The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif

The human protein apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), also known as CEM-15, mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates. Because DNA deamination takes place after virus entry into target cells, APOBEC3G function is dependent on its association with the viral nucleoprotein complexes that synthesize cDNA and must therefore be incorporated into virions as they assemble in infected cells. Here we show that the HIV-1 virion infectivity factor (Vif) protein protects the virus from APOBEC3G-mediated inactivation by preventing its incorporation into progeny virions, thus allowing the ensuing infection to proceed without DNA deamination. In addition to helping exclude APOBEC3G from nascent virions, Vif also removes APOBEC3G from virus-producing cells by inducing its ubiquitination and subsequent degradation by the proteasome. Our findings indicate that pharmacologic strategies aimed at stabilizing APOBEC3G in HIV-1 infected cells should be explored as potential HIV/AIDS therapeutics.     

APOBEC3G DNA deaminase acts processively 3' --> 5' on single-stranded DNA

Akin to a 'Trojan horse,' APOBEC3G DNA deaminase is encapsulated by the HIV virion. APOBEC3G facilitates restriction of HIV-1 infection in T cells by deaminating cytosines in nascent minus-strand complementary DNA. Here, we investigate the biochemical basis for C --> U targeting. We observe that APOBEC3G binds randomly to single-stranded DNA, then jumps and slides processively to deaminate target motifs. When confronting partially double-stranded DNA, to which APOBEC3G cannot bind, sliding is lost but jumping is retained. APOBEC3G shows catalytic orientational specificity such that deamination occurs predominantly 3' --> 5' without requiring hydrolysis of a nucleotide cofactor. Our data suggest that the G --> A mutational gradient generated in viral genomic DNA in vivo could result from an intrinsic processive directional attack by APOBEC3G on single-stranded cDNA.     

Influence of primate lentiviral Vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of APOBEC3G

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral evasion of the host antiviral protein APOBEC3G, also known as CEM15. Vif mutant but not wild-type HIV-1 viruses produced in the presence of APOBEC3G have been shown to undergo hypermutations in newly synthesized viral DNA upon infection of target cells, presumably resulting from C-to-U modification during minus-strand viral DNA synthesis. We now report that HIV-1 Vif could induce rapid degradation of human APOBEC3G that was blocked by the proteasome inhibitor MG132. The efficiency of Vif-induced downregulation of APOBEC3G expression depended on the level of Vif expression. A single amino acid substitution in the conserved SLQXLA motif reduced Vif function. Vif proteins from distantly related primate lentiviruses such as SIVagm were unable to suppress the antiviral activity of human APOBEC3G or the packaging of APOBEC3G into HIV-1 Vif mutant virions, due to a lack of interaction with human APOBEC3G. In the presence of the proteasome inhibitor MG132, virion-associated Vif increased dramatically. However, increased virion packaging of Vif did not prevent virion packaging of APOBEC3G when proteasome function was impaired, and the infectivity of these virions was significantly reduced. These results suggest that Vif function is required during virus assembly to remove APOBEC3G from packaging into released virions. Once packaged, virion-associated Vif could not efficiently block the antiviral activity of APOBEC3G.     

HIV-1 Viral Infectivity Factor (Vif) Alters Processive Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (−)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of Vif_IIIB- and Vif_HXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, Vif_HXB2 inhibited the jumping and Vif_IIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.     

Antiviral defense by APOBEC3 family proteins

APOBEC3G is a potent antiretroviral factor, which belongs to the APOBEC superfamily of cytidine deaminases. It deaminates cytidine to uridine in nascent minus-strand viral DNA, inducing G-to-A hypermutation in the plus-strand viral DNA. HIV-1 Vif protein overcomes the antiviral activity of APOBEC3G by targeting it for ubiquitin-dependent degradation. Recent accumulating evidences that other members of APOBEC proteins also show antiviral activity on a wide variety of viruses suggest that APOBEC family proteins play a crucial role in an antiviral defense as an innate immunity. Here, we review recent progress in research on APOBEC3 proteins.     

Absence of H186R polymorphism in exon 4 of the APOBEC3G gene among North Indian individuals

AIDS restriction genes have been defined in which allelic variations have been shown to influence infection or disease progression. Members of the APOBEC family of cellular polynucleotide cytidine deaminases (e.g., APOBEC3G) have been identified as a host factor that inhibits HIV-1 replication. It deaminates cytidine to uridine in nascent minus-strand viral DNA, inducing G-to-A hypermutation in the plus-strand viral DNA. The impact of codon-changing variant APOBEC3G H186R polymorphism on HIV-1 susceptibility and progression is not clear. We conducted genetic risk association study in HIV-1-exposed seronegative (HES; n = 50) individuals, HIV-1 seronegative (HSN; n = 320) healthy control, and HIV-1 seropositive patients (HSP; n = 190). The APOBEC3G H186R genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in DNA extracted from peripheral blood and confirmed by direct sequencing the randomly selected 58 samples. Frequency of rare homozygous RR (mutant type) and HR (heterozygous mutant) genotype was 0% while HH (wild type) was 100% among North Indians. In conclusion, we demonstrated that no genetic H186R polymorphism in exon 4 of APOBEC3G gene is found and therefore neither associated with differential susceptibility to HIV-1 infection/progression among North Indians.     

Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.     

APOBEC3D and APOBEC3F Potently Promote HIV-1 Diversification and Evolution in Humanized Mouse Model

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.     

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).     

APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo

APOBEC3G (CEM15 ) deaminates cytosine to uracil in nascent retroviral cDNA. The potency of this cellular defense is evidenced by a dramatic reduction in viral infectivity and the occurrence of high frequencies of retroviral genomic-strand G --> A transition mutations. The overwhelming dinucleotide hypermutation preference of APOBEC3G acting upon a variety of model retroviral substrates is 5'-GG --> -AG. However, a distinct 5'-GA --> -AA bias, which is difficult to attribute to APOBEC3G alone, prevails in HIV-1 sequences derived from infected individuals (e.g., ). Here, we show that APOBEC3F is also a potent retroviral restrictor but that its activity, unlike that of APOBEC3G, is partially resistant to HIV-1 Vif and results in a clear 5'-GA --> -AA retroviral hypermutation preference. This bias is also apparent in a bacterial mutation assay, suggesting that it is an intrinsic APOBEC3F property. Moreover, APOBEC3F and APOBEC3G appear to be coordinately expressed in a wide range of human tissues and are independently able to inhibit retroviral infection. Thus, APOBEC3F and APOBEC3G are likely to function alongside one another in the provision of an innate immune defense, with APOBEC3F functioning as the major contributor to HIV-1 hypermutation in vivo.