Figure 6
SYNCRIP regulates the ratio of NS1 mRNA to NS2 mRNA and the effect on viral replication. A Laser confocal identification of the effect of SYNCRIP on viral entry in the presence of the translation inhibitor cycloheximide. B NS1 mRNA and its alternatively spliced products are depicted and the arrowheads show primer positions for detection of various mRNA in the following experiments. Cells were infected with PPV, and total RNA was isolated and analyzed by q-PCR with primers specific to NS1, NS2, NS3, VP1 and VP2 mRNA. Ratios of NS2 mRNA to NS1 (C) or to NS3 (D), or VP2 to VP1 (E) at the indicated times are presented. F Diagram of NS2 knockout Y-PPVNS2−. The NS2 protein knockout Y-PPV clone (Y-PPVNS2−) is diagrammed and shown with mutations of three translation initiation codons from CTC to TAG. G Transmission electron microscopy of rescued Y-PPVNS2− virus. Viral particle morphology was observed using transmission electron microscopy. H Q-PCR analysis of parental PPV and Y-PPVNS2− relative DNA copies at different infection time points. I SYNCRIP knockout decreases viral DNA replication via reduction of NS2 protein expression. PK-15 cells were infected with Y-PPV or Y-PPVNS2−, and NS2 proteins were in part of these cells as indicated. PK-15SYNCRIP−/− cells or NS2 protein expressing PK-15SYNCRIP−/− cells were infected with Y-PPV. At 24 h post-infection, the supernatants from each infected sample was collected and used for virion quantification by TCID50. The results are shown as the mean ± SD (n = 3). ns: no significant difference; #p < 0.05 versus PK-15 cells infected with PPV at the same time points. **p < 0.01 versus PK-15 cells with the same treatment.