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The APOBEC3 deaminases are potent inhibitors of virus replication and barriers to cross-species transmission. For simian immunodeficiency virus (SIV) to transmit to a new primate host, as happened multiple times to seed the ongoing HIV-1 epidemic, the viral infectivity factor (Vif) must be capable of neutralizing the APOBEC3 enzymes of the new host. Although much is known about current interactions of HIV-1 Vif and human APOBEC3s, the evolutionary changes in SIV Vif required for transmission from chimpanzees to gorillas and ultimately to humans are poorly understood. Here, we demonstrate that gorilla APOBEC3G is a factor with the potential to hamper SIV transmission from chimpanzees to gorillas. Gain-of-function experiments using SIVcpzPtt Vif revealed that this barrier could be overcome by a single Vif acidic amino acid substitution (M16E). Moreover, degradation of gorilla APOBEC3F is induced by Vif through a mechanism that is distinct from that of human APOBEC3F. Thus, our findings identify virus adaptations in gorillas that preceded and may have facilitated transmission to humans., Author summary Humans are exposed continuously to a menace of viral diseases such as Ebola virus and coronaviruses. Such emerging/re-emerging viral outbreaks can be triggered by cross-species viral transmission from wild animals to humans. HIV-1, the causative agent of AIDS, most likely originated from related precursors found in chimpanzees and gorillas (SIVcpzPtt or SIVgor), approximately 100 years ago. Additionally, SIVgor most likely emerged through the cross-species jump of SIVcpzPtt from chimpanzees to gorillas. However, it remains unclear how primate lentiviruses successfully transmitted among different species. To limit cross-species lentiviral transmission, cellular "restriction factors", including tetherin, SAMHD1, and APOBEC3 proteins potentially inhibit lentiviral replication. In contrast, primate lentiviruses have evolutionary acquired their own "arms" to antagonize the antiviral effect of restriction factors. Here we show that gorilla APOBEC3G potentially plays a role in inhibiting SIVcpzPtt replication. To our knowledge, this is the first report suggesting that a great ape APOBEC3 protein can potentially restrict the cross-species transmission of great ape lentiviruses and how lentiviruses overcame this species barrier.

APOBEC3 (A3) family proteins are DNA cytosine deaminases recognized for contributing to HIV-1 restriction and mutation. Prior studies have demonstrated that A3D, A3F, and A3G enzymes elicit a robust anti-HIV-1 effect in cell cultures and in humanized mouse models. Human A3H is polymorphic and can be categorized into three phenotypes: stable, intermediate, and unstable. However, the anti-viral effect of endogenous A3H in vivo has yet to be examined. Here we utilize a hematopoietic stem cell-transplanted humanized mouse model and demonstrate that stable A3H robustly affects HIV-1 fitness in vivo. In contrast, the selection pressure mediated by intermediate A3H is relaxed. Intriguingly, viral genomic RNA sequencing reveled that HIV-1 frequently adapts to better counteract stable A3H during replication in humanized mice. Molecular phylogenetic analyses and mathematical modeling suggest that stable A3H may be a critical factor in human-to-human viral transmission. Taken together, this study provides evidence that stable variants of A3H impose selective pressure on HIV-1.

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached., Author Summary The APOBEC3 enzymes protect cells by inhibiting the spread of retroelements, including HIV-1, by blocking reverse transcription and mutating cytosines in single-stranded DNA replication intermediates. HIV-1 Vif counteracts restriction by marking APOBEC3 proteins for proteasomal degradation. APOBEC3H is the most diverse member of this protein family. Humans have seven distinct APOBEC3H haplotypes with three producing stable and four producing unstable proteins upon forced overexpression. Here, we examine the stability phenotype of endogenous APOBEC3H in donors with different haplotypes and address how these stability differences, as well as natural viral diversity, combine to determine HIV-1 infectivity. We found that endogenous APOBEC3H haplotypes yield stable or unstable proteins and that stable APOBEC3H is induced during viral infection and restricts the replication of isolates with naturally occurring hypo-functional but not hyper-functional Vif alleles. We also found that the global distribution of stable APOBEC3H alleles correlates with the prevalence of HIV-1 Vif alleles capable of mediating its degradation, strongly suggesting that the viral Vif protein is capable of adapting to the APOBEC3H restriction potential of an infected individual. Thus, the combination of human APOBEC3H haplotypes and virus Vif alleles may help account for some of the observed disparities in disease progression and virus transmission.

Lentiviruses are a unique class of retroviruses that infect a subset of mammalian species, including humans. Human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), maedi-visna virus (MVV), and caprine arthritis encephalitis virus (CAEV) infections result in immunodeficiency syndromes, neurological diseases, and other conditions. Disease is chronic, lifelong, and often fatal. Pathology is due in part to a capacity to immortalize by integrating into a host’s genomic DNA and a remarkable genetic plasticity that enables escape from potent adaptive immune responses without compromising vital viral functions. Lentiviruses encode three universal retroviral proteins (Gag, Pol, and Env), one lentivirus-specific protein called Vif (virus infectivity factor), and a handful of other less conserved accessory factors. Vif is required to protect lentiviruses from restriction by several members of the APOBEC3 family of DNA cytosine deaminases by forming a cellular polyubiquitin ligase complex to degrade these enzymes (Fig 1A). Viruses lacking Vif are inactivated because cytoplasmic APOBEC3 enzymes remain abundant, package into assembling virus particles, and prevent the production of a viable DNA copy of the viruses’ RNA genome by catalyzing the deamination of viral cDNA cytosines to uracils. Viral DNA genomes that make it through this gauntlet are rendered nonfunctional by the resulting guanine to adenine hypermutations (cDNA uracils template the insertion of genomic strand adenines that become immortalized as G-to-A mutations). In this Pearl, we review recent progress in understanding the Vif-APOBEC3 interaction and discuss a model that provides a molecular explanation for past zoonotic transmission events as well as present-day, likely ongoing optimizations that enable lentiviruses to thrive. Fig 1 Lentiviral Vif and APOBEC3 diversity and functionality in mammals. CBF-β is a specific co-factor for SIV and HIV Vif function Although HIV-1 was identified in 1983, Vif function evaded discovery for nearly 20 years, until APOBEC3G (A3G) emerged as a dominant cellular factor that strongly inhibited vif-deficient virus replication [1]. This advance led rapidly to elucidation of the proteasome-dependent APOBEC3 degradation mechanism, including the identification of cellular factors that Vif recruits to form the degradation complex (CUL5, ELOB, ELOC, and RBX2) (Fig 1A) [2, 3]. However, HIV-1 Vif still resisted biochemical and structural studies for nearly another decade, suggesting that the complex may be incomplete. This puzzle was solved in 2011, when the transcription cofactor CBF-β proved to be an essential part of the HIV-1 Vif ubiquitin ligase complex [4, 5]. CBF-β enabled the biochemical reconstitution of the Vif ubiquitin ligase complex [4] and, shortly thereafter, structural determination by X-ray crystallography [6]. Vif and CBF-β heterodimerize and form a scaffold for further assembly of an active ligase complex (Fig 1A and 1B). Surprisingly, CBF-β is only essential for HIV and SIV Vif function (Fig 1C) [7]. The APOBEC3 degradation activity of non-primate lentiviral Vif proteins is unaffected by cellular CBF-β [4, 5, 8–11]. These differing genetic and biochemical requirements were initially puzzling given that all known Vif proteins function to counteract host APOBEC3 enzymes (e.g., [12]). However, the HIV/SIV Vif requirement for CBF-βmay be reconciled by postulating that this adaptation occurred when an ancestral lentivirus transmitted from a non-primate mammal into a primate. Such an event likely took place millions of years ago, because an endogenous lentivirus with a putative Vif protein exists in the present-day genomes of at least two evolutionarily diverged lemur genera [13, 14]. Because exogenously spreading lentiviruses evolve too quickly to date based on cumulative genetic diversity, endogenization events such as this effectively fossilize a virus and provide rare opportunities for estimating the age of a particular viral species and making comparisons with present day descendants such as SIV and HIV.

The DNA deaminase APOBEC3G converts cytosines to uracils in retroviral cDNA, which are immortalized as genomic strand G-to-A hypermutations by reverse transcription. A single round of APOBEC3G-dependent mutagenesis can be catastrophic, but evidence suggests that sublethal levels contribute to viral genetic diversity and the associated problems of drug resistance and immune escape. APOBEC3G exhibits an intrinsic preference for the second cytosine in a 5′CC dinucleotide motif leading to 5′GG-to-AG mutations. However, an additional hypermutation signature is commonly observed in proviral sequences from HIV-1 infected patients, 5′GA-to-AA, and it has been attributed controversially to one or more of the six other APOBEC3 deaminases. An unambiguous resolution of this problem has been difficult to achieve, in part due to dominant effects of protein over-expression. Here, we employ gene targeting to dissect the endogenous APOBEC3 contribution to Vif-deficient HIV-1 restriction and hypermutation in a nonpermissive T cell line CEM2n. We report that APOBEC3G-null cells, as predicted from previous studies, lose the capacity to inflict 5′GG-to-AG mutations. In contrast, APOBEC3F-null cells produced viruses with near-normal mutational patterns. Systematic knockdown of other APOBEC3 genes in an APOBEC3F-null background revealed a significant contribution from APOBEC3D in promoting 5′GA-to-AA hypermutations. Furthermore, Vif-deficient HIV-1 restriction was strong in parental CEM2n and APOBEC3D-knockdown cells, partially alleviated in APOBEC3G- or APOBEC3F-null cells, further alleviated in APOBEC3F-null/APOBEC3D-knockdown cells, and alleviated to the greatest extent in APOBEC3F-null/APOBEC3G-knockdown cells revealing clear redundancy in the HIV-1 restriction mechanism. We conclude that endogenous levels of APOBEC3D, APOBEC3F, and APOBEC3G combine to restrict Vif-deficient HIV-1 and cause the hallmark dinucleotide hypermutation patterns in CEM2n. Primary T lymphocytes express a similar set of APOBEC3 genes suggesting that the same repertoire may be important in vivo., Author Summary HIV uses its accessory protein Vif to shield itself from lethal mutagenesis by cellular APOBEC3 proteins. The APOBEC3-Vif interaction is therefore a promising target for drug development, and a clear definition of all proteins involved on both sides of this critical host-pathogen conflict is crucial. However, apart from APOBEC3G, conflicting over-expression studies have been published on the six other human APOBEC3s, some supporting and some refuting each protein's involvement in HIV restriction. Here, we use a combination of gene targeting and RNA interference to define the endogenous sources of Vif-deficient HIV restriction and G-to-A hypermutation in a nonpermissive T cell line. As anticipated, APOBEC3G contributed partially to restriction and proved to be the source of hypermutations at 5′GG dinucleotides. However, a major and unexpected role for APOBEC3D became evident in the APOBEC3F-null background, with both proteins combining to suppress viral infectivity and form hypermutations at 5′GA dinucleotides. We conclude that endogenous levels of APOBEC3D, APOBEC3F, and APOBEC3G act redundantly to restrict Vif-deficient HIV and produce the two hallmark dinucleotide hypermutation patterns observed in patient-derived viral sequences. Therapeutic strategies that unleash this full ‘swarm’ of restrictive APOBEC3 proteins are likely to be more effective than those that focus on enabling a single enzyme.

The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV., Author Summary The APOBEC3 family of editing enzymes catalyzes lethal hypermutation of retroviral genomes to block spread of virus in host. HIV Vif targets APOBEC3 family members for destruction by a cellular ubiquitin ligase containing CUL5. A major goal in the design of the next generation of antiretroviral therapies is to find an inhibitor of Vif so that the activity of the APOBEC3 family of antiretroviral enzymes can be restored. We define a three-enzyme cascade that is required to activate Vif by addition of the ubiquitin-like NEDD8 protein to CUL5. MLN4924, an anti-cancer compound currently in phase 1 clinical trials, inhibits the NEDD8 cascade, blocks the action of Vif, and thus has potent anti-HIV activity. Furthermore, our studies define downstream drug targets in the NEDD8 cascade more selective for inhibition of HIV Vif. We demonstrate pharmacological inhibition of HIV replication through a mechanism that restores the innate immunity provided by APOBEC3 enzymes by targeting a host pathway, providing additional candidates that could be further exploited for therapeutic development. Inhibition of this NEDD8 cascade alone, or in combination with existing antiretroviral drugs could prove to be a useful treatment for HIV.

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Human APOBEC3G inhibits the replication of Vif-deficient HIV-1 by hypermutating nascent viral cDNA (reviewed in [1]–[3]). Zheng and colleagues recently reported that the HIV-1 restriction activity of APOBEC3G requires a cellular co-factor [4]. Their conclusion depended on three critical observations: i) CEM-T4 cells support the replication of Vif-deficient HIV-1, ii) CEM-T4 cells express restrictive levels of APOBEC3G, and iii) CEM-T4 cells engineered to express more APOBEC3G still permitted Vif-deficient HIV-1 replication [4]. These observations suggested that APOBEC3G alone is insufficient for restriction, and together with subsequent cell fusion experiments, that a recessive cellular co-factor is required. However, the fact that APOBEC3G is capable of restricting a broad number of retroelements, including yeast Ty elements, strongly suggests that other human cellular proteins are not absolutely required for restriction (e.g., [5],[6] and reviewed in [1]–[3]). An alternative explanation that could account for the observed permissive phenotype of the CEM-T4 line is that it is mixed, composed of a population of cells expressing low and/or variable levels of APOBEC3G. If this were the case, then the permissive phenotype could simply be due to virus replication in the subset of cells expressing low levels of APOBEC3G. To address this hypothesis, we generated subclones of the CEM-T4 line by serial dilution and determined the level of APOBEC3G expression by immunoblotting (Figure 1A). First, we observed that CEM-T4 cells expressed levels of APOBEC3G that were considerably lower than those in the non-permissive line CEM, regardless of whether they were obtained from the AIDS Research and Reference Reagent Program (CEM-T4-A) or directly from the Zheng laboratory (CEM-T4-Z). Second, we found that representative CEM-T4 subclones, regardless of source, expressed both low and variable APOBEC3G levels. It is further notable that none of the subclones expressed fully non-permissive, CEM-like APOBEC3G levels. This heterogeneity is reflected by the kinetics of Vif-deficient virus replication, with some subclones being fully permissive and others being semi-permissive (Figure 1B and 1C). Nevertheless, these data demonstrated that APOBEC3G levels are both low and variable within individual cells of the CEM-T4 line, thus providing a reasonable molecular explanation for the permissive phenotype of this cell line. Figure 1 CEM-T4 cells express low APOBEC3G levels and subclones show additional heterogeneity. Curiously, Zheng and coworkers [4] also showed that CEM-T4 cells retrovirally transduced with APOBEC3G were still permissive for Vif-deficient HIV-1 replication. This result may be due to the distinct possibility that APOBEC3G itself inactivated some of the transducing viral cDNAs. Such an attempt at complementation would probably result in a CEM-T4 line that is mixed for APOBEC3G expression. To clarify this important point, we used electroporation to generate panels of HA-tagged and untagged APOBEC3G-expressing CEM-T4 clones and used them for virus replication experiments (Figures 2 and ​and3,3, respectively). Figure 2 Expression of exogenous APOBEC3G-HA is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1. Figure 3 Increasing levels of untagged APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. Contrary to the findings of Zheng and colleagues [4], our APOBEC3G-expressing CEM-T4 clones were fully restrictive for Vif-deficient HIV-1 replication. The observed non-permissive phenotype was not due to gross overexpression of APOBEC3G because many of the restrictive CEM-T4 clones stably expressed APOBEC3G to levels slightly lower to those of CEM, the original parent of CEM-T4 (e.g., clone G4 in Figure 3). It is also notable that some vector control clones such as V9, like the subclones in Figure 1, expressed low and non-restrictive levels of APOBEC3G. These data suggest that cells must pass an APOBEC3G expression “threshold” before they become fully non-permissive for Vif-deficient HIV-1. Nevertheless, together these virus replication data clearly demonstrated that expression of exogenous APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. We conclude that this cell line does not require a specific endogenous APOBEC3G “co-factor” to restrict Vif-deficient HIV-1. In this report, we question the obligate APOBEC3G co-factor hypothesis put forth by Zheng and colleagues [4]. Our data clearly provide an alternative explanation that—rather than lacking a co-factor for APOBEC3G—the CEM-T4 line is mixed with individual cells expressing heterogeneous and less-than-restrictive levels of APOBEC3G. Importantly, when transfections were used to generate stable APOBEC3G-expressing CEM-T4 clones, the resulting lines were non-permissive for Vif-deficient HIV-1 replication. Based on these results, we conclude that CEM-T4 cells do not lack an APOBEC3G co-factor and, more generally, that APOBEC3G may not strictly require another cellular protein for HIV-1 restriction. However, we would like to emphasize that neither study rules out the attractive possibility that APOBEC3G, like most other proteins, is likely to be regulated by a variety of cellular processes.