Figure 2
Release and interaction characteristics of various PFV Gag – Env protein combinations. (A) Representative Western blot analysis of 293T cell lysates (cells) and subtilisin-digested 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. Cells were transfected with pczHFVenvEM002 and lane 2, 14: pcziCLEGHH (EGHH); lane 3, 15: pcziPG4 CLHH (wt CLHH); lane 4, 16: pcziPG4 130–648 CLEGHH (130–648 CLEGHH); lane 5, 17: pcziPG4 130–295 CLEGHH (130–295 CLEGHH); lane 6, 18: pcziPG4 CLEGHH (wt CLEGHH); lane 7, 19: pcziPG4 1–621 CLEGHH (1–621 CLEGHH); lane 8, 20: pcziPG4 1–450 CLEGHH (1–450 CLEGHH); lane 9, 21: pcziPG4 1–350 CLEGHH (1–350 CLEGHH);; lane 10, 22: pcziPG4 1–310 CLEGHH (1–310 CLEGHH); lane 11, 23: pcziPG4 1–295 CLEGHH (1–295 CLEGHH); lane 12, 24: pcziPG4 1–129 CLEGHH (1–129 CLEGHH); or lane 1, 13: only pUC19 (mock). (B-C) 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 left, samples of proteins eluted by boiling in SDS-PAGE loading buffer from pelleted glutathione-sepharose beads (pull-down) are shown to the right. Prey (Gag and ctrls) 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: EGHH: pcziCLEGHH; wt CLHH: pcziPG4 CLHH; wt CLEGHH: pcziPG4 CLEGHH; 1–621 CLEGHH: pcziPG4 1–621 CLEGHH; 1–450 CLEGHH: pcziPG4 1–450 CLEGHH; 1–350 CLEGHH: pcziPG4 1–350 CLEGHH; 1–310 CLEGHH: pcziPG4 1–310 CLEGHH; 1–295 CLEGHH: pcziPG4 1–295 CLEGHH; 1–180 CLEGHH: pcziPG4 1–180 CLEGHH; 1–155 CLEGHH: pcziPG4 1–155 CLEGHH; 1–129 CLEGHH: pcziPG4 1–129 CLEGHH. Baits: mock: pCAGGS; GST: pCAG GST; wt: pCAG Env1-60 CLGST; W/A: pCAG Env1-60 CLGST W/A.