The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism.

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity.

Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity.

APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination.IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

Recurrent Loss of APOBEC3H Activity during Primate Evolution.

Genes in the APOBEC3 family encode cytidine deaminases that provide a barrier against viral infection and retrotransposition. Of all the APOBEC3 genes in humans, APOBEC3H (A3H) is the most polymorphic: some genes encode stable and active A3H proteins, while others are unstable and poorly antiviral. Such variation in human A3H affects interactions with the lentiviral antagonist Vif, which counteracts A3H via proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions, as well as its evolution in Old World monkeys, we characterized A3H variation within four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of function was partially related to protein expression levels but was also influenced by amino acid mutations in the N terminus. Moreover, we demonstrate that the evolution of A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests that the activity of A3H is evolutionarily dynamic and may have a negative effect on host fitness, resulting in its recurrent loss in primates.IMPORTANCE Adaptation of viruses to their hosts is critical for viral transmission between different species. Previous studies had identified changes in a protein from the APOBEC3 family that influenced the species specificity of simian immunodeficiency viruses (SIVs) in African green monkeys. We studied the evolution of a related protein in the same system, APOBEC3H, which has experienced a loss of function in humans. This evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken place during primate evolution, suggesting that APOBEC3H places a fitness cost on hosts. The variability of APOBEC3H activity between different primates highlights the differential selective pressures on the APOBEC3 gene family.

The Structural Interface between HIV-1 Vif and Human APOBEC3H.

Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif ß-sheet (ß2-ß5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif ß-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

Role of APOBEC3H in the Viral Control of HIV Elite Controller Patients.

Background APOBEC3H (A3H) gene presents variation at 2 positions (rs139297 and rs79323350) leading to a non-functional protein. So far, there is no information on the role played by A3H in spontaneous control of HIV. The aim of this study was to evaluate the A3H polymorphisms distribution in a well-characterized group of Elite Controller (EC) subjects. Methods We analyzed the genotype distribution of two different SNPs (rs139297 and rs79323350) of A3H in 30 EC patients and compared with 11 non-controller (NC) HIV patients. Genotyping was performed by PCR, cloning and Sanger sequencing. Both polymorphisms were analyzed jointly in order to adequately attribute the active or inactive status of A3H protein. Results EC subjects included in this study were able to maintain a long-term sustained spontaneous HIV-viral control and optimal CD4-T-cell counts; however, haplotypes leading to an active protein were very poorly represented in these patients. We found that the majority of EC subjects (23/30; 77%) presented allelic combinations leading to an inactive A3H protein, a frequency slightly lower than that observed for NC studied patients (10/11; 91%). Conclusions The high prevalence of non-functional protein coding-genotypes in EC subjects seems to indicate that other innate restriction factors different from APOBEC3H could be implicated in the replication control exhibited by these subjects.

Polymorphisms in Human APOBEC3H Differentially Regulate Ubiquitination and Antiviral Activity.

The APOBEC3 family of cytidine deaminases are an important part of the host innate immune defense against endogenous retroelements and retroviruses like Human Immunodeficiency Virus (HIV). APOBEC3H (A3H) is the most polymorphic of the human APOBEC3 genes, with four major haplotypes circulating in the population. Haplotype II is the only antivirally-active variant of A3H, while the majority of the population possess independently destabilizing polymorphisms present in haplotype I (R105G) and haplotypes III and IV (N15del). In this paper, we show that instability introduced by either polymorphism is positively correlated with degradative ubiquitination, while haplotype II is protected from this modification. Inhibiting ubiquitination by mutating all of the A3H lysines increased the expression of haplotypes III and IV, but these stabilized forms of haplotype III and IV had a strict nuclear localization, and did not incorporate into virions, nor exhibit antiviral activity. Fusion chimeras with haplotype II allowed for stabilization, cytoplasmic retention, and packaging of the N15del-containing haplotype III, but the haplotype III component of these chimeras was unable to restrict HIV-1 on its own. Thus, the evolutionary loss of A3H activity in many humans involves functional deficiencies independent of protein stability.

The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species. RNA binding is required for localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles and ultimately for effective virus restriction activity. Here we compared wild-type human A3H and RNA binding-defective mutants to ask whether RNA may be a factor in the functional interaction with HIV-1 Vif. We used structural modeling, immunoblotting, live cell imaging, and split green fluorescence protein (GFP) reconstitution approaches to assess the capability of HIV-1 Vif to promote the degradation of wild-type A3H in comparison to RNA binding-defective mutants. The results combined to show that RNA is not strictly required for Vif-mediated degradation of A3H, and that RNA and Vif are likely to bind this single-domain DNA cytosine deaminase on physically distinct surfaces. However, a subset of the results also indicated that the A3H degradation process may be affected by A3H protein structure, subcellular localization, and differences in the constellation of A3H interaction partners, suggesting additional factors may also influence the fate and functionality of this host-pathogen interaction.

RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids.

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.

APOBEC3B Activity Is Prevalent in Urothelial Carcinoma Cells and Only Slightly Affected by LINE-1 Expression.

The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity in vitro. While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.

APOBEC3H Subcellular Localization Determinants Define Zipcode for Targeting HIV-1 for Restriction.

APOBEC enzymes are DNA cytosine deaminases that normally serve as virus restriction factors, but several members, including APOBEC3H, also contribute to cancer mutagenesis. Despite their importance in multiple fields, little is known about cellular processes that regulate these DNA mutating enzymes. We show that APOBEC3H exists in two distinct subcellular compartments, cytoplasm and nucleolus, and that the structural determinants for each mechanism are genetically separable. First, native and fluorescently tagged APOBEC3Hs localize to these two compartments in multiple cell types. Second, a series of genetic, pharmacologic, and cell biological studies demonstrate active cytoplasmic and nucleolar retention mechanisms, whereas nuclear import and export occur through passive diffusion. Third, APOBEC3H cytoplasmic retention determinants relocalize APOBEC3A from a passive cell-wide state to the cytosol and, additionally, endow potent HIV-1 restriction activity. These results indicate that APOBEC3H has a structural zipcode for subcellular localization and selecting viral substrates for restriction.

Biochemical Characterization of APOBEC3H Variants: Implications for Their HIV-1 Restriction Activity and mC Modification.

APOBEC3H (A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif.

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFß. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1's survival in vivo.

Correlation of APOBEC3 in tumor tissues with clinico-pathological features and survival from hepatocellular carcinoma after curative hepatectomy.

OBJECTIVE: This study aimed to evaluate the relationships between members of APOBEC3 in tumor tissues and hepatocellular carcinoma (HCC) aggressiveness and prognosis. METHODS: Using the expression profile GSE36376 from Gene Expression Omnibus (GEO), we compared APOBEC3 expression between tumor and non-tumor tissues, and correlated this with clinico-pathological features and outcomes of HCC patients. RESULTS: A3B, A3D, A3F and A3H were overexpressed in HCC tumor tissues compared to non-tumor tissues (all P≤0.001). Cox regression shown that A3G was negatively associated with overall survival of HCC patients (HR=2.277, 95% CI=1.324-3.915, P=0.033), in contrast, A3C level in tumor tissues might play a positive role in HCC overall survival (HR=0.364, 95% CI=0.182-0.727, P=0.004). Interestingly, A3F contributed to a poor disease-free survival of HCC (HR=3.383, 95% CI=1.249-9.715, P=0.017), while A3H may be a positive factor associated with HCC disease-free survival (HR=0.25, 95% CI=0.098-0.636, P=0.004). Cirrhosis, tumor size and intrahepatic metastasis were associated with HCC poor disease-free survival (HR=1.838, 95% CI=1.308-2.583, P<0.001; HR= 1.095, 95% CI=1.042-1.15, P<0.001 and HR=3.669, 95% CI=2.447-5.5, P<0.001; respectively). Logistic regression analysis indicated that up-regulation of A3F in tumor tissues promoted HCC vascular invasion, intrahepatic metastasis and AFP elevation (all P<0.05). In contrast, A3H might decrease these risks (all P<0.05). CONCLUSIONS: APOBEC3G and APOBEC3F might be risk factors for HCC development and survival, while APOBEC3C and APOBEC3H should play positive roles in HCC aggressiveness and prognosis. Further investigation for APOBEC3 mechanisms are needed in the future.

Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more extensive analysis and in vitro experiments.

Multiple APOBEC3 restriction factors for HIV-1 and one Vif to rule them all.

Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients.