FIG 3.

Effects of immunoproteasome deficiency on adaptive immune responses to MAV-1 in adult mice. (A) Adult B6 and TKO mice were infected i.n. with MAV-1. Control animals were mock infected with conditioned media. Mice were reinfected i.n. with MAV-1 at 28 dpi. Lungs were harvested at 7 dpi following reinfection. RT-qPCR was used to quantify TPL mRNA levels in the lungs. mRNA levels are expressed in arbitrary units standardized to GAPDH. Data from mice at 7 dpi. following primary infection (also depicted in Fig. 1B) are presented to the left of the dotted line for reference but are not included in statistical analysis. Individual circles represent values for individual mice (n = 4 to 6 mice per reinfected group), and horizontal bars represent means for each group. Statistical comparisons were made using two-way ANOVA, followed by Bonferroni’s multiple-comparison tests. No statistically significant differences were identified. (B) Neutralization assays were performed in which serum obtained from infected B6 and TKO mice (n = 10 per group) at 21 dpi was used at the indicated dilutions to neutralize in vitro infection of 3T12 mouse fibroblasts by MAV-1.pIXeGFP, a recombinant GFP-expressing MAV-1. Serum from mock-infected mice (n = 4) was used as a control. Accumulating fluorescence expressed as relative fluorescence units (RFU) was used as an indicator of viral replication. The bottom horizontal dashed line represents background fluorescence in wells with uninfected cells. The top horizontal dashed line represents fluorescence in wells with cells infected with virus in the absence of any serum. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple-comparison tests. ***, P < 0.001, comparing sera from mock-infected and infected mice (both B6 and TKO). No statistically significant differences between sera from infected B6 and TKO mice were identified.