Fig 3. Trimer molecules containing mutant forms of gO fail to block HCMV entry.

(A) Trimer molecules purified from 293 6E cells were loaded and separated by SDS-PAGE under reducing conditions followed by staining the gels with GelCode Blue (Thermo). Fibroblasts cells (panel A) were either left untreated (no trimer) or incubated with trimer that contained: gO-wt (5 μg/ml), gO-251 (5 μg/ml), or gO-417 (10 μg/ml). ARPE-19 epithelial cells (panel B) were either left untreated (no trimer) or incubated with trimer that contained: gO-wt (25 μg/ml), gO-251(25 μg/ml), or gO-417 (50 μg/ml). in both cases, soluble proteins were incubated with cells for 1 h at 4°C then HCMV BADrUL131 that expresses GFP was added to the cells in the presence of the trimers and incubated for an additional 1 h at 4°C. The cells were then shifted to 37° C and incubated for an additional 2 h (with trimer present) then the virus inoculum was removed, the cells washed, and then incubated in fresh growth media at 37° C for 24 h. The level of virus entry was determined by assessing GFP expression comparing to the no trimer conditions. The data was determined from three separate wells for each condition.