Expression of viral proteins results from alterations of splicing pattern. 293T cells (2 × 105 per well) were transfected with 1 μg of HIV-1 pNL4.3 in the presence of increasing amounts of plasmid encoding either ASF/SF2, SC35 or 9G8 (ratios indicate molar amounts of HIV-1 DNA vs SR-expressing vector). DNA concentrations were maintained constant by supplementation with the pCLacZ control plasmid which also served to monitor transfection efficiency. A: Viral production was monitored by CAp24 antigen ELISA and expressed as ng of p24 per ml of medium (see methods). Results are representative of 3 independent experiments. Note that the effect of ASF/SF2 on virion production was already drastic at a HIV/SR molar ratio of 1:0.5. B: The pelleted viral particles were tested for their content in Gag, Pol, Env and Vpr proteins. Equivalent amounts of CAp24 antigen measured by ELISA were subjected to Western blotting. The same membrane was alternatively probed with the respective antibodies as indicated on the right: anti-CAp24 for Gag, anti-RT for p66 and p51, anti-Vpr for p15 and anti-TMgp41 for Env. The viral Gag, RT, Vpr and Env proteins are indicated according to their molecular weights in kDaltons. Note that fully mature CAp24 and RTp66/p51 were abundant in all virion preparations. ASF caused an indirect increase of Vpr incorporation in virions (lane 2) whereas SC35 and 9G8 had an opposite effect (lanes 3–4). All Env levels were low (lanes 2–4) except in the control (lane 1).