Release and interaction characteristics of PFV Gag proteins with small N-terminal deletions or point mutations. (A) Schematic outline of the PFV Gag expression constructs. On top the outline of the wild type full-length PFV Gag coding sequences are shown as grey boxes with different structural and functional domains indicated in differentially shaded boxes as described in the legend of Figure 1. (B) Coiled-coil domain prediction using COiLS v2 algorithm (embnet.vital-it.ch/software/coils)  for different PFV Gag proteins. Shown are the graphic displays of the analysis for full-length wild type protein as well as the N-terminal domains (aa 1–200) of wild type (wt), as well as mutants L17A (L17A) and L17S (L17S). (C-D) Representative Western blot analysis of 293T cell lysates (cells) and viral particles preparations (particles) purified by ultracentrifugation through 20% sucrose, transiently transfected with different combinations of Gag and Env expression constructs as indicated. Gag proteins were detected using a polyclonal anti-HA serum (α-HA), Env proteins using a polyclonal anti-PFV Env LP serum (α-LP). Cells were transfected with pczHFVenvEM002 and lane 1: pcziPG4 6–450 CLEGHH (∆N5); lane 2: pcziPG4 11–450 CLEGHH (∆N10); lane 3: pcziPG4 16–450 CLEGHH (∆N15); lane 4: pcziPG4 21–450 CLEGHH (∆N20); lane 5: pcziPG4 31–450 CLEGHH (∆N30); lane 6: pcziPG4 41–450 CLEGHH (∆N40); lane 7: pcziPG4 1–450 CLEGHH (wt); lane 9: pcziPG4 1–450 L17A CLEGHH (L17A); lane 10: pcziPG4 1–450 L17S CLEGHH (L17S); lane 11: pcziPG4 1–450 P30A CLEGHH (P30A); lane 12: pcziPG4 1–450 H32A CLEGHH (H32A); lane 13: pcziPG4 1–450 R39A CLEGHH (R39A); lane 14: pcziCLEGHH (EGHH); or lane 15: only pUC19 (mock). (E-F) Representative Western blot analysis of pull-down assays using mixed lysates of 293T cells transiently transfected with different GST-tagged Env or EGFP-HH-tagged Gag expression constructs as indicated. Samples of Gag expressing cell lysates corresponding to 10% of the input (input) for the pull-down assay are shown to the right, samples of proteins eluted by boiling in SDS-PAGE loading buffer from pelleted glutathione-sepharose beads (pull-down) are shown to the left. Prey (Gag) proteins were detected using a polyclonal anti-HA serum (α-HA), bait (Env and ctrls) proteins using a polyclonal anti-GST serum (α-GST). Shown are pull-down assays combining 293T cell lysates transiently transfected with various prey or bait expression constructs as indicated. Preys: wt: pcziPG4 1–450 CLEGHH; ∆N5: pcziPG4 6–450 CLEGHH; ; ∆N10: pcziPG4 11–450 CLEGHH; ; ∆N15: pcziPG4 16–450 CLEGHH; ; ∆N20: pcziPG4 21–450 CLEGHH; ; ∆N30: pcziPG4 31–450 CLEGHH; ; ∆N40: pcziPG4 41–450 CLEGHH; wt: pcziPG4 1–450 CLEGHH; L17A: pcziPG4 1–450 L17A CLEGHH; L17S: pcziPG4 1–450 L17S CLEGHH; P30A: pcziPG4 1–450 P30A CLEGHH; L17A: pcziPG4 1–450 H32A CLEGHH; R39A: pcziPG4 1–450 R39A CLEGHH; mock: pUC19. Baits: mock: pCAGGS; GST: pCAG GST; wt: pCAG Env1-60 CLGST; W/A: pCAG Env1-60 CLGST W/A.