FIG. 6.

Phe residue at position 40 is required for high-level virus binding to NMuMG cells. Equal amounts of MMTV pseudotypes prepared with either the wild-type (WT) envelope or envelope containing the Ser₄₀ mutation (inset, panel A) were incubated on ice with NMuMG cells. The cells were stained with anti-MMTV antiserum, followed by FITC-labeled secondary antibodies, and subjected to FACS analysis. Dead cells were excluded by their forward scatter/side scatter properties. The experiment was performed three to four times with similar results; a representative experiment is shown. (A) Histograms showing the fluorescence of cells incubated with wild-type (thick line) or Ser₄₀ mutant virus (thin line) or without virus (dotted line). The inset shows a Western blot of 10 μl of each concentrated virus preparation or milk-borne MMTV (MMTV) as a positive control, with polyclonal anti-MMTV SU antiserum used for detection. NV, no virus. (B) Histograms showing the fluorescence of cells incubated with wild-type or mutant virus in cells pretreated with 100 μg of heparan sulfate per ml.