Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro
Figure 3
Modification of the ERT reaction components.
(A) Sucrose-cushion purified virus was further purified by velocity-gradient ultracentrifugation. (B) The presence of a contaminating RNase was assessed by quantitative RT-PCR on the viral RNA after incubation at 37°C for 1 h. (C) Gel filtration chromatography on the Jurkat cell lysate (S100 fraction). (D) SDS-PAGE on the S100 (S) and peak gel filtration (P) fraction. (E) Comparison of the ability of sucrose-cushion and velocity-gradient purified virus to be stimulated to generate “second-strand transfer” late reverse transcription products by S100, PGF fraction or control lysis buffer. The data shown are representative of at least two independent experiments. The experiment was performed in triplicate reactions. The mean value and standard deviation of the mean is shown.