Single-round infection analysis phenotype of PFV Gag proteins with small N-terminal deletions and point mutations. 293T cells were co-transfected with equal amounts of PFV transfer vector puc2MD9, Env packaging vector pcziPFVenvEM002, Pol packaging vector pcziPol and various Gag packaging constructs (wt: pcziPG4; ∆N5: pcziPG4 6–648; ∆N10: pcziPG4 11–648; ∆N15: pcziPG4 16–648; ∆N20: pcziPG4 21–648; ∆N30: pcziPG4 31–648; ∆N40: pcziPG4 41–648; L17A: pcziPG4 L17A; L17S: pcziPG4 L17S; P30A: pcziPG4 P30A; H32A: pcziPG4 H32A; R39A: pcziPG4 R39A) or only with pUC19 (mock) as indicated. Western blot analysis of (A) cell lysates (cells) and (B) pelleted viral supernatants (particles) with polyclonal antibodies specific for PFV Gag (α-Gag), and PFV Env-LP (α-LP). The identity of the individual proteins is indicated on the right. C) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay was determined 3 days post infection. The values obtained using the wild type Gag packaging vector pcziPG4 were arbitrarily set to 100%. Absolute titers of these plain supernatants were 5.3 ± 3.5 × 105 EGFP ffu/ml. Means and standard deviations of three independent experiments are shown.