In this study, we investigated whether A3G is able to contribute to HIV-1 variation by sublethal mutation. To quantify the effects of A3G on HIV replication and genetic diversity, we used single-cycle env− HIV-1 produced from cell lines that stably expressed A3G at near-physiologic levels. A3G hot spots were identified and were identical within Vif + and Vif − viruses. These data strongly indicate that A3G can cause sublethal levels of G-to-A mutation in Vif+ viruses.
In addition to A3G-catalyzed editing, there are clearly other sources of G-to-A mutations in the HIV-1 replication cycle. This is evident in the virus produced in the absence of A3G, which had G-to-A transition mutations scattered among many sites (Fig. ). These mutations were likely due to mistakes made during reverse transcription (31
), although cellular RNA polymerase II may also cause a minor fraction (35
). Moreover, both RT and RNA polymerase II are clearly influenced by cellular dNTP concentrations (41
In addition to deaminase-dependent G-to-A mutational activity, A3G can inhibit HIV-1 RT (3
), tRNA annealing or processing (15
), integration (28
), and/or DNA strand transfer (27
). The role of these alternative activities in preventing HIV-1 pathogenesis is still under investigation. However, regardless of these other potential functions, our data clearly support the role of cytosine deaminase activity as a major factor in diminishing viral infectivity by G-to-A hypermutation, which has been directly addressed elsewhere (4
). The mutational load in the HSA gene ranged from single base mutations to hypermutation (multiple G-to-A changes within the same sequence). A rescue experiment was done to determine if HIV could sustain an attack by A3G and maintain infectivity (Fig. ). We found evidence of single and multiple G-to-A mutations at A3G hot spots that correlated with A3G exposure during the first round of vector virus replication. This was observed across a range of A3G expression levels and in the presence or absence of Vif. While other studies have shown the presence of A3G-mediated mutations in Vif−
virus, their infectivity was not determined (34
Two models can be proposed to explain the hypermutated sequences identified in Fig. . First, the hypermutated sequences could have been introduced into the proviral sequence via recombination. A recent study showed that recombination could rescue hypermutated viral sequences and accelerate the rate of developing drug resistance (34
). Second, A3G may be processive in concentrated regions, such as the HSA reporter gene, which is located near the polypurine tract. This viral DNA region remains single stranded for a longer period of time during viral DNA synthesis. It has been proposed that A3G acts on particular areas of the DNA, which are flanked by regions which cause A3G to disengage (4
). Also, in patients with predominantly hypermutated env sequences, vif
was found to be largely normal, further suggesting that different sections of the viruses may be differentially susceptible to hypermutation (26
). Nucleotide sequencing of larger regions of viable proviruses in our study may help provide further insight into the origins of the hypermutated genomes.
Computational modeling supports the ability of A3G to cause sublethal mutations (21
). This is in contrast to reports that have suggested essentially an all-or-none model, whereby a single molecule (3
) or dimer (4
) of A3G causes hypermutation and loss of infectivity. The average A3G incorporation into Vif−
virions produced from PBMCs is thought to be 7 ± 4 (mean ± standard deviation) molecules (with an A3G/Gag ratio of 1:439), a level known to extinguish replicating Vif−
). Our results indicate that there is a wide range of mutational potential within a single HIV-1 replication cycle, such that all virions produced in the presence of A3G do not contain identical mutational loads. This suggests that either there is a range of A3G molecules incorporated per particle and/or that the A3G mutation efficiency differs in target cells during individual infection events. In summary, our data provide support for A3G-mediated sublethal mutagenesis of HIV-1. Further studies will be needed to evaluate the impact of sublethal mutagenesis on HIV-1 evolution, pathogenesis, drug resistance, and immune evasion.