N protein of SVCV inhibits p53 expression.

(A) SVCV infection decreases the expression of endogenous p53 protein. ZFL cells were seeded in 6-well plates and uninfected (null) or infected with SVCV (MOI = 1) at various time points, and the whole-cell lysates (WCL) were detected by IB, with the anti-p53 and anti-β-actin Abs. (B) High dose of SVCV infection attenuates the expression of endogenous p53 protein. ZFL cells were seeded in 6-well plates and infected with SVCV at the dose (MOI = 10, or 1, or 0.1) for 24 h prior to being harvested for IB analysis of WCL with the Abs indicated. (C) SVCV infection inhibits exogenous p53 expression in a dose-dependent manner. EPC cells were seeded in 6-well plates and transfected with 2 μg p53-Myc for 24 h, then cells were infected with SVCV (MOI = 10, or 1, or 0.1) for 24 h prior to being harvested for IB analysis of WCL with the anti-Myc and anti-β-actin Abs. (D) p53 is downregulated by SVCV in the stably expressed cell line. p53-stably expressed ZFL cells were seeded in 6-well plates and infected with SVCV (MOI = 10, or 1, or 0.1) for 24 h, then harvested for IB analysis with the anti-p53 and anti-β-actin Abs. (E) The expression of SVCV N and P protein at protein level. ZFL cells were seeded in 6-well plates and uninfected (null) or infected with SVCV (MOI = 0.1) at various time points, and the WCL were detected by IB, with the anti-N protein, anti-P protein and anti-β-actin Abs. (F) p53 associates with N protein in a mammalian overexpression system. HEK 293T cells seeded in 10 cm2 dishes were transfected with the indicated plasmids (5 μg each). After 24 h, cell lysates were IP with anti-Flag-affinity gels. Then the immunoprecipitates and WCL were analyzed by IB with the Abs indicated. (G) Overexpression of N protein declines the expression of p53. EPC cells were seeded in 6-well plates and transfected with 1 μg p53-Myc together with1 μg empty vector, or N-Flag for 24 h. Then the WCL were subjected to IB with the Abs indicated. (H) N protein blocks the expression of p53 in a dose-dependent manner. EPC cells were seeded in 6-well plates and transfected with 1 μg p53-Myc and 1 μg empty vector, or N-Flag (1, 0.5, or 0.25 μg) for 24 h. Then the WCL were subjected to IB with the Abs indicated. (I) Increase of virus replication by overexpression of the N protein. EPC cells seeded in 24-well plates overnight were transfected with 0.5 μg N-Myc or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 10) for 48 h, the culture supernatants were collected and then the viral titer was measured by standard plaque assays. Error bars are the SDs obtained by measuring each sample in triplicate. Asterisks indicate significant differences from control (*, p < 0.05). All experiments were repeated for at least three times with similar results.