Fig 6.
Dependence of viral infectivity on PR structure. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9, Env packaging vector pcoPE, and various Pol packaging constructs harboring expression-optimized ORFs for the full-length wild-type PFV Pol protein (coPP, lanes 1, 10, and 11), the mature PR-RT and IN subunits (coRT+coIN, lanes 2, 12, and 13), the mature PR-RT subunit with enzymatically inactive PR and IN subunit (coRT iPR+coIN, lanes 3, 14, and 15), the full PR domain-deleted PR-RT subunit and IN subunit (coRT1+coIN, lanes 4, 5, and 16 to 19), and the core PR-deleted PR-RT subunit and IN subunit (coRT2+coIN, lanes 6, 7, and 20 to 23) as well as different ratios of expression-optimized PFV Gag packaging constructs coding for p71Gag and p68Gag as indicated. As a control, the Env packaging construct was omitted in a sample containing the wild-type full-length Pol packaging construct (coPP +ΔEnv, lanes 9, 26, and 27), or cells were transfected only with pUC19 DNA (mock, lanes 8, 24, and 25). The total amount of transfected PFV Gag packaging was kept constant in all samples. The ratios of p71Gag (p71) to p68Gag (p68) packaging constructs are indicated on top. The total amount of transfected DNA (16 μg) was kept constant in all samples by addition of pUC19 DNA. (A to C) Western blot analysis of Pol and Gag expression in cell lysates (cell) and concentrated virus (virus) either digested with subtilisin (+) or mock (−) incubated using a polyclonal antibody mixture specific for SFV-1 PR-RT and PFV IN (α-RT+α-IN) (A), a rabbit polyclonal antibody specific for PFV Gag (α-Gag) (B), and a rabbit polyclonal antibody specific for PFV Env-LP (α-Env-LP) or mouse monoclonal antibodies specific for PFV Env-SU (α-Env-SU) (C). (D) At 3 days postinfection, transduction rates of HT1080 cells were measured using an egfp marker gene transfer assay. The values obtained using full-length expression-optimized pol ORF expression plasmid (coPP) were arbitrarily set to 100%. Absolute titers of these plain supernatants were (4.38 ± 0.96) × 106 EGFP-positive FFU/ml. Means and standard deviations of at least four independent experiments are shown. In this assay the sample coPP+ΔEnv was not determined (nd).