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. 2004 May 5;101(20):7721–7726. doi: 10.1073/pnas.0401312101

Fig. 2.

Fig. 2.

Cellular factor(s) stimulate ASLV fusion/uncoating in the cell-free system. (A) The 293 S10 fraction enhances both the rate and extent of ASLV early viral DNA production. 293(TVA800) cells were challenged with RCASBP(A)-EGFP at an estimated MOI of 0.4 EGFP transducing unit, and fusion/uncoating reactions were set up at 37° C with 3 mM NH4Cl and with different amounts (0–350 μg) of 293 S10 fraction added. For control purposes, samples containing the maximum amount of 293 S10 fraction (350 μg) were also incubated under nonpermissive fusion conditions (63 mM NH4Cl or at 4°C). Reactions were then stopped at the indicated times and early viral DNA products were measured as in Fig. 1B. A representative example of an experiment that was performed two independent times, each time in triplicate, is shown, with the standard deviation of the data indicated with error bars. (B) ASLV early viral DNA production in the cell-free system is specifically enhanced by a 293 S10 fraction. 293(TVA800) cells were challenged with RCASBP(A)-EGFP at an estimated MOI of 0.1 EGFP transducing unit. The ASLV fusion/uncoating reaction was then set up at 37°C with 3 mM NH4Cl and with different amounts of the 293 S10 fraction, BSA, or yeast cell S10 fraction added. Early viral DNA products were then measured as in Fig. 1B. A representative example of an experiment that was performed two independent times, each time in triplicate, is shown with the standard deviation of the data indicated with error bars. (C) The 293 cell factor(s) are soluble and >5 kDa in size. 293(TVA800) cells were challenged with RCASBP(A)-EGFP at an estimated MOI of 0.15 EGFP transducing unit. Fusion/uncoating reactions were then set up at 37°C with either 150 μg of 293 S10 fraction or equivalent amounts of either 293 P100 or S100 fractions, or 293 S10 fractions that had been subjected to gel filtration (Mr > 5 kDa), dialysis (molecular mass cutoff, 3.5 kDa) or membrane filtration (molecular mass cutoff, 5 kDa). Early viral DNA products were measured as described in Fig. 1B. The results are shown as a percentage of the data obtained with 150 μg of 293 S10 fraction added (designated as 100%). A representative example of an experiment that was performed twice, both times in triplicate, is shown, with the standard deviation of the data indicated with error bars.