FIG. 4.

Activation and/or upregulation of expression of antiviral innate immune factors in MVMp-infected MEF and A9 cells. The stimulation of different elements of the antiviral machinery was investigated over time in MVMp-infected A9 or C57BL/6 MEF cultures grown in 9.5-cm dishes. Cells were mock treated or infected at an MOI of 10 PFU/cell and incubated for the times indicated before being subjected to Western blotting and RT-PCR analysis. (A) Western blot analysis of the activation and induction of expression of innate antiviral factors as well as levels of expression of viral proteins were performed on total protein extracts (100 μg/sample) obtained in complete RIPA buffer from infected or mock-treated cells. Extracts were subjected to 10% SDS-PAGE and blotted, and membranes were probed with antibodies specific for phosphorylated and total isoforms of STAT₁ and STAT₂ as well as for PKR and the viral polypeptides NS1 and NS2. GAPDH was used as a loading control. Each blot shown is representative of three additional blots which all gave similar results. (B) Assessment of IFN-β, IFN-non-α4, 2′-5′-OAS, and viral NS mRNA levels by RT-PCR in MVMp-infected or mock-treated cells. Total RNA was isolated at the indicated times by using an RNeasy kit, and 1 μg was reverse transcribed into cDNA. Ten percent of this product was then subjected to PCR with sets of primers specific to each mRNA. GAPDH was used as a housekeeping gene control. No signal was detected when reverse transcriptase was omitted. The presented data are representative of four independent experiments which gave, under the same experimental conditions, identical results.