Fig. 2.

Inhibition of antiviral T cell responses by MPV is not mediated by MHC down-regulation. (A) After 16 h of infection, virus-infected CD14⁺ cells were identified by intracellular staining for OPV antigens as described in ref. 40. Surface expression of MHC class I (HLA-A, -B, and -C) on virus-infected primary human monocytes was down-regulated by CPV, but not by VV or MPV. Surface expression of MHC class II (HLA-DR) was unaltered by infection with CPV, VV, or MPV. (B) MHC class I assembly and transport was measured in OPV-infected cells. HeLa cells were uninfected (uninfected* indicates a lighter scanned image of the same experiment) or infected with VV, MPV, or CPV for 5 h (starved for the last 1 h) and pulse-labeled for 20 min, and the labels were chased for 0, 30, or 60 min as indicated. More than 90% of HeLa cells were infected (data not shown), and cell lysates were immunoprecipitated with anti-MHC class I antibody, W6/32. The precipitated material was treated with EndoH and separated by SDS/PAGE. HC, heavy chain; E^R, EndoH-resistant band; E^S, EndoH-sensitive band. These data show one of four independent experiments, each with comparable results. (C) Stability of MHC class I heavy chain was determined in OPV-infected cells. HeLa cells were infected with VV or MPV for 5 h; starved for the last 1 h in the presence or absence of proteasomal inhibitor, MG132; and pulse-labeled for 20 min, and the labels were chased for 0, 30, or 60 min in the continued presence or absence of MG132. The lysates were then immunoprecipitated with polyclonal anti-MHC class I antibody, K455, followed by EndoH treatment and SDS/PAGE. The open arrowheads indicate the presence of nonspecific low molecular weight proteins that were immunoprecipitated with either anti-MHC class I or anti-CD44 antibodies (see Fig. S1).