FIG. 5.

Nucleic acid composition and reverse transcriptase activities of mutant PFV particles. Mutant PFV particles as indicated were generated by transient transfection of 293T cells using the four-plasmid PFV vector system. (A) Relative nucleic acid composition of mutant particles. Following DNase I digestion of intact, purified particles, nucleic acids were isolated, and the relative amounts of vector RNA and DNA copies (expressed as percentages) were determined in comparison to that for the wild type by real-time PCR. Mean relative RNA and DNA contents and standard deviations (n = 3), normalized for the differential particle releases of the individual mutants, are shown. (B) Relative reverse transcriptase activities of mutant particles. Following subtilisin digestion of intact, purified particles, particle lysates were generated after an additional ultracentrifugation step and analyzed for PFV reverse transcriptase activity using a RetroSys C-type RT enzyme-linked immunosorbent assay. Mean relative reverse transcriptase activities (n = 3) and standard deviations, normalized for the differential particle releases of the individual mutants and expressed as percentages, are shown. 293T cells were cotransfected with pMD9, pcziPol, pczHFVenvEM002, and either pcziPG2 (wt), pcziPG L3 (L3), pcziPG Y464A (Y₄₆₄A), pcziPG E465A (E₄₆₅A), pcziPG I466A (I₄₆₆A), pcziPG L467A (L₄₆₇A), pcziPG G468A (G₄₆₈A), or pcziPG L469A (L₄₆₉A). As a control, cells were transfected with pMD9, pcziPG2, pczHFVenvEM002, and an RT-deficient Pol expression vector, pGiPolΔRT (ΔRT).