Analysis of the GR-rich PFV Gag C-terminus in context of a replication-deficient vector system. 293T cells were co-transfected with pMD9, pcziPol, pcoPE and either pcoPG4 (wt), pcoPG4 μgR (μgR), or pcoPG4 GR R/A (GR R/A). As controls, cells were transfected with pcoPE, pcoPG4 and pcziPol (wt ΔvgRNA), with pMD9, pcoPE, pcoPG4 and pcziPol iRT (wt iRT), with pMD9, pcoPG4 and pcziPol (wt ΔEnv) or only with pcDNA3.1 zeo + (mock). (A, B) Representative Western blot analysis of viral particles (virus) purified from 293T cell culture supernatant by ultracentrifugation through 20% sucrose and 293T cell lysates (cell). PFV proteins were detected using antibodies specific for PFV Gag (α-Gag), for PFV Pol PR/RT and IN (α-PR/RT + α-IN), or for PFV Env SU (α-SU). (C) Viral particle release was determined by quantitative Western blot analysis of viral particles. Mean values and standard deviations (n = 3-6) are shown as relative values compared to the wild type control and normalized for cellular expression levels. (D) Infectivity analysis of PFV particle containing cell culture supernatants. The values obtained using wild type PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations from six independent experiments are shown. Absolute titers of wt supernatants ranged between 2 × 106 and 1.3 × 107 eGFP ffu/ml. (E) Viral particle nucleic acid content was determined by qPCR using specific primer - probes sets for PFV Pol, as well as human PGK1, ASB1 and PLEKHB2 mRNAs as summarized in Table 1. Mean values and standard deviation (n = 3-6) are shown as relative values compared to the wild type control. Values were not normalized for Gag content. Differences between means of the wild type and the individual mutants in (C)-(E) were analyzed by Welch’s t test (*, p < 0.05; **, p < 0.01).