Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
Over‑expression of SYNCRIP leads to the reduction of NS1
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- Điều hướng trang này:
- SYNCRIP regulates the ratio of NS1 mRNA to NS2 mRNA and the replication of PPV
| Over‑expression of SYNCRIP leads to the reduction of NS1
mRNA and protein expression To detect whether the interaction between SYN- CRIP and NS1 mRNA will affect the expression of NS1, we established a cell line stably expressing pCDNA-His-SYNCRIP, named as PK-15 SYNCRIP (Fig- ures 5 A, B). The cell viability assay shows that the viability of overexpressing SYNCRIP cells was not significantly different from PK-15 or PK-15 V (Fig- ure 5 C). When recombinant plasmids (pEGFP-NS1, pEGFP-NS2, pEGFP-VP1 and pEGFP-VP2) express- ing NS1, NS2, VP1, or VP2 were transfected into PK-15 SYNCRIP , PK-15 or PK-15 V cells, the level of NS1 protein decreased in PK-15 SYNCRIP cells, while the protein level of VP1, VP2 or NS2 were not changed, compared to those in PK-15 or PK-15 V control cells (Figure 5 D). We further tested whether transient overexpression of SYNCRIP could also decrease NS1 levels, and found that SYNCRIP overexpression did decrease the amount of NS1 protein in PK-15 cells (Figure 5 E). This was consistent with the results that the expression of NS1 decreased gradually in PK-15 cells or ST cells, with the increase of the expression of SYNCRIP plasmid (Figures 5 F, G). Consistently, we noted that NS1 mRNA was significantly decreased in the cells that overexpressed SYNCRIP compared to the cells without overexpression (Figures 5 H, I), sug- gesting that SYNCRIP could regulate NS1 expression at the mRNA and protein levels. SYNCRIP regulates the ratio of NS1 mRNA to NS2 mRNA and the replication of PPV To investigate the function of SYNCRIP in the PPV life cycle, we first assessed whether SYNCRIP deple- tion affected viral entry by examining the localiza- tion of viral capsid protein (VP) in SYNCRIP-depleted cells after PPV infection in the presence of the trans- lation inhibitor cycloheximide. Compared to the con- trol, SYNCRIP knockout did not decrease nuclear VP staining at 2 hpi (Figure 6 A), indicating that SYNCRIP deficiency did not affect PPV entry. To investigate the potential effect on the splice of mRNA, the mRNA lev- els of NS2 and NS1 were measured by qPCR as shown in Figure 6 B. Indeed, the ratio of NS2 to NS1 mRNA decreased in PPV-infected SYNCRIP depleted cells (Figure 6 C). As NS2 mRNA, NS3 mRNA was also derived from the alternative splicing of NS1 mRNA seg- ment, but we did not find a significant difference in the ratio of NS3 to NS1 mRNA among PK-15, PK SYNCRIP+/+ and PK SYNCRIP−/− cells (Figure 6 D). However, for another viral mRNA segment, VP mRNA, which could generate VP1 and VP2 mRNA through alterna- tive splicing, no differences were observed between the control and SYNCRIP depleted cells in the ratio of VP2 to VP1 mRNA (Figure 6 E). Taken together, these find- ings revealed that SYNCRIP could regulate the produc- tion of NS2 mRNA through alternative splicing of NS1 mRNA. To further determine the role of NS2 in PPV rep- lication, we used PPV infectious clone Y-PPV as the operating platform to construct the NS2 mutant strain using the overlap technique for site-directed mutagen- esis (Figure 6 F), and further observed whether NS2 mutant could package into virion by transmission elec- tron microscopy. The results show that the NS2 mutant could package into virion (Figure 6 G), but the NS2 mutant replication rate significantly decreased com- pared to parental PPV (Figure 6 H). To confirm whether SYNCRIP knockout decreased viral DNA replication via inhibition of the NS2 protein, we tested the relative viral titer of PPV or Y-PPV NS2− in PK-15 cells and SYN- CRIP deficient cells depending on whether they express NS2 or not via infection with NS2 protein expressing lentivirus. The results show that NS2 protein comple- mentation significantly increased PPV production in PK-15 cells infected by Y-PPV NS2− (Figure 6 I, compare lane 2 and 3). Similarly, NS2 protein complementa- tion also significantly increased PPV production in Y-PPV NS2 -infected PK-15 SYNCRIP−/− cells (Figure 6 I, compare lane 5 and 7). Meanwhile, we noted that PPV replication levels were reduced in Y-PPV NS2− infected PK-15 cells, PPV or Y-PPV NS2 -infected PK-15 SYNCRIP−/− cells, but did not show a significant difference among these three cells (Figure 6 I, compare lane 2, 4 and 5). Similarly, PPV replication levels did not show a signifi- cant difference among Y-PPV NS2 -infected PK-15 cells, PPV or Y-PPV NS2 -infected PK-15 SYNCRIP−/− cells when these cells exogenously expressed NS2 protein (Fig- ure 6 I, compare lane 3, 6 and 7). However, PPV repli- cation levels were higher in Y-PPV NS2 -infected PK-15 cells, PPV or Y-PPV NS2 -infected PK-15 SYNCRIP−/− cells when these cells exogenously expressed NS2 protein as compared to those in these cells without NS2 exog- enous expression (Figure 6 I). Taken together, these results confirm that SYNCRIP plays an important |