Inhibition of IFN-β mRNA expression by MP-12 NSs mutants.

<p>(A) MRC-5 cells were infected with MP-12 or its mutants at an m.o.i of 3. Total RNA was collected at various time p.i. as indicated. IFN-β mRNA was detected by Northern blotting analysis using the IFN-β mRNA-specific RNA probe. (B) Growth kinetics of MP-12 and its mutants in MRC-5 cells. MRC-5 cells were infected with each virus at an m.o.i. of 0.01, and the culture supernatants were collected at various times p.i. Virus titers were determined by using a plaque assay in Vero E6 cells. (C) Co-immunostaining for NSs and SAP30. HeLa cells were transfected with plasmid expressing human SAP30 carrying an N-terminal V5 tag and infected with MP-12 carrying a Flag-tagged NSs or mutant MP-12 carrying Flag-tagged NSs mutant at 16 h post transfection. At 6 h p.i., the cells were fixed, immunostained with an anti-V5 tag and anti-Flag tag antibodies, and subjected to fluorescent microscopic examination. (D) Co-immunoprecipitation of NSs and SAP30. HeLa cells were transfected with the plasmid encoded V5-tagged SAP30 or V5-tagged Venus protein. At 16 h post transfection, cells were mock infected or infected with the MP-12 carrying Flag-tagged NSs or mutant MP-12 carrying Flag-tagged NSs mutant. At 8 h p.i., the cells were lysed and subjected to immunoprecipitation by using anti-V5 antibody. Precipitates were examined by using Western blotting with anti-V5 tag antibody (top and third panels) or anti-Flag tag antibody (second and bottom panels). The top two panels show Western blot analysis of the input lysates.