FIG. 9.

Cell-cell fusion assay of Lys 1 mutant in 3T3 cells. NIH 3T3 cells expressing Env A, Env Acl, or Lys 1 were seeded in six-well dishes (10⁵ cells per well) and incubated overnight. Cells were then infected with MVA at a multiplicity of infection of 1 as described in Materials and Methods and incubated overnight at 31°C in the presence of 100 μg of Ara-C per ml and sodium butyrate. Cells were then either analyzed for surface expression of Env proteins or used in the cell-cell fusion assay. (A) One well of a six-well dish of infected 3T3 cells was biotinylated with NHS-LC-biotin, lysed, and immunoprecipitated with anti-A tail antibody. Samples were analyzed as described in the legend to Fig. 2B. (B) Infected 3T3 cells were overlaid with 293T cells which had been cotransfected with pOS8 and pCB6-Tva 2 days earlier. After 24 h cells, were lifted off the dish, lysed and analyzed in the MUG β-galactosidase fluorometric assay. The data represent the averages of three replicates; bars show standard deviations.