Analysis of the GR-rich PFV Gag C-terminus in context of replication-competent proviral expression constructs. 293T cells were transfected with pczHSRV2 (wt), pczHSRV2 μgR (μgR), or pczHSRV2 GR R/A (GR R/A). As controls, cells were transfected with variants pczHSRV2 iRT (iRT), expressing a Pol protein with enzymatically inactive RT domain; pczHSRV2 iEnv (ΔEnv), with inactivated Env translation start; pczHSRV2 M78 (μgag), with inactivated Gag translation start; or only with pUC19 (mock). (A, B) Representative Western blot analysis of viral particles (virus) 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 proviral expression plasmids were arbitrarily set to 100%. Relative means and standard deviations from three independent experiments are shown. Absolute titers of wt supernatants ranged between 6 × 103 and 7 × 104 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 ACTB, GAPDH and PLEKHB2 mRNAs as summarized in Table 1. Mean values and standard deviation (n = 4-6) 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 < 0.01).