Viral and non-viral RNA encapsidation by Gag mutants with individual GR box deletions. 293T cells were co-transfected with puc2MD9, pcoPP, pcoPE and either pcoPG4 (wt), pcoPG μgRI (μgRI), pcoPG μgRII (μgRII), pcoPG μgRIII (μgRIII), pcoPG4 μgR (μgR), or pcoPG4 GR R/A (GR R/A). The amount of packaging constructs was kept constant whereas different amounts of transfer vector puc2MD9 (5, 0.5, 0.05 μg) were used as indicated. The total amount of DNA used for transfection was kept constant by filling up with pUC19. Subsequently extracted particle-associated nucleic acids and total cellular RNA samples were subjected to qPCR analysis using different primer-probe sets as summarized in Table 1. (A) Cellular and viral particle-associated levels of vgRNA. Viral particle and cellular nucleic acid content was determined by qPCR using specific primer - probes sets for PFV Pol. Mean values and standard deviation (n = 3-6) are shown as relative values compared to the wild type control. Viral particle values were normalized for Gag content, cellular values were normalized per ng of total RNA. (B) Viral particle-associated levels of vgRNA and selected cellular mRNAs. Viral nucleic acid content was determined by qPCR using specific primer - probes sets for PFV Pol, as well as human PGK1 and GAPDH mRNAs. Mean values and standard deviation (n = 3-6) are shown as relative values compared to the wild type control in each set of transfections varying in their amount of transfer vector as indicated. Viral particle values were normalized for Gag content. Differences between means of the wild type and the individual mutants were analyzed by Welch’s t test (*, p < 0.05; **, p < 0.01).