Effect of APOBEC3 protein expression on the level of mutation detected in bacteria. This analysis measures the ability of APOBEC3 proteins to induce a mutator phenotype in bacteria, as determined by acquisition of resistance to the antibiotic rifampicin (Rif^R). Plasmids that encode the indicated APOBEC3 proteins were introduced into E. Coli strain BW310 and their expression activated using isopropyl β-D-thiogalactoside. The level of mutation induced by each protein was then quantified by plating the bacteria on media containing rifampicin and counting the number of resistant colonies. The parental pTrc99A expression plasmid served as the negative control. A) Analysis of the mutator phenotype of wild-type human APOBEC3 proteins. Average of three experiments. The Western blot analyzing the expression of each protein in bacteria was performed using an HA-tag specific antibody. APOBEC3 proteins containing 2 CDAs, i.e. A3B, A3F and A3G, are detected in both their full-length, ~42 kDA form and as a breakdown product of ~22 kDA that likely represents the HA-tagged carboxy-terminal CDA domain. B) Analysis of the mutator phenotype of CDA mutants of A3G or A3B. Data are given as a percentage of the mutator activity seen with the wild-type A3G or A3B protein, which was set at 100%. Average of three experiments with standard deviation indicated. A Western blot showing the expression level of the full-length, ~42 kDA forms of each A3B or A3G variant in bacteria is presented at the bottom of the figure.

Consensus editing sequences for A3B CDA mutants. This figure presents the sequence context of the C to T mutations tabulated in Fig. 4 for each of the indicated APOBEC3 proteins. In particular, this figure shows the 5′ and 3′ flanking nucleotide around each edited C residue.

Effect of A3G and A3B mutants on HIV-1 infectivity and virion incorporation. A) Inhibition of HIV-1 infectivity by wild-type and mutant A3G variants was measured by co-transfecting 293T cells with the HIV-1 indicator vector pNL-LucΔ Vif and an APOBEC3 expression plasmid. At 48 h post-transfection, supernatant virions were collected and used to infect naïve 293T cells. At 24 h later, these infected cells were lysed and induced luciferase levels determined. The data are presented relative to virus derived from control cultures transfected with the parental pK vector in place of an APOBEC3 expression vector (NEG), which was set at 100% infectivity. These data derive from three independent experiments with standard deviations shown. B) Similar to panel A, except that 293T cells were co-transfected with plasmids expressing wild-type or mutant A3B proteins. C) 293T cells were co-transfected with the HIV-1 proviral plasmid pNL4-3Δ EnvΔ Vif and a plasmid expressing carboxy-terminally HA-tagged A3G or an HA-tagged cytoplasmic control protein, β-arrestin (CONT). At 48 h post-transfection, the supernatant media were harvested and filtered and virions collected by pelleting through a 20% sucrose cushion. At the time the supernatant media were harvested, we also collected the virion producing 293T cells. We then performed Western analyses of the producer cell lysates and the lysed, pelleted virions using antibodies specific for the HA-tag or specific for the HIV-1 capsid protein. D) Similar to panel C, except that the intracellular expression and virion incorporation of A3B mutants was analyzed.

Analysis of C to T editing of HIV-1 reverse transcripts by A3G and A3B CDA mutants. To analyze the level of C to T editing by the various CDA mutants, we generated HIV-1 virions in the presence of each protein and used these to infect naïve 293T cells. At 16 h after infection, total cellular DNA was harvested, cleaved with DpnI and then a region of the env gene of HIV-1 amplifed using a high fidelity Taq polymerase. After cloning and sequencing, the number of introduced missense mutations were quantified and are here presented in tabular form for each wild-type or mutant APOBEC3 protein. The original HIV-1 sequence is given at left and the new sequence across the top. The total number of bases sequenced is shown at the lower right of each box.