Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA doublestrand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. (Blood. 2012; 120(2):366-375) IntroductionIonizing radiation and the majority of anticancer agents inflict deleterious DNA damage on tumor cells, predominantly DNA double-strand breaks (DSBs) and covalent DNA crosslinks. DNA DSBs are highly genotoxic lesions, constituting the most disruptive form of DNA damage. Cells use an intricate set of mechanisms to repair genomic DSBs, based on nonhomologous end-joining (NHEJ) or homology-directed repair. 1,2 The choice of DSB repair pathway is governed mainly by the cell-cycle phase and the nature of the DSB lesion. [3][4][5] NHEJ operates throughout the cell cycle, resolving approximately 75% to 85% of DSBs induced by ionizing radiation (IR). 6,7 Homologous recombination (HR), in contrast, functions predominantly in the S/G 2 phase, after synthesis of a homologous DNA template. 8,9 The complexity of the DSB lesion determines the extent of DNA end-processing required for repairing the break. Whereas simple DSBs may be repaired by direct ligation via the NHEJ machinery, complex DSBs, often introduced by IR, may require end-processing to reveal 3Ј ssDNA overhangs by 5Ј-3Ј nucleolytic end-resection. 5 These ssDNA tails, which may be several kilobases long, are substrates for HR factors, such as replication protein A (RPA), RAD51, and RAD52. 5,9,10 The human APOBEC3 locus encodes 7 homologous genes expanded in tandem on chromosome 22. 11 APOBEC3 (A3) proteins are potent cytidine deaminases acting to restrict retroviral replication and retrotransposition. 12,13 APOBEC3G (A3G) is incorporated into assembling HIV-1 virions in the cytoplasm of infected cells and lead...

In the absence of HIV-1 Vif protein, the host antiviral deaminase APOBEC3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative ssDNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity, and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here we show that: (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif virus associated A3G; (ii) Encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) Purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes (PICs) following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G anti-viral activity.

The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in dsDNA damage, such as ionizing irradiation (IR) and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases sensitivity of lymphoma cells to IR. In the current study, we show that additional peptides derived from Vif, A3G and A3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, while, replacing a single amino acid in the LYYF motif completely abrogate inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break (DSB) repair after radiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit DSB repair halts their propagation. These results suggest that A3G may be a potential therapeutic target amenable to peptide and peptidomimetic inhibition.

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription.