The human APOBEC3 proteins are DNA cytidine deaminases that impede the replication of many different transposons and viruses. The genes that encode APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H were generated through relatively recent recombination events. The resulting high degree of inter-relatedness has complicated the development of specific quantitative PCR assays for these genes despite considerable interest in understanding their expression profiles. Here, we describe a set of quantitative PCR assays that specifically measures the mRNA levels of each APOBEC3 gene. The specificity and sensitivity of each assay was validated using a full matrix of APOBEC3 cDNA templates. The assays were used to quantify the APOBEC3 repertoire in multiple human T-cell lines, bulk leukocytes and leukocyte subsets, and 20 different human tissues. The data demonstrate that multiple APOBEC3 genes are expressed constitutively in most types of cells and tissues, and that distinct APOBEC3 genes are induced upon T-cell activation and interferon treatment. These data help define the APOBEC3 repertoire relevant to HIV-1 restriction in T cells, and they suggest a general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction.

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication1-3. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host’s antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation4-10. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β–Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.