP2 exerted their effects on PoRV replication.Apply pan-GTPase enzyme inhibitor(CID-1067700)to probe the effect of GTPase enzyme activity of GBP1 and GBP2 on PoRV.The expression of GBP1 and GBP2 proteins were detected by Western blot,quantitative PCR and viral TCID50 assays,and their antiviral functions were investigated.The results showed that pCDNA3.1(+)-GBP1-full-length-HIS and pCDNA3.1(+)-GBP2(47aa-592aa),(131aa-592aa)-HIS recombinant plasmids were successfully constructed and verified to be expressd.PoRV significantly promoted up-regulations of GBP1 and GBP2 at protein and mRNA expressions.Overexpression of GBP1 and GBP2 significantly inhibited PoRV replication,and silencing the expression of GBP1 and GBP2 genes significantly promoted PoRV replication.The pCDNA3.1(+)-GBP 1/GBP 2-G,M,E,ME-HIS structural domain truncations of GBP1 and GBP2 proteins were successfully constructed and verified to be expressed.Overexpression of the truncator gene revealed that the G region of GBP1 and GBP2 exhibited an inhibitory effect on PoRV replication.It was verified by pan-GTPase inhibitory enzyme activity that GTPase enzyme activity is required for GBP1 and GBP2 to inhibit PoRV replication.The results suggest that the GBP1 and GBP2 proteins are able to inhibit PoRV replication at replication phase of the virus,and the inhibitory effect dependents on its G region containing the GTPase enzyme active sites.The results of the study provide a theoretical basis for finding new drug targets for PoRV and developing novel vaccines.
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