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. 2007 Apr 18;81(13):6785–6797. doi: 10.1128/JVI.00198-07

FIG. 1.

FIG. 1.

CVB3 gene expression correlates with the downregulation of surface MHC class I. All experiments were repeated at least three times, and results are shown for a single representative experiment. (A) HeLa (RW) cells were mock infected or infected with wild-type CVB3 at an MOI of 10. Following virus adsorption, cells were harvested, washed, and incubated in suspension at 37°C with 5% CO2. At the indicated times postharvest, an aliquot of cells from each culture was stained with mouse anti-human β2M antibody, fixed, and analyzed by flow cytometry to assess surface MHC class I levels (MFI β2M). (B) During the experiment depicted in panel A, cell lysates from the CVB3 infection time course were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis to examine the onset of viral protein expression. A rabbit anti-3A antibody detected the mature 3A protein as well as the immediate precursor polypeptide 3AB (indicated to the right of the panel). A longer exposure of this region of the blot is shown below. (C) Comparison of uninfected and infected cell surface MHC class I expression within a single-cell population. HeLa (RW) cells were infected at a low MOI (0.1) with a recombinant CVB3 that expresses EGFP, and surface staining (as in panel A) was carried out to assess MHC class I levels on the surfaces of uninfected (black bars) versus infected (white bars) cells, which were distinguished based on the expression of GFP, which became detectable at ∼3 h postinfection. (D) HeLa (RW) cells were mock infected or infected with CVB3 at an MOI of 10. Following virus adsorption, both cell populations were harvested, washed, and incubated in suspension at 37°C with 5% CO2. Two μg/mL brefeldin A (BrA) was added to the uninfected population immediately postharvest, and at the indicated times, an aliquot of cells from each population was stained with mouse anti-human β2M antibody. The cells were then fixed and analyzed by flow cytometry to assess the percentage of cells that were positive for β2M expression.