FIG 4.

LCMV blocks virus-induced IFN-β production but not apoptosis. (A) A549 cells were infected with LCMV (MOI of 0.1), and NP-positive cells were detected at 48 h postinfection by flow cytometry. Solid line, MAb 113 to LCMV NP combined with anti-mouse IgG-phycoerythrin; dashed line, secondary antibody only. One representative example of three independent experiments is shown. (B) LCMV infection prevents VSV- and SeV-induced IFN-I response. A549 cells were infected with LCMV at an MOI of 0.1 or mock infected (u) for 48 h and then superinfected with VSV (MOI of 3) or SeV (80 hemagglutinin units/ml). At 16 h postinfection, levels of IFN-β mRNA were determined by RT-qPCR as described in the legend of Fig. 3B. Data represent fold induction above values for uninfected (u) cells (n = 3; means ± standard deviations). (C) Detection of IFN-α protein. Supernatants from the experiment described in panel B were analyzed in a multisubtype IFN-α ELISA. Induction of IFN-α protein was calculated over the level in mock-treated uninfected (u) cells (n = 3; means ± standard deviations). (D) LCMV infection does not prevent virus-induced caspase activation. A549 cells were infected with LCMV (MOI of 0.1) or mock infected for 48 h, followed by superinfection with VSV (MOI of 3) (C) or SeV (80 hemagglutinin units/ml) (D) as described for panel B. At 16 h postinfection, activity of caspases 9 and 3/7 was measured as described in the legend of Fig. 1A. Data presented are normalized to levels of uninfected (u) cells (n = 3; means ± standard deviations). For statistical analysis, LCMV-infected and mock-infected specimens were compared at each time point by one-way ANOVA. P values are indicated. n.s., not significant (P > 0.05). (E) LCMV infection does not prevent VSV-induced cytochrome c release. A549 cells were infected with LCMV or mock infected, followed by superinfection with VSV. At the indicated time points, cells were stained for cytochrome c (green) with a specific antibody, and nuclei were visualized with DAPI (blue). (F) Cytochrome c release in LCMV-infected and staurosporine-treated cells was quantified as described in the legend of Fig. 1C. (G) LCMV does not prevent VSV-induced PARP cleavage. Cells were LCMV infected or mock infected and superinfected with VSV for 16 h. PARP cleavage was assessed by Western blotting. Tubulin was used as a loading control. The increased PARP cleavage in LCMV-infected versus mock-infected cells was consistently observed.