Fig. 4

The NS38, S9 segment-encoded protein of GCRV, inhibits SVCV proliferation. A, B Effect of overexpression of GCRV segments on the SVCV proliferation. EPC cells were seeded in 6-well plates and transfected with indicated plasmids (1 μg each) for 24 h, then cells were infected with SVCV (MOI = 1) for 24 h prior to being harvested for RNA extraction and quantitative PCR (qPCR). The β-actin was used as an internal control for normalization. The relative expressions of SVCV genes were represented as fold induction relative to the expression level in control cells (set to 1). Data were expressed as mean ± standard error of the mean (SEM), n = 3. Statistical analysis was performed by the Student's t-test. Asterisks indicate significant differences from control (^∗P < 0.05). The expressions of SVCV proteins were determined by immunoblotting (IB) with the anti-N protein, anti-P protein, and anti-β-actin antibodies (Abs). C, D Detection of the viral titer of SVCV effected by overexpression of NS38. EPC cells were seeded in 24-well plates overnight and transfected with 2 μg NS38-Myc or vector. After 24 h, cells were infected with SVCV (MOI = 1) for 48 h. The cells were fixed with 4% PFA and stained with 1% crystal violet for visualizing cellular cytopathic effect (CPE) (C), and the viral titers of the supernatants were determined by 50% tissue culture infective dose (TCID₅₀) assays (D). E NS38 degrades SVCV on a dose-dependent manner. EPC cells were seeded in 6-well plates and transfected with 0.5, 1, 1.5, or 2.0 μg NS38-Myc or empty vector for 24 h, then cells were infected with SVCV (MOI = 1) for 24 h prior to being harvested for IB analysis of WCLs with the indicated Abs. F–I NS38 inhibits proliferation SVCV. Immunofluorescence (IF) analysis was used to indicate amplification of SVCV, with green fluorescence representing the N protein (F). The mRNA levels of SVCV genes were detected upon overexpression and knockdown of S9 (G, I). H Transcript levels of S9 tested the efficiency of knockdown. Data were expressed as mean ± SEM, n = 3. Statistical analysis was performed by the Student's t-test. Asterisks indicate significant differences from control (^∗P < 0.05).