FIG. 1.

Loss of cell surface MHC-I in the early phase of MCMV infection. (A) Cell surface expression of K^d molecules determined on murine embryonic fibroblasts (MEFs) by flow cytometry after infection with Δm138-MCMV and after treatment with cycloheximide (CH) or brefeldin A (BFA). Results are presented as percentages of initial expression. Data are averages of data from four independent experiments ± standard deviations (SD). (B) Detection of K^d by indirect immunofluorescence on PFA-fixed (cell surface) and PFA-fixed and Triton X-100-permeabilized (intracellular) cells. Bars, 10 μm. (C) Cell surface level of K^d after 8 h of infection with wt MCMV, Δm138-MCMV, Δie1-MCMV, Δie2-MCMV, and Δie3-MCMV. (D) Cell surface levels of D^d and K^d after infection with Δm138-MCMV under conditions of selective and enhanced expression of immediate-early (IE) and early (E) genes. The protocol used for the definition of the period of E gene transcription (10) required for the synthesis of the protein(s) that downregulates cell surface MHC-I is presented above the data. (Top) Infection in the presence of CH results in enhanced transcription of IE genes, which is followed by selective IE protein synthesis after the replacement of CH with actinomycin D (Act D). (Bottom) After the removal of CH, the synthesized IE proteins activate the transcription of E genes until Act D is added. (E) Effect of ganciclovir (GC) (8 μM) on cell surface levels of K^d (left axis and circles) and DNA replication (right axis and lines) in Δm138-MCMV-infected fibroblasts. The incorporation of [³H]thymidine was determined in 2-h intervals.