FIG. 4.

Infectivity analysis of PFV Gag L3 domain motif point mutants. Mutant PFV particles as indicated were generated by transient transfection of 293T cells using the four-plasmid PFV vector system in duplicate. (A and B) During the sodium butyrate induction step, one plate per construct was shifted to 30°C (shaded bars) whereas the other plate was incubated at 37°C (solid bars) until virus was harvested from the culture supernatant (extracellular) (A) or freeze-thaw cell lysates (intracellular) (B). Subsequently, HT1080 target cells were incubated for 6 h with viral supernatants or dilutions thereof; after removal of the viral supernatants, target cells were incubated for an additional 4 h at the same temperature as before. Then all target cells were shifted to 37°C, and they were analyzed by FACS 48 h after addition of the viral supernatants to the target cells. (C and D) Reciprocal incubation at 37°C (C) or 30°C (D) during extracellular virus production and subsequent infection of HT1080 target cells using selected PFV Gag L3 motif mutants. Mean relative infectivities and standard deviations (n = 3) of cell supernatants (extracellular) and freeze-thaw cell lysates (intracellular) by the EGFP marker gene transfer assay are shown. The values obtained using the wild-type PFV Gag expression plasmid (wt) were arbitrarily set to 100%. 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 controls, cells were transfected either with pMD9 only (pMD9) or with pcDNA3.1+zeo only (pcDNA).