Figure 3.

Endogenous reverse transcription in cell free particles and proviral DNA synthesis in cells infected with WT or CA mutants expressing HIV Env or VSV-G. (A) Competence for ERT of WT and mutant cell free particles after permeabilization and incubation with dNTPs. Efficiency of DNA synthesis is estimated as the ratio of second-strand transfer to strong-stop DNA detected by qPCR. Data presented are representative of three different experiments performed in duplicate. (B) MAGIC-5B cells were infected with normalized amounts of DNAse-treated WT, S₁₀₉A, S₁₄₉A or S₁₇₈A viruses. Twenty-four hours post-infection, cells were lysed and RT intermediates were quantified by qPCR using primers specific for strong-stop or first-strand transfer or full-length minus strand or second-strand transfer DNAs. Results are the mean of three separate experiments performed in duplicate and are expressed as a percentage of values obtained with WT ± standard deviation. 2-LTR circle formation was detected by amplification of the LTR-LTR junctions in total DNA extracted from MAGIC-5B cells infected with WT or CA mutants expressing HIV Env (C) or pseudotyped with VSV-G (E). Results are the mean of three separate experiments performed in duplicate and are expressed as a percentage ± standard deviation of the values obtained with WT. (D) Efficiency of RT progression was evaluated in MAGIC-5B cells infected with WT or CA mutants expressing either HIV-Env or VSV-G. Values are expressed on a logarithmic scale and are representative of three separate experiments performed in duplicate. For each virus studied, RT efficiency in cells infected through VSV-G relative to that observed in cells infected with the corresponding virus expressing HIV-Env is indicated as a ratio.