FIG. 8.

Effects of inhibition of type I IFN activity on the MVMp life cycle in MEFs and A9 cells. Cells grown in 9.5-cm dishes were treated or not with an IFN-β-neutralizing (7FD3) or an IFN-α-neutralizing (4EA1) antibody at a 1:30 dilution starting 24 h before their mock treatment or infection with MVMp (MOI, 5 PFU/cell). They were left to grow further in the presence or absence of antibody for the indicated times before being processed for Western blotting, Southern blotting, indirect immunofluorescence, or LDH and MTT assays. (A) Western blot analysis of JAK/STAT pathway activation and NS1 expression in RIPA buffer extracts from MEF and A9 cells infected or not for 40 h with MVMp and pretreated or not with 7FD3 or 4EA1. A 100-μg aliquot of total protein extract/sample was subjected to 10% SDS-PAGE and blotted, and membranes were probed with antibodies specific to the phosphorylated and total isoforms of STAT₁ and STAT₂ as well as to PKR and NS1. GAPDH detection was included as a loading control. The data presented are representative of three experiments performed, all of which showed similar results. (B) Southern blot analysis of the kinetics and extent of MVMp replication in infected MEF or A9 cultures, pretreated or not with 7FD3, was performed at the times indicated as described for Fig. 1 and in Materials and Methods. One representative experiment of three is shown. (C) Assessment by immunofluorescence of the effect of IFN-β neutralization on the amount of cells showing NS1 expression in infected A9 or MEF cultures. Cells grown on spot slides (3,000 cells/spot) were treated or not with 7FD3 starting 24 h before their further infection with MVMp for 24 h. They were then fixed with paraformaldehyde and labeled with a primary antibody directed against NS1 (3D9) and a secondary Alexa Fluor 594 donkey anti-mouse IgG. Nuclei were visualized with Hoechst solution. The fraction of NS1-expressing cells was then determined by microscopy. Total and NS1-positive cells were scored for at least five frames per spot under a 20× objective. Values shown are means with standard deviation bars from five independent experiments that were analyzed. In the case of MEF cells, an unpaired t test was performed to determine statistical significance (*, P < 0.05). (D) Analysis by LDH assays of the effects of IFN-β neutralization on the lytic activity of MVMp in A9 or MEF cultures. Cells grown in 96-well plates were treated or not with 7FD3 at a 1:30 dilution starting 24 h before being infected, or not, with MVMp (5 PFU/cell) for a further 72 h. They were then analyzed for virus-induced lysis through quantification of the amount of LDH released into the culture medium as described in Materials and Methods. LDH levels are expressed as a percentage of total LDH present in the respective cultures. The data shown are means with standard deviation bars from three independent experiments, each carried out in triplicate. In the case of MEF cells, an unpaired t test was performed to determine statistical significance (**, P < 0.01). (E) Proliferation of A9 and MEF cultures was investigated by MTT assay (see Materials and Methods) in 96-well plates over a period of 48 h. The data shown are means with standard deviation bars from five independent experiments, each carried out in triplicate.