Figure 3

Influence of SR proteins on HIV-1 protein synthesis. 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. DNA concentrations were maintained constant by supplementation with the pCLacZ control plasmid which also served to monitor transfection efficiency. Values reported here correspond to assays carried out with a HIV to SR DNA molar ratio of 1:05. Cells were recovered two days after DNA transfection. A: Levels of Gag production were assessed by CAp24 antigen ELISA and expressed as pg of CA per μg of total cellular proteins. Note that ASF had a clear negative impact on Gag accumulation in cells whereas 9G8 had an opposite effect. B: Equivalent amounts of CAp24 antigen as 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-Vpr for p15, anti-NEF for p27 and anti-TMgp41 for Env. The viral Gag, Vpr, NEF and Env proteins are indicated according to their molecular weights in kDaltons. Note that SR proteins did not change the Gag processing pattern (compare lanes 2–4 and 1). ASF caused an indirect increase of Vpr cellular accumulation (lane 2) in agreement with its positive effect on Vpr mRNA level (Figure 1). On the other hand SC35 and 9G8 had an opposite effect (lanes 3–4). All Env levels were low (lanes 2–4) except in the control (lane 1).