Fig 9.

SHFL suppresses the –2 PRF of PRRSV. (A and B) SHFL had no significant effect on the novel –2/−1 PRF during PRRSV infection. MARC-145 cells were transfected with Monkey-siSHFL or siNC (70 pmol) for 48 h. Subsequently, cells were infected with rTA-EGFP (MOI = 1). Cell lysates were collected at 24 hpi for Western blot using an anti-nsp2 antibody to detect the expression of nsp2, nsp2TF, and nsp2N (A). Densitometric analysis of the relative ratios of nsp2, nsp2TF, and nsp2N (B). Data are presented as means ± SD from three replicates. Statistical significance was analyzed using the two-tailed unpaired Student’s t-test. ns, not significant. (C) Schematic diagram of the dual-luciferase reporter carrying –2/−1 PRF signal from PRRSV (WT) of IFC. (D and E) Knockdown of endogenous SHFL stimulated the −2 PRF of PRRSV. HEK-293T cells were first transfected with Monkey-siSHFL or siNC. Twelve hours later, the WT or IFC plasmid and FLAG-tagged nsp1β were introduced. Cell lysates were collected at 24 hpt for dual-luciferase assays (D) and Western blot (E) using an anti-FLAG antibody to detect nsp1β and an anti-RyDEN antibody to detect endogenous SHFL expression. (F and G) The effect of SHFL and its mutants on the −2 PRF of PRRSV. The WT or IFC plasmid and FLAG-tagged nsp1β plasmid were cotransfected with SHFL or its mutant plasmids in HEK-293T cells. Cell lysates were harvested at 24 hpt and analyzed by dual-luciferase assay (F) and Western blot (G) using an anti-FLAG antibody to detect nsp1β and SHFL mutant expression. β-Tubulin was detected as a loading control. Data are presented as means ± SD from three replicates. Statistical significance was analyzed using the two-tailed unpaired Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.