Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
Figure 7 SYNCRIP affects NS1 expression by acting on the 3
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| Figure 7 SYNCRIP affects NS1 expression by acting on the 3′‑terminal site of NS2 mRNA. A A schematic diagram of NS1 mRNA mutation
site. B Western blotting analysis of the expression of different NS1 mutants in PK-15 SYNCRIP−/− cells. C Western blotting analysis of viral parental NS1, NS1 pNmut , NS1 pCmut and NS1 pNpCmut protein expression levels in PK-15 cells with SYNCRIP over-expression. D Western blotting analysis of NS1 pCmut protein levels (upper) and mRNA levels (lower) in PK-15 cells co-transfected with different doses of SYNCRIP for 24 h. E Western blotting analysis of NS1 pNpCmut protein levels (upper) and mRNA levels (lower) in PK-15 cells co-transfected with different doses of SYNCRIP for 24 h. F Western blotting analysis of NS1 pNmut protein levels (upper) and mRNA levels (lower) in PK-15 cells co-transfected with different doses of SYNCRIP for 24 h. G The effects of NS1 pCmut mutation on PPV DNA copy number were determined by q-PCR assay. (H) The effects of NS1 pCmut mutation on PPV viral titer were determined by TCID 50 assay. The results are shown as the mean ± SD (n = 3). **p < 0.01 versus PK-15 cells with the same treatment at the same time points. Page 14 of 15 Chen et al. Vet Res (2021) 52:73 SYNCRIP targets NS1 mRNA to affect the production of NS2-mRNA and regulated replication of PPV during PPV infection. To further identify how SYNCRIP regulates NS1 mRNA splicing, we constructed different NS1 3′-termi- nal and 5′-terminal splicing site mutants in NS1 mRNA. The results show that the expression of NS1 in the Flag- NS1 and Flag-NS1 pNmut were significantly reduced by SYNCRIP co-expression, while the expression of NS1 in Flag-NS1 pCmut and Flag-NS1 pNpCmut were not decreased by SYNCRIP co-expression. The increasing of SYN- CRIP expression also did gradually decrease the expres- sion of Flag-NS1 and Flag-NS1 pNmut , but did not affect the expression of Flag-NS1 pCmut and Flag-NS1 pNpCmut . Consistently, we noted that NS1 mRNA was obviously decreased in the cells that transfected with Flag-NS1 and Flag-NS1 pNmut expressing vectors, but not in the cells that were transfected with Flag-NS1 pCmut and Flag-NS1 pNpC- mut expressing vectors. When these cells overexpressed SYNCRIP, these results demonstrate that SYNCRIP tar- gets the 3′-terminal of NS1 mRNA to regulate the pro- duction of NS2 mRNA. Meanwhile, a 3′-terminal site mutant of NS2 infectious clone (Y-PPV pCmut ) shows a lower replication rate compared to parental PPV, further demonstrating that the 3′-terminal of NS1 mRNA as the splicing target of SYNCRIP is important for NS2 expres- sion and PPV replication. In summary, the data presented here demonstrate that a porcine RNA binding protein SYNCRIP can directly interact with PPV NS1 mRNA to modulate PPV repli- cation by targeting the 3′-termial site of NS1 mRNA to regulate NS2 expression. This finding will provide one possible antiviral target for porcine parvovirus disease. tải về 5.65 Mb. Chia sẻ với bạn bè của bạn:
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