Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
Figure 5 Over‑expression of SYNCRIP leads to the reduction of NS1 mRNA and protein expression. A
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- SYNCRIP affects the NS1 expression by acting on the 3 ′‑terminal sites of NS2 mRNA
| Figure 5 Over‑expression of SYNCRIP leads to the reduction of NS1 mRNA and protein expression. A Western blotting analysis of SYNCRIP
protein levels in PK-15 cells with SYNCRIP over-expression using anti-His monoclonal antibodies. PK-15 as the cells without transfection, PK V as the cells transfected with blank vector, PK SYNCRIP as the cells transfected with the vector expressing His-SYNCRIP. B Western blotting analysis of SYNCRIP protein levels in PK-15 cells with SYNCRIP over-expression using SYNCRIP monoclonal antibodies. C Cell viability of cell lines stably overexpressing SYNCRIP. D Western blotting analysis of viral NS1, NS2, VP1 and VP2 proteins expression levels in PK-15 cells with SYNCRIP over-expression. E Western blotting analysis of NS1 protein expression levels in PK-15 cells of SYNCRIP over-expression. F Western blotting analysis of NS1 protein levels in PK-15 cells co-transfected with different doses of SYNCRIP (0, 1, 2, 4 or 8 μg) for 24 h.G Western blotting analysis of NS1 protein levels in ST cells co-transfected with different doses of SYNCRIP (0, 1, 2, 4 or 8 μg) for 24 h. H Q-PCR analysis of NS1 mRNA levels in PK-15 cells co-transfected with different doses of SYNCRIP (0, 1, 2, 4 or 8 μg) for 24 h. I Q-PCR analysis of NS1 mRNA levels in ST cells co-transfected with different doses of SYNCRIP (0, 1, 2, 4 or 8 μg) for 24 h. The results are shown as the mean ± SD (n = 3). *p < 0.05 versus untreated cells with the vector pCDNA-His-SYNCRIP over-expression. Page 11 of 15 Chen et al. Vet Res (2021) 52:73 role in PPV replication via regulation of NS2 protein expression. SYNCRIP affects the NS1 expression by acting on the 3 ′‑terminal sites of NS2 mRNA To further identify how SYNCRIP regulates NS2 mRNA splicing, we constructed different NS1 5′-terminal and 3′-terminal splicing site mutants in NS1 mRNA (Fig- ure 7 A), and detected and compared the levels of NS1 protein when cells were transfected with the different mutant constructs together with pCDNA-His-SYNCRIP or without. The results show that the expression of NS1 did not show a difference among these cells transfected with Flag-NS1, Flag-NS1 pNmut , Flag-NS1 pCmut or Flag- NS1 pNpCmut when these cells lacked SYNCRIP expression (Figure 7 B). However, when cells were expressed with SYNCRIP, the expression of NS1 significantly reduced in the cells transfected with Flag-NS1 or Flag-NS1 pNmut compared to the cells transfected with Flag-NS1 pCmut and Flag-NS1 pNpCmut (Figure 7 C). With the increase of SYN- CRIP expression, the expression of Flag-NS1 pCmut and Flag-NS1 pNpCmut did not gradually decrease in cells, but Flag-NS1 and Flag-NS1 pNmut gradually decreased in cells (Figures 7 D–F and Figure 5 F). Consistently, we noted that NS1 mRNA did not decrease in the cells transfected with Flag-NS1 pCmut or Flag-NS1 pNpCmut with the increase of SYNCRIP, but decreased in the cells transfected with Flag-NS1 pNmut when SYNCRIP expression gradually increased (Figures 7 D–F), suggesting that SYNCRIP tar- geted the 3′-terminal site of NS1 mRNA to regulate the production of NS2 mRNA. To further detect the effects of the 3′-terminal mutation site of NS1 mRNA on PPV replication, we constructed a 3′-terminal site mutant of NS1 infectious clone (Y-PPV pCmut ) using PPV infectious clone Y-PPV by site-directed mutagenesis. The results show that the replication rate of Y-PPV pCmut mutants significantly decreased compared to parental PPV (Fig- ures 7 G, H). tải về 5.65 Mb. Chia sẻ với bạn bè của bạn:
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