APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure.

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

Recruitment of the nuclear form of uracil DNA glycosylase into virus particles participates in the full infectivity of HIV-1.

The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.

APOBEC3G contributes to HIV-1 variation through sublethal mutagenesis.

The mammalian APOBEC3 proteins are an important component of the cellular innate immune response to retroviral infection. APOBEC3G can extinguish HIV-1 infectivity by its incorporation into virus particles and subsequent cytosine deaminase activity that attacks the nascent viral cDNA during reverse transcription, causing lethal mutagenesis. It has been suggested, but not formally shown, that APOBEC3G can also induce sublethal mutagenesis, which would maintain virus infectivity and contribute to HIV-1 variation. To test this, we developed a novel model system utilizing an HIV-1 vector and a panel of APOBEC3G-expressing cells. We observed proviruses with single APOBEC3G-mediated mutations (in the presence or absence of Vif), occurring at distinct hot spots and which could be rescued and shown to have infectivity. These data indicate that APOBEC3G-dependent restriction of HIV-1 can result in viable viral progeny that harbor sublethal levels of G-to-A mutations. Such mutations have the potential to contribute significantly to HIV-1 evolution, pathogenesis, immune escape, and drug resistance.