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. 2005 Jan;79(2):823–833. doi: 10.1128/JVI.79.2.823-833.2005

FIG. 6.

FIG. 6.

Functional relationship between Vif2 proteins and APOBEC3G. (A) Expression analysis of HA-Vif wild-type or HA-Vif mutant proteins and APOBEC3G in cotransfected 293 cells. Cell lysates were immunoprecipitated, and HA fusion proteins were detected by Western blotting with horseradish peroxidase-conjugated anti-HA monoclonal antibody and by enhanced chemiluminescence. Cells were transfected with 1 μg of DNA plasmid as follows: lane 1, HA-APOBEC3G; lane 2, HA-Vif2 wild type (wt); lane 3, HA-APOBEC3G and HA-Vif2 wt; lane 4, HA-APOBEC3G and HA-MD1A; lane 5, HA-APOBEC3G and HA-MD1B; lane 6, HA-APOBEC3G and HA-M2; lane 7, HA-APOBEC3G and HA-MD5; lane 8, HA-APOBEC3G and HA-M6; lane 9, HA-APOBEC3G and HA-M7; lane 10, HA-APOBEC3G and MD8; and lane 11, HA-Vif2STOP. (B) Titers of APOBEC3G concentrations necessary for inhibition of HIV-1 vif-minus were determined by single-cycle infectivity assay. Briefly, 293T cells were transfected with proviral HIV-1NL43 vif-minus and HIV-2ROD vif-minus DNA plus increasing concentrationsAPOBEC3G, with plasmid ratios of 1:0, 1:1, 1:4, and 1:10. Viruses in the supernatant were collected, and its virus titers were normalized by using p26 antigen to infect P4 cells. Infectivity was measured based on the level of β-galactosidase expression as detailed in Materials and Methods. Values shown are percentages of infectivity relative to HIV vif-minus complemented with HA-Vif wild-type. The results are representative of three independent experiments. (C) Single-cycle infectivity assay was used to evaluate the effect of APOBEC3G in infectivity of HIV-2. 293T cells were cotransfected with proviral HIV-2ROD vif-minus DNA and plasmids expressing HA-Vif2 wild-type or HA-Vif2 mutant proteins together with APOBEC3G. The ratio of proviral DNA to APOBEC3G was 1:1. As controls, cells were transfected with HIV-1NL43 vif-minus transcomplemented by HA-Vif1 and HA-Vif2 wild type (wt; right side of the graphic). Viruses in the supernatant were collected, and its virus titers were normalized by using p26 antigen to infect P4 cells. Infectivity was measured based on the level of β-galactosidase expression as detailed in Materials and Methods. Values shown are percentages of infectivity relative to HIV vif-minus complemented with HA-Vif wild-type. The results are representative of four independent experiments.