G42E enhances virus binding to human cells. (A) Equal amounts of MMTV-based vector particles carrying either wild-type or mutant (G42E) MMTV(C3H) Env were incubated on ice with either Hs578T or NMuMG cells. The cells were stained with anti-MMTV antiserum, followed by FITC-conjugated secondary antibodies and subjected to FCM. Dead cells were excluded by their forward scatter/side scatter properties. Binding levels were made relative to the value obtained for MMTV-based vector particles carrying wild-type Env and expressed as a percentage of the controls. The values shown are mean ± standard errors from three independent experiments. (B) Supernatants (left panel) from HEK293T cells cotransfected with packaging construct (pCMgpRRE17), an egfp-containing MMTV vector plasmid (pRRpCeGFPWPRE25), a Rev expression construct (pLP2) and either the wild-type or mutant MMTV(C3H) Env were harvested before and after subtilisin A treatment and virions pelleted by ultracentrifugation. 10 μl of the resuspended pellets was subjected toWestern blotting with anti-MMTV-CA antibodies. Cell lysates (right panel) of the virus producers before subtilisin A treatment were analysed by Western blotting. Equivalent protein loading was verified by Coomasie staining (bottom panel). The experiment was performed three times with similar results; a representative experiment is shown.