FIG. 1.

Downregulation of HVEM from the cell surface induced by gD. (A) Downregulation of HVEM expression in cocultures with gD-expressing cells. Target B78-HVEM cells were labeled with Qtracker655 and mixed with effector B78 cells or cells expressing gD. Cells were stained with MAb CW10-PE to detect HVEM by FACS. Target cells were positively selected based on Qtracker655 fluorescence for measurement of HVEM expression. Bar graphs represent PE fluorescence of Qtracker655-positive HVEM-expressing cells as a percentage of the PE fluorescence in cocultures with control B78 cells. The averages from three experiments ± standard errors (SE) are shown. (B) gD on HSV virions downregulates HVEM. HSV KOS (MOI = 50) or KOSgDβ (equivalent of MOI = 50) was added to B78-HVEM cells and left for 45 min at 4°C before shifting to 37°C for 30 min. Cells were then stained with CW10-PE to detect HVEM and analyzed by FACS. The level of HVEM detected on the cell surface when no virus was added was set to 100%. (C) Protease protection assay on HVEM cells. The indicated cell lines were incubated with virus at 4°C before being shifted to 37°C for 15 min. Cells were then treated with proteinase K (+PK) or mock digested (−PK). Cells were lysed, and gB was immunoprecipitated with MAb DL16 and detected by Western blotting (PAb R68). (D) Downregulation of HVEM-ΔCT and HVEM-GPI detected by FACS. Target cells were labeled with Qtracker655 and mixed with effector cells expressing no gD, gD(wt) (white bars), gD(W294A) (gray bars), or gD(D30A) (black bars). Bar graphs represent PE fluorescence of Qtracker655-positive HVEM-expressing cells as a percentage of the PE fluorescence in cocultures with control B78 cells (no gD). The averages from three experiments ± SE are shown.