Fig. 2.

PIV5-VΔC DIs can activate IRF3 and the IFN-β promoter in the absence of virus protein synthesis. (a) A549 cells were mock-infected or infected with 10 p.f.u. PIV5 wt vM0 or PIV5-VΔC vM2 per cell in the presence or absence of cycloheximide (CHX). At various times p.i., the cells were harvested and the presence of phosphorylated IRF3 (p-IRF3), viral NP and actin was detected by immunoblot analysis. Uninfected (UI) cells were included as a negative control. Note: for PIV5-VΔC vM2 infections, the amount of NP corresponds to input virions only, as PIV5-VΔC DIs inhibit ND virus replication significantly in infected cells. As a result, although CHX markedly reduces PIV5 wt NP synthesis, it does not significantly affect NP expression in PIV5-VΔC vM2-infected cells. (b, c) MRC-5 (b) or HSF (c) cells were mock-infected or infected with 10 p.f.u. PIV5-VΔC vM2 per cell in the presence or absence of CHX. Sixteen hours p.i., cells were harvested and lysates were immunoblotted for p-IRF3, total IRF3, viral P protein and actin. (d) A549 cells were infected with 10 p.f.u. PIV5-VΔC vM2 per cell in the presence of CHX and/or the proteasome inhibitor MG132. Sixteen hours p.i., cells were harvested and lysates were immunoblotted for p-IRF3, total IRF3 and actin. (e) A549/pr(IFN-β).GFP cells were infected with 10 p.f.u. PIV5-VΔC vM2 per cell and cultured in the presence of CHX for the times indicated, after which the CHX block was removed and any further transcription was prevented by culturing the cells in medium containing actinomycin D (ActD). At 12 h p.i., GFP-positive cells were visualized by fluorescence microscopy. (f) A549 cells were infected with 10 p.f.u. PIV5-VΔC vM2 per cell in the presence of CHX or ActD for 12 h, or 6 h CHX followed by 6 h ActD treatment. Monolayers were harvested and ISG56 and actin were detected by immunoblot analysis.