Figure 1
From: Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication
Analysis of PFV Pol particle association in virus samples of different origin. A) Western blot analysis of viral particle preparations of different origin, concentrated by ultracentrifugation through 20% sucrose and digested by subtilisin (+) or mock incubated (-) prior to lysis, using antibodies specific for PFV p85PR-RTand PFV p40IN (α-RT + α-IN), PFV Gag (α-Gag), PFV Env LP (α-LP), PFV Env SU (α-SU), or mouse GAPDH (α-GAPDH) as indicated. Cell culture supernatants (30 ml total) were harvested after transient transfection of 293T cells (six 10 cm dishes per sample) with 16 μg wild type proviral expression construct pczHSRV2 wt (provirus transf., lane 3+4 [15 ml sup], lane 12 [1/30 10 cm dish]), transient co-transfection with 4-plasmids for a replication-deficient PFV vector system (4 μg puc2MD9, 4 μg p6iGag4, 4 μg p6iPol, 4 μg pczHFVenv EM002) (4-component, lane 5+6 [15 ml sup], lane 13 [1/30 10 cm dish]), transient transfection with the Pol expression construct p6iPol (4 μg + 12 μg pUC19) alone (Pol, lane 7+8 [15 ml sup], lane 14 [1/30 of a 10 cm dish]), or from infected BHK/LTR(HFV)lacZ cells (provirus inf., lane 1+2 [11 ml d9 MOI 1 infection sup], lane 11 [1/8 of a 175 cm2 flask]). B-F) Linear velocity sedimentation gradient centrifugation analysis of PFV particles generated by transient transfection of 293T cells with the wild type proviral expression construct pczHSRV2 wt (forty-two 10 cm dishes, 210 ml supernatant total), concentrated by ultracentrifugation through 20% sucrose and prior pretreatment either by subtilisin digestion (+Sub, 60 ml supernatant equivalents), with 1% NP40 (+NP40, 90 ml supernatant equivalents), or mock incubated (mock, 60 ml supernatant equivalents). B) Infectious titer and density of the individual fractions from top to bottom (1-16). C-F) Western blot analysis of the load (lane 1, 1/12 of total) and the individual fractions F1-F16 (lane 2-17, 3/4 of total) using C) monoclonal antibodies specific for PFV Pol p85PR-RT and p40IN subunits (α-RT + α-IN), D) polyclonal antibodies specific for PFV Gag (α-Gag), E) monoclonal antibodies specific for PFV Env SU (α-SU), and F) polyclonal antibodies specific for PFV Env LP (α-LP). Subtilisin protein crossreacting with the PFV Env LP antiserum is marked with an asteriks. L: load.