M78 relocalizes MHC II.

<p><b>a</b>. Cloned RAW-C2TA cells were transduced with vector alone or with M78 (line 1 and line 2), or infected with MCMV-GFP (+ MCMV, 1 p.f.u. / cell, 72h), then stained for MHC II (dashed lines). Solid lines show uninfected, untransduced RAW-C2TA cells. The biphasic population with MCMV corresponds to GFP⁺ (MHC II^-) and GFP^- (MHC II⁺) cells. M78 transfection had much less effect than MCMV infection on cell surface MHC II expression. <b>b</b>. MCMV-GFP-infected RAW-C2TA cells were stained for M78. GFP was visualized directly. Nuclei were stained with DAPI. Arrows show example infected cells with M78 in vesicles. <b>c</b>. Untransduced (control) and M78-transduced RAW-C2TA cells were stained for M78 and CD44. Arrows show M78 staining, which did not co-localize with CD44. <b>d</b>. M78-transduced RAW-C2TA cells were stained for M78 and MHC II. M78 occupied intracellular vesicles, as did MHC class II in M78⁺ cells. White arrows show examples of co-localization. Grey arrows show M78^- cells with MHC II on the plasma membrane. <b>e</b>. Macrophages grown from bone marrow were infected with WT MCMV (1 p.f.u. / cell, 24h), then fixed and stained for M78 and MHC II. The white arrow shows a typical uninfected MHC II⁺ cell. The yellow arrow shows a typical infected cell with MHC II in vesicles. >80% of vesicles were M78⁺MHC II⁺, <20% were M78⁺MHC II^- and <1% were M78^-MHC II⁺ (n>300). Thus there was significant endosomal M78 / MHC II co-localization (p<10^-4 by Fisher's exact test). <b>f</b>. RAW-C2TA cells were transfected with a plasmid expressing HA-tagged M78. 3d later they were fixed, permeabilized and stained for M78 and MHC II. Nuclei were stained with DAPI. Arrows show examples of M78 and MHC II co-localizing in internal vesicles. <b>g</b>. Cloned RAW-C2TA-M78 cells were stained for MHC II and M78. White arrows show M78⁺ cells with MHC II in vesicles; grey arrows show cells with diffuse M78 staining and MHC II not relocalized. M78/MHC II co-localization was observed in >30% of transduced cells (n>500) and not in untransduced controls. <b>h</b>. RAW-C2TA cells were infected with HA-M78⁺ MCMV (4h, 37°C), incubated with rabbit anti-HA and rat anti-MHC II IgG (1h, 4°C), then washed and incubated at 37°C for the times indicated before fixation, permeabilization and staining with anti-rat (red) and anti-rabbit (green) antibodies. Nuclei were stained with DAPI. Stainings show the change in distribution of surface-labelled M78 from 0 to 30min, and at 30min M78 co-localization with internalized MHC II (yellow, arrows). <b>i</b>. Summary of staining results from <b>h</b> shows similar kinetics of surface-labelled M78 and MHC II loss. Cells were scored as stained or not regardless of internalization. Each point shows mean ± SEM of 5 fields of view and >100 cells counted. <b>j</b>. RAW-C2TA cells were transfected with plasmid expressing HA-tagged M78, or HA-tagged CCR5 as a control. 3d later they were incubated with rabbit anti-HA IgG (1h, 4°C), then washed and either fixed and permeabilized at once (0 min) or first incubated at 37°C (60 min). All cells were then stained with for rabbit IgG (green). Nuclei were stained with DAPI. HA-CCR5 detection was maintained over 60min, whereas HA-M78 detection was lost.