Figure 3
From: Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication
Analysis of expression constructs for different Gag-Pol fusion proteins. 293T cells were transiently transfected with the individual proviral expression constructs (A-E) or Gag/Pol packaging constructs in context of the replication-deficient 4-component PFV vector system (F) as indicated. Cell lysates (cell, lane 1-10, 1/25 of total) as well as viral particle preparations (virus, lane 11-29, 10 ml supernatant equivalents), concentrated by ultracentrifugation through 20% sucrose and either digested with subtilisin (+) or mock incubated (-) prior to lysis, were analyzed by Western blot. Antibodies or antisera used were specific for A) PFV Gag (α-Gag), B) PFV p85PR-RT and PFV p40IN (α-RT + α-IN), C) PFV Env SU (α-SU), D) PFV Env LP (α-LP) E) PFV Bet (α-Bet), F) rabbit GAPDH (α-GAPDH). The identity of the individual proteins is indicated in the middle, the molecular weight of the protein standard on both sides. 293T cells were transfected with: pczHSRV2 wt (lane 1, 11, 12; wt); pczHSRV2 iPR (lane 2, 13, 14; iPR); pczHSRV2 PGfP1 (lane 3, 15, 16; PGfP1); pczHSRV2 PGfP1 iPR (lane 4, 17, 18; PGfP1 iPR); pczHSRV2 PGP1 (lane 5, 19, 20; PGP1); pczHSRV2 PGP1 iPR (lane 6, 21, 22; PGP1 iPR); pczHSRV2 PGP2 (lane 7, 23, 24; PGP2); pczHSRV2 PGP3 (lane 8, 25, 26; PGP3); pczHSRV2 PGP4 (lane 9, 27, 28; PGP4); pcDNA3.1+zeo (lane 10, 29; mock). G) Relative titers of the individual 293T supernatants on BHK/LTR(PFV)lacZ cells. Means and standard deviations (n = 4) are shown. H) Relative infectivity of extracellular culture supernatants of 293T cells transfected with Gag/Pol packaging constructs in context of the replication-deficient 4-component PFV vector system using an eGFP marker gene transfer assay determined 3 days post infection. Means and standard deviations (n = 4-8) are shown. Identical amounts of Gag, Pol and Gag-Pol fusion protein packaging constructs were used. In case of the Gag-Pol fusion protein packaging constructs the total amount of transfected DNA was kept constant by the addition of empty pcDNA3.1zeo vector. Cotransfection of empty pcDNA3.1+zeo vector (plain), p6iGag4 (+Gag wt), or p6iPol (+ Pol wt).