Fig. 1.

A cell-free system to study ASLV fusion and uncoating. (A) A schematic diagram of the cell-free system described in the text. (B) Low pH-dependent membrane fusion and added magnesium ions and dNTPs are critical for ASLV early DNA production. 293(TVA800) cells were challenged with RCASBP(A)-EGFP at an estimated MOI of 0.15 EGFP transducing unit. The ASLV fusion/uncoating reaction was then set up at 37°C as described in Materials and Methods with 150 μg of 293 S10 fraction and was performed under permissive (3 mM NH₄Cl) or nonpermissive (63 mM NH₄Cl) conditions for ASLV Env-dependent fusion. Samples were incubated with or without 3 mM MgCl₂ and 50 μM dNTPs. Viral DNA was then quantified by using a real-time PCR amplification method. The results are shown as the number of viral DNA molecules synthesized per cell that was used to make the VPNS. A representative example of an experiment that was performed at least three independent times, each time in triplicate, is shown, with the standard deviation of the data indicated with error bars.