Effect of HIV-1 virion protein and RNA composition on APOBEC3G incorporation into viral particles. (A) Western blot (WB) analyses of cell lysates (left panels) and extracellular VLPs (right panels) generated following transfection of 293T cells with plasmids expressing HIV-1 Gag-Pol and either myc-tagged APOBEC3G or myc-tagged GFP. A plasmid expressing a packageable RNA genome was included where indicated. The upper panels show Western blots probed with an anti-HIV-1 CA antibody, and the positions of the Gag precursor (Pr55 Gag) and processed p24 capsid (p24 CA) are indicated. The blots in the lower panels were probed with an anti-myc epitope antibody. Note that the VLP blots probed with the anti-myc antibody were exposed approximately 10-fold longer than the cell lysate blots. (B) Schematic representation of plasmids used to express HXB Gag from viral RNA-derived sequences (pCR/V1/HXB Gag; upper diagram) or a synthetic codon-optimized RNA (pCR3.1/synGag; lower diagram). (C) Western blot analyses of cell lysates (left panels) and extracellular VLPs (right panels) generated following transfection of 293T cells with plasmids expressing synGag or HXB Gag and myc-tagged APOBEC3G. α, anti; BGH, bovine growth hormone; CMV, cytomegalovirus.

NC binds to APOBEC3G in an RNA-dependent manner. (A) Schematic representation of intact and fragmented Gag-GST fusion proteins. MHR, major homology region. (B) Gag-GST fusion proteins were coexpressed with myc-APOBEC3G (left panels) or myc-GFP (right panels). The upper and middle panels show Western blot (WB) analyses of total cell lysates. The blots in the upper panels were probed with an anti-GST antibody, and the blots in the middle panels were probed with an anti-myc antibody. The lower panels show a Western analysis (blots probed with an anti-myc antibody) of proteins precipitated from each of the cell lysates by glutathione-Sepharose beads. (C) The same experiment illustrated in the left panels of panel B was done, and Western blot analyses of the glutathione-bound proteins are shown. myc-APOBEC3G was precipitated by Gag-GST fusion proteins from duplicate aliquots of cell lysates that were left untreated (upper panel) or treated with RNase A (lower panel).

NC, but not the majority of Gag, is required for APOBEC3G incorporation into HIV-1 VLPs. (A) Schematic representation of the intact HIV-1 Gag precursor and the deletion mutants used in this study. The positions of the major homology region (MHR) and the various Gag cleavage sites are shown. (B) Western blot (WB) analyses of cell lysates (left panels) and extracellular VLPs (right panels) generated following transfection of 293T cells with plasmids expressing HIV-1 Gag or the deletion mutants shown in panel A and myc-tagged APOBEC3G. Blots were probed with anti-HIV-1 CA or anti-myc antibodies, as described in the legend to Fig. 1. (C) Schematic representation of the intact HIV-1 Gag precursor and its derivative, Zwt-p6. (D) Western blot analyses of cell lysates (left panels) and extracellular VLPs (right panels) generated following transfection of 293T cells with plasmids expressing HIV-1 Gag or Zwt-p6 and either myc-tagged APOBEC3G or myc-tagged GFP. The upper panels show Western blots probed with an anti-HIV-1 CA antibody, and the positions of the Gag and Zwt-p6 proteins are indicated. The blots in the lower panels were probed with an anti-myc epitope antibody. (E) Quantitation of RNA in wild-type Gag and Zwt-p6 VLPs, as determined by Ribogreen analysis of one-fifth of the VLPs pelleted from 30 ml of 293T cell supernatants. Pelletable material released from control-vector-transfected 293T cells was also analyzed as a control. A fourfold dilution series of the same wild-type Gag and Zwt-p6 VLPs was analyzed by Western blotting (right) to verify that equivalent amounts of VLPs were included in the Ribogreen analyses. α, anti.