MCMV m166 expression.

<p>(A) Primary sequence of the m166 mRNA transcript as determined by RACE analysis. Black Bold: start and stop codon, gray bold: polyadenylation signal. (B) 3T3 cells were infected with MCMV-WT and expression of MCMV <i>m166</i> and <i>ie1</i> mRNA was determined by qPCR at the indicated times. All mRNA levels are normalized to L32 (×10⁴). (C) 3T3 cells were mock infected or infected with MCMV-WT, MCMV-Δm166 or MCMV-m166HA, and cell surface levels of TRAIL-DR were assessed 24 h later by FACS. Black histograms, mock; gray histograms, MCMV; shaded histograms, isotype control. (D–F) 3T3 cells were infected with MCMV-m166HA and harvested at the indicated times for Western detection of (D) m166HA protein in untreated cells, or (E) after treatment with foscarnet or actinomycin D/cycloheximide. (F) Infected 3T3 cells were co-stained with anti-HA antibody (red) along with anti-GRP94, ER marker (top panels, blue) or anti-GM130, cis-golgi marker (bottom panels, blue) antibodies at indicated times. (G) 3T3 cells were infected with MCMV-WT, and expression of TRAIL-DR mRNA was determined by qPCR at indicated times. All mRNA levels are normalized to L32 (×10⁴). All infections were performed at an MOI of ∼1. (H) 3T3 cells were transfected with an m166-GFP expression plasmid, and TRAIL-DR expression in GFP- (black histogram) and GFP+ (gray histogram) was determined by flow cytometry 72 h later. Shaded histogram, isotype control antibody.