Figure 3

Characterization of single and double-tagged FV particles. (A) Cellular and particle-associated protein expression analysis of either PFV or SFVmac Env constructs. Representative immunoblots of 293T cell lysates (cell) and purified viral particles (virus). For PFV particles, 293T cells were cotransfected with puc2MD9, pcoPP and (1–4) pcoPG4, (5–8) pcoPG4 CeGFP, (9–12) pcoPG4 : pcoPG4 CeGFP (3:1) as well as (1, 5, 9) pcoPE, (2, 6, 10) pcoPE iCS, (3, 7, 11) pcoPE Ch, (4, 8, 12) pcoPE Ch iCS. For the production of SFVmac Env pseudotyped particles, 293T cells were cotransfected with puc2MD9, pcziPol and (14–17) pcziGag4, (18–21) pcziGag4 CeGFP, (22–25) pcziGag4 : pcziGag4 CeGFP (3:1) as well as (14, 18, 22) pciSE, (15, 19, 23) pciSE iCS, (16, 20, 24) pciSE Ch, (17, 21, 25) pciSE Ch iCS. As a control, cells were only transfected with pUC19 (13, 26). The viral proteins were detected using antibodies specific for PFV Gag (α-Gag), PFV or SFVmac Env LP (α-LP) and PFV Env SU (α-SU). (B) Transduction efficiency shown as relative infectivity of cell culture supernatants containing PFV (bar 1–13) or SFVmac (bar 14–26) Env harboring foamy viral particles. The obtained relative values of one representative experiment are shown. The sample with untagged Gag and Env was arbitrarily set to 100%. (C) 2D colocalization analysis of spotted FV particles. Exemplary wide-field images were obtained with purified Gag-eGFP and Ch-Env (PE Ch) tagged particles. Scale bar 10 μm. (D) Summary of the obtained colocalization percentages of the indicated viruses