FIG. 4.

HCMV gB recycles between the TGN and the cell surface. To determine whether retrieval from the cell surface played a role in mediating TGN localization of gB, we performed antibody uptake experiments. U373 cells expressing recombinant gB were incubated with MAb M1 for a period of 4 h prior to fixation and staining. The gB that cycled to the cell surface and had been retrieved into the cytoplasm was then visualized using an M1 isotype-specific secondary antibody. Total gB was visualized by incubation of fixed cells with MAb M2 and the respective isotype-specific antibody. Cells expressing recombinant gB that had been incubated with MAb M1 prior to fixation show a vesicular perinuclear distribution of M1 reactivity (cell surface-retrieved gB) that was superimposable upon the postfixed M2 staining pattern (total gB). These results demonstrate that retrieval from the cell surface plays a role in the TGN localization of HCMV gB.