Figure 3

Correlation between cellular and viral RNA encapsidation and APO3G packaging. HeLa cells were co-transfected with pcDNA-APO3G-MycHis together with vif-defective variants of either pNL4-3 (43ΔVif), pC-Help (C-HelpΔVif), or mS.1 (mS.1ΔVif). Viruses were harvested 24 h after transfection and purified as described in Methods. (A) Virus production and packaging of APO3G was monitored by immunoblot analysis using an aliquot of the purified, concentrated virus preparations. APO3G encapsidation was identified using a polyclonal APO3G-specific peptide antibody. Viral capsid proteins (CA) were identified using an HIV-positive human patient serum (APS). (B) APO3G-specific bands in panel A were quantified by densitometric scanning and corrected for fluctuations in capsid levels. Results were calculated relative to APO3G associated with NL4-3ΔVif particles, which was defined as 100%. (C) RNAs were extracted from purified, concentrated viruses and amplified by RT-PCR using primer pairs specific for HIV-1 RNA or host RNAs as indicated on the left and detailed in table 1. RT-PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide.