Expression and packaging of A3G variants into vif -deficient HIV-1 virions. (A) HeLa cells were transfected with pcDNA-APO3G (1 μg), pcDNA-APO3G-MycHis (1 μg), pcDNA-APO3G-W127A (1 μg), pcDNA-APO3G-W127A-MycHis (2 μg), pCMV4-APO3G (1 μg), pCMV4-APO3G-HA (2 μg), pCMV4-APO3G-W127A (2 μg), pCMV4-APO3G-W127A-HA (2 μg), pcDNA-APO3G-Y124A (2 μg), and pcDNA-APO3G-Y124A-MycHis (1 μg), together with the vif-defective proviral construct pNL43Vif(-) (3 μg). Total amounts of transfected DNA were adjusted to 5 μg using empty vector DNA as appropriate. Cells and virus-containing supernatants were harvested 24 h post transfection and processed for immunoblotting as described in Methods. Blots were probed with antibodies to A3G or an HIV-positive patient serum (APS) to identify viral capsid (CA) protein. Samples in lanes 1 & 3 and 5 & 7 are replicates derived from independent transfections. (B) A3G and capsid-specific bands in panel A were quantified by densitometric scanning of the gel and the encapsidation efficiency of A3G was calculated for each variant taking into consideration fluctuations in intracellular A3G expression and viral capsid protein. Results are expressed relative to untagged A3G wt (Fig. 1B, lane 1), which was defined as 100%. Actual values are shown above each column.