Fig 7.

SHFL suppresses PRRSV −1 PRF. (A–D) ISG mediated the inhibitory effect of IFN on PRRSV −1 PRF. After pretreatment with 10-fold serially diluted IFN-α2b for 12 h, the −1 PRF reporter plasmid or IFC was transfected into HEK-293T (A and B) and STAT1-knockout HEK-293T cells (C and D). At 24 hpt, cell lysates were analyzed by dual-luciferase assay and Western blot analysis. (E, F, I, J) SHFL overexpression suppressed PRRSV −1 PRF in a dose-dependent manner. The −1 PRF reporter plasmid was cotransfected with increasing amounts of FLAG-tagged SHFL plasmid in HEK-293T (E and F) and BHK-21 (I and J) cells. At 24 hpt, cell lysates were analyzed by dual-luciferase assay and Western blot. (G, H, K, L) Knockdown of endogenous SHFL enhanced PRRSV −1 PRF. HEK-293T (G and H) and PK1 (K and L) cells were first transfected with siSHFL or siNC. Twelve hours later, the −1 PRF reporter plasmid or IFC was introduced. Cell lysates were collected at 24 hpt for dual-luciferase assays and Western blot using an anti-FLAG antibody to detect FLAG-tagged SHFL, an anti-RyDEN antibody to detect endogenous SHFL expression, and an anti-Renilla antibody to detect –1 PRF products. 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 < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.