Figure 2
Cellular and particle associated protein expression- and infectivity analysis of PFV Gag-FPs. PFV particles were generated by transient transfection of 293T cells using the 4-plasmid PFV vector system. (A-C) Representative Western Blot analysis of 293T cell lysates (cell) (A) and viral particles (virus) purified by ultracentrifugation through 20% sucrose for N- or C-terminal Gag-FPs (B, C). PFV proteins were detected by using (A, B) a polyclonal anti-PFV Gag (α-Gag) or (C) an anti-PFV Env LP (α-LP) specific antiserum. (B) In addition subtilisin- and mock-treated samples were compared according to their particle associated Gag expression by α-Gag immunoblot. (D) Relative infectivities of extracellular cell culture supernatants using EGFP marker gene transfer assay. The values obtained using wild-type PFV Gag expression plasmids (lane 1, 8) were arbitrarily set to 100%. Mean values and standard deviations from three independent experiments are shown. 293T cells were cotransfected with puc2MD9, pcziPol, pczHFVenv EM002 and either (lane 1, 8) pcoPG4 (wt), (lane 2) pcoPG4 NHA, (lane 3) pcoPG4 NeGFP, (lane 4) pcoPG4 NeYFP, (lane 5) pcoPG4 NCerulean, (lane 6) pcoPG4 NmCherry, (lane 9) pcoPG4 CHA, (lane 10) pcoPG4 CeGFP, (lane 11) pcoPG4 CeYFP, (lane 12) pcoPG4 CCerulean, (lane 13) pcoPG4 CmCherry or wtGag cotransfected at a ratio of 1:1 (lane 14) pcDNA 3.1zeo+, (lane 15) pcoPG4 CHA, (lane 16) pcoPG4 CeGFP, (lane 17) pcoPG4 CeYFP, (lane 18) pcoPG4 CCerulean, (lane 19) pcoPG4 CmCherry. As control, cells were only transfected with pcDNA3.1 zeo+ (lane 7, 20). (E) Comparison of relative infectivities of C-terminal Gag-GFP (Gag-C-GFP) fusion proteins either transfected alone or cotransfected with untagged Gag (wt-Gag) with the EGFP marker gene transfer assay, as depicted. The values obtained using wild-type PFV Gag expression plasmids (1:0) were arbitrarily set to 100%. Mean values and standard deviations from two independent experiments are shown. 293T cells were cotransfected with puc2MD9, pcziPol, pczHFVenv EM002, pcoPG4 (wt) or/and pcoPG4 CeGFP at different ratios as indicated.