Fig 5.

SHFL inhibits PRRSV RNA synthesis. (A and B) SHFL suppressed the synthesis of negative-sense and positive-sense viral RNAs. MARC-145 cells transfected with Monkey-siSHFL or siNC were infected with rTA-EGFP. Cells were harvested for total cellular RNA extraction. The accumulation of viral RNAs, including positive-strand genomic RNA (+gRNA) and subgenomic RNAs (+sgRNA) (A) and negative-strand genomic RNA (−gRNA) and subgenomic RNAs (−sgRNA) (B), was quantified by RT-qPCR as described previously (47). The expression of viral RNA was normalized to the expression of β-actin. (C and D) SHFL had no significant effect on the relative expression levels of both negative-sense (C) and positive-sense subgenomic RNAs (D), which were calculated by normalizing their expression values to those of genomic RNA. (E–H) The effect of SHFL on viral biosynthesis was evaluated using an HP-PRRSV replicon (47). The endogenous SHFL was knocked down in HEK-293T (Monkey-siSHFL, 5 pmol) and BHK-21 cells (Pig-siSHFL-1, 50 pmol). Twelve hours later, cells were transfected with the PRRSV replicon. Cell supernatants were collected at 24 hpt and analyzed by the GLuc assay (E, G). Cell lysates were subjected to Western blot analysis of nsp9 and endogenous SHFL. β-Tubulin was detected as a loading control (F, H). (J and K) SHFL suppressed viral translation at the early stage of PRRSV infection. MARC-145 cells transfected with Monkey-siSHFL or siNC were infected with rTA-NLuc (MOI = 3) expressing nsp2-NLuc. At the indicated times, cell lysates were harvested for the NLuc assay and Western blot analysis. (A–J) 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; ns, not significant.