Antiviral Mechanism and Biochemical Basis of the Human APOBEC3 Family

High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.

The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1. APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing. A signature sequence for APOBEC1 has been used to identify other members of this family. One of these genes, designated APOBEC2, is found on chromosome 6. Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12. Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22. Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family. The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1. Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.

HIV-1 deleted for the vif accessory gene encapsidates the cellular cytidine deaminase APOBEC3G. Upon infection, the encapsidated APOBEC3G induces G-->A mutations in the viral reverse transcripts. The G-->A mutations result either from C-->U deamination of the minus strand or deamination of both strands followed by repair of the plus strand. We report here that minus-strand deamination occurred over the length of the virus genome, preferentially at CCCA sequences, with a graded frequency in the 5'-->3' direction. APOBEC3G induced previously undetected C-->T mutations in the 5' U3 and the primer-binding site, both of which become transiently single-stranded during reverse transcription. In vitro, APOBEC3G bound and deaminated single-stranded DNA (ssDNA) but not double-stranded DNA (dsDNA) or DNA-RNA hybrids. We propose that the requirement for ssDNA accounts for the minus-strand mutations, the 5'-->3' graded frequency of deamination and the rare C-->T mutations.