Figure 4

Packaging incompetent A3G variants are defective for binding viral RNA. (A-C) HeLa cells were transfected with 4 μg of empty vector DNA (lane 1), 2 μg of either Myc-tagged or untagged A3G wt (lanes 2 & 3, respectively), 4 μg of A3G-Myc W127A (lane 4), 2 μg of A3G W127A (lane 5), 2 μg of Y124A (lane 6), or 3 μg of A3G-Myc Y124A (lane 7). All samples were co-transfected with 1 μg of vif-defective pNL4-3Vif(-) as a source of genomic RNA. Total amounts of DNA were adjusted to 5 μg using empty vector DNA as appropriate. Cells were harvested 24 h after transfection and divided into four fractions. Fraction 1 was used for immunoblot analysis of whole cell extracts (panel A, top); fraction 2 was used for total RNA extraction and qRT-PCR (panel B). Fractions 3 & 4 were first immunoprecipitated as a pool with an A3G-specific rabbit antibody as described in Methods. Part of the immunoprecipitate (fraction 3) was then used for immunoblotting (panel A, bottom); the other part (fraction 4) was used for RNA extraction and qRT-PCR. (A) Fractions 1 & 3 were analyzed by immunoblotting for the presence of A3G using an A3G specific rabbit polyclonal antibody. Proteins are identified on the right. IgG = rabbit immunoglobulin heavy chain. Total cellular RNA (B) or RNA present in the immune complexes (C) was extracted and subjected to qRT-PCR analysis of 7SL and genomic RNA as described in Methods. 7SL and genomic RNA levels detected in the presence of untagged A3G wt were used as reference and defined as 1.0. RNA levels from all other samples were calculated relative to the reference sample. An RNA sample treated with DNase-free RNaseA (1 mg/ml; 30 min, 37°C) was used as a control for the absence of contaminating DNA.