Characterization of m155 expression and its correlation with CD40 downregulation. (A) RAW264.7 or DC2.4 cells were infected with the indicated viruses and RNA corresponding to the m155 transcript identified by Northern blotting. (B) RAW264.7 cells were infected as indicated, and HA-tagged proteins were detected by immunoblotting cell lysates. Lysates were treated with endoglycosidase H (EndoH) where shown. + or −, with or without indicated virus or EndoH. (C) DC2.4 cells were infected with MCMV m155-HA and analyzed 10 h p.i. by confocal microscopy. m155-HA and the ER marker calnexin are shown. Cell nuclei were detected using Hoechst 33342 DNA stain and are indicated. The overlap between m155-HA (red) and calnexin (green) is shown in yellow. (D) RAW264.7 cells were infected with MCMV-GFP wild type, and CD40 surface expression was measured at the indicated times postinfection. (E) RAW264.7 cells were infected with MCMV m155-HA, and intracellular m155-HA expression was measured as indicated. (F) Lysates of RAW264.7 cells infected with MCMV-GFPm155HA for the times shown were analyzed by immunoblotting.

MCMV-specific CD4 T cell frequency increases in the absence of m155. (A) Splenocytes from mice infected with wild-type (black squares) or m155 deletion (white circles) viruses were restimulated with the indicated peptides at 8 days p.i., and CD8 (A) or CD4 (B) T cell cytokine production was analyzed by intracellular cytokine staining. Graphs represent the percentage of gated splenic CD4 T cells producing IFN-γ (left panel) and IFN-γ and TNF (right panel). Representative results from two experiments are shown. Statistical significance was determined with the two-tailed Mann-Whitney test. Asterisks indicate significance (P < 0.05); numbers under vertical lines are P values.

(A) Schematic depiction of MCMV mutants utilized in this study. MCMV-GFPm155 was designed based on the MCMV-Δ6 backbone inserting the m155 gene under the control of the HCMV major immediate early promoter. Boxes, indicated ORFs; dashed lines, deleted areas replaced by kanamycin resistance gene; solid lines, distance between ORFs. WT, wild type. (B) RAW264.7 cells were infected with MCMV-GFP, and CD40 surface expression was analyzed at 24 h p.i. by microscopy. Red, CD40; green, MCMV-derived GFP. (C and D) RAW264.7 or DC2.4 cells were infected with the indicated viruses. CD40 or CD86 surface expression was determined at 18 h p.i. Black lines, indicated antibody; gray lines, isotype control. Gated for GFP⁺ (MCMV-GFP-infected) cells.

CD40 downregulation results from a loss of protein expression. (A) RAW264.7 cells were infected with the MCMV-GFP wild type or were left uninfected, and cytoplasmic RNA was extracted and analyzed for the presence of CD40 and actin transcripts. +, reverse transcriptase (RT) treated; −, without reverse transcriptase. RAW264.7 (B) and DC 2.4 (C) cells were mock infected or infected with MCMV-GFP, and CD40 (top) and β-actin (bottom) proteins were detected by immunoblotting.