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The main protease, M pro , of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development.

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.

HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Macrophage cells are also thought to provide a reservoir for HIV-1 replication but less is known in this cell type about virus restriction and counteraction mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes involved in HIV-1 restriction in macrophages have yet to be delineated fully. We show that multiple APOBEC3 genes including APOBEC3G are expressed in myeloid cell lines such as THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus, and proviral DNA resulting from such Vif-deficient infections shows strong G to A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses with selective sensitivity to APOBEC3G show Vif null-like infectivity levels and similarly strong APOBEC3G-biased mutation spectra. Importantly, APOBEC3G-null THP-1 cells yield Vif-deficient particles with significantly improved infectivities and proviral DNA with background levels of G to A hypermutation. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells.

Background The APOBEC3 (A3) family of DNA cytosine deaminases provides an innate barrier to infection by retroviruses including HIV-1. A total of five enzymes, A3C, A3D, A3F, A3G and A3H, are degraded by the viral accessory protein Vif and expressed at high levels in CD4+ T cells, the primary reservoir for HIV-1 replication in vivo. Apart from A3C, all of these enzymes mediate restriction of Vif-deficient HIV-1. However, a rare variant of human A3C (Ile188) was shown recently to restrict Vif-deficient HIV-1 in a 293T-based single cycle infection system. The potential activity of this naturally occurring A3C variant has yet to be characterized in a T cell-based spreading infection system. Here we employ a combination of Cas9/gRNA disruption and transient and stable protein expression to assess the roles of major Ser188 and minor Ile188 A3C variants in HIV-1 restriction in T cell lines. Results Cas9-mediated mutation of endogenous A3C in the non-permissive CEM2n T cell line did not alter HIV-1 replication kinetics, and complementation with A3C-Ser188 or A3C-Ile188 was similarly aphenotypic. Stable expression of A3C-Ser188 in the permissive T cell line SupT11 also had little effect. However, stable expression of A3C-Ile188 in SupT11 cells inhibited Vif-deficient virus replication and inflicted G-to-A mutations. Conclusions A3C-Ile188 is capable of inhibiting Vif-deficient HIV-1 replication in T cells. Although A3C is eclipsed by the dominant anti-viral activities of other A3s in non-permissive T cell lines and primary T lymphocytes, this enzyme may still be able to contribute to HIV-1 diversification in vivo. Our results highlight the functional redundancy in the human A3 family with regards to HIV-1 restriction and the need to consider naturally occurring variants.

Tumor cells frequently evade applied therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor that restricts the potency of oncolytic vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFN-β-dependent manner, which was responsible for the evolution of virus-resistant cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. Knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV both in vitro and in vivo. Finally, we demonstrate that APOBEC3B expression had a direct effect on the fitness of VSV, an RNA virus that has not previously been identified as restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of viral or broader nucleic acid-based therapeutic platforms. Keywords: APOBEC3, VSV, oncolytic virus, viral resistance, therapeutic resistance

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded (ss)DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes—APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new pathogen-host conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.

Accessory proteins are a key feature that distinguishes primate immunodeficiency viruses such as human immunodeficiency virus type I (HIV-1) from other retroviruses. A prime example is the virion infectivity factor, Vif, which hijacks a cellular co-transcription factor (CBF-β) to recruit a ubiquitin ligase complex (CRL5) to bind and degrade antiviral APOBEC3 enzymes including APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H). Although APOBEC3 antagonism is essential for viral pathogenesis, and a more than sufficient functional justification for Vif’s evolution, most viral proteins have evolved multiple functions. Indeed, Vif has long been known to trigger cell cycle arrest and recent studies have shed light on the underlying molecular mechanism. Vif accomplishes this function using the same CBF-β/CRL5 ubiquitin ligase complex to degrade a family of PPP2R5 phospho-regulatory proteins. These advances have helped usher in a new era of accessory protein research and fresh opportunities for drug development.

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here we demonstrate a quantitative reporter for Mpro function in living cells, in which protease inhibition by genetic or chemical methods results in strong eGFP fluorescence. This robust gain-of-function system readily distinguishes between inhibitor potencies and can be scaled-up to high-throughput platforms for drug testing.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G(2)/M cell cycle arrest. Vif initiates G(2)/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-β/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G(2)/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G(2)/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

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.

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade., DNA mutations induced by dysregulated APOBEC3 expression are associated with tumour-progression and therapeutic resistance, but also with the generation of neoepitopes. Here, the authors show that APOBEC3 function can be exploited in a vaccine setting to generate heteroclitic neoepitopes, enhancing sensitivity to immunotherapy.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. Significance: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution. This article is highlighted in the In This Issue feature, p. 2355

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1-4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1-4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers5, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2'-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

APOBEC3G is a cytidine deaminase with a well-characterized anti-viral role against HIV and other retroviruses. Its deaminase activity yields G to A edits in ssDNA to disrupt viral coding sequences and inhibits cDNA production during reverse transcription. However, APOBEC3G is also highly expressed in cells and tumors of B-lymphocyte lineage. In this study we aimed to evaluate the biological and clinical relevance of APOBEC3G in Diffuse Large B Cell Lymphomas (DLBCL), where it is expressed in absence of viral infection. We first confirmed the high expression of APOBEC3G in a panel of DLBCL cell lines. Interestingly, immunofluorescence of APOBEC3G in DLBCL cells demonstrated localization to p-bodies. SILAC mass-spectrometry of Flag-APOBEC3G further revealed its association with several RNA binding proteins (RBPs) involved in RNA metabolism. Using the enhanced crosslinking and immunoprecipitation (eCLIP) assay for identification of bound RNA species, we demonstrated that APOBEC3G binds to 3' UTR and exonic RNA sequences, mostly of genes encoding ribosomal components. To study the functional effects of APOBEC3G in DLBCL, we generated multiple DLBCL knockout (KO) lines for APOBEC3G loss using CRISPR-Cas9. Whole transcriptome sequencing in SC1 cells revealed that knockout of APOBEC3G led to a global reduction in the RNA transcripts for ribosomal proteins. Together, these results suggest a role for APOBEC3G in binding/regulating cellular RNA species and RNA processing in DLBCL, in the absence of viral infection. We then evaluated the clinical relevance of APOBEC3G expression in DLBCL using publicly available gene expression datasets. We noted a correlation between APOBEC3G and ribosomal protein mRNAs in DLBCL. Interestingly however the expression of APOBEC3G was linked to survival only in male patients of germinal center subtype DLBCL. On further study of our cell line knockout models (n=2 each of M/F) we noted a sex specific role of APOBEC3G in modulating cellular stress. In male cells, the loss of APOBEC3G led to decreased protein synthesis, differential expression of Y-chromosomal genes, endogenous retroviruses and interferon stimulated genes. These effects were not noted in female KO cells KO. As APOBEC3G loss did not affect viability following exposure to chemotherapeutics in-vitro, we postulate that the sex-specific association of APOBEC3G with survival is a function of possible immunomodulatory effects of its binding/processing complex RNA species. The biological significance of this sex-specific role of APOBEC3G in normal B-cell development and maturation is as yet unclear, and represents an important area for future research. Citation Format: Joanna D. Wardyn, Michal M. Hoppe, Allison S. Chan, Jia Li, Omer An, Shruthi Venguidessane, Dominic P. Sheng, Henry Yang, Gene W. Yeo, Daniel J. Hodson, Reuben S. Harris, Anand D. Jeyasekharan. APOBEC3G is a sex-specific determinant of post-transcriptional RNA processing and survival in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2443.

The APOBEC3 DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several APOBEC3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) shows strong virus restriction activities in a variety of experimental systems, and is subjected to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity and active nuclear import. Here we ask whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells.

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use co-immunoprecipitation and immunofluorescent microscopy approaches to ask whether this mechanism is shared with the closely related γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) and the more distantly related α-herpesvirus, herpes simplex virus-1 (HSV-1). The large RNR subunit of KSHV, ORF61, co-precipitated multiple APOBEC3s including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also co-precipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. These results combine to indicate that both γ- and α-herpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and, further, suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.ImportanceThe APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counter-restriction mechanism has recently been uncovered for the γ-herpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related γ-herpesvirus, KSHV, and to a more distantly related α-herpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

APOBEC3A (A3A) inhibits the replication of a range of viruses and transposons and might also play a role in carcinogenesis. It is a single-domain deaminase enzyme that interacts with single-stranded DNA (ssDNA) and converts cytidines to uridines within specific trinucleotide contexts. Although there is abundant information that describes the potential biological activities of A3A, the interplay between binding ssDNA and sequence-specific deaminase activity remains controversial. Using a single-molecule atomic force microscopy spectroscopy approach developed by Shlyakhtenko et al. [(2015) Sci. Rep. 5, 15648], we determine the stability of A3A in complex with different ssDNA sequences. We found that the strength of the complex is sequence-dependent, with more stable complexes formed with deaminase-specific sequences. A correlation between the deaminase activity of A3A and the complex strength was identified. The ssDNA binding properties of A3A and those for A3G are also compared and discussed.

Purpose: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value. Experimental Design: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types. Results: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types. Conclusions: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746–55. ©2016 AACR.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat ( Felis catus ) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif -defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif -defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif -proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary “arms race” between the domestic cat and its cognate lentivirus. IMPORTANCE Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian immunodeficiency virus [SIV]) if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat, which was likely influenced by lentiviral infection.

SummaryRestriction factors including APOBEC3 family proteins have the potential prevent cross-species lentivirus transmissions. Such events as well as ensuing pathogenesis require the viral Vif protein to overcome/neutralize/degrade the APOBEC3 enzymes of the new host species. Previous investigations have focused on the molecular interaction between human APOBEC3s and HIV-1 Vif. However, the evolutionary interplay between lentiviruses and great ape (including human, chimpanzee and gorilla) APOBEC3s has not been fully investigated. Here we demonstrate that gorilla APOBEC3G plays a pivotal role in restricting lentiviral transmission from chimpanzee to gorilla. We also reveal that a sole amino acid substitution in Vif is sufficient to overcome the gorilla APOBEC3G-mediated species barrier. Moreover, the antiviral effects of gorilla APOBEC3D and APOBEC3F are considerably weaker than those of human and chimpanzee counterparts, which can result in the skewed evolution of great ape lentiviruses leading to HIV-1.HighlightsSIVcpz requires M16E mutation in Vif to counteract gorilla A3GAcidic residue at position 16 of Vif is crucial to counteract gorilla A3GGorilla A3D and A3F poorly suppress lentiviral infectivitySIVgor and related HIV-1s counteract human A3D and A3F independently of DRMR motif

Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient human immunodeficiency virus type 1 (HIV-1) by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire, and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, simian immunodeficiency virus SIV mac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include degradation of several human APOBEC3s and even human APOBEC3B, a non-HIV-1-restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIV mac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIV mac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap HIV-1 Vif surface residues that interact with human APOBEC3G and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIV mac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to the growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution. IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIV mac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire, including APOBEC3B. Understanding the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIV mac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes.

BK polyomavirus (BKV) causes nephropathy in kidney transplant recipients (KTRs). The virus has also been implicated as a possible cause of bladder and kidney cancers. Two KTRs who developed BKV nephropathy followed by renal carcinoma both showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major coat protein. The appearance and disappearance of these mutations over time suggests intra-patient evolution of the virus. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans the virus engages during the infectious entry process. Nearly all observed mutations are consistent with DNA damage caused by APOBEC3B, an antiviral cytosine deaminase. This is intriguing in light of a recent report showing that BKV induces APOBEC3B expression. The results indicate that polyomaviruses can employ APOBEC3B to acquire beneficial site-specific mutations, conceivably with carcinogenic consequences for the host.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the α1-β1, β2-α2 and β4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.

APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by Vif (viral infectivity factor) of lentiviruses, which binds APOBEC3s and promotes their degradation. CBF-β (core binding factor subunit β) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-β in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here, we determined the sequence of feline CBFB and performed phylogenetic analyses. These analyses revealed that mammalian CBFB is under purifying selection. Moreover, we demonstrated that CBF-β is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggested that primate lentiviruses have adapted to use CBF-β, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses.

Human DNA cytosine-to-uracil deaminases catalyze mutations in both pathogen and cellular genomes. APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H restrict human immunodeficiency virus-1 (HIV-1) infection in cells deficient in the viral infectivity factor (Vif), and have the potential to catalyze sub-lethal levels of mutation in viral genomes in Vif proficient cells. At least two APOBEC3 enzymes, and in particular APOBEC3B, are sources of somatic mutagenesis in cancer cells that drive tumor evolution and may manifest clinically as recurrence, metastasis, and/or therapy resistance. Consequently, APOBEC3 enzymes are tantalizing targets for developing chemical probes and therapeutic molecules to harness mutational processes in human disease. This review highlights recent efforts to chemically manipulate APOBEC3 activities.

APOBEC3G (A3G) is a restriction factor that provides innate immunity against HIV-1 in the absence of viral infectivity factor (Vif) protein. However, structural information about A3G, which can aid in unraveling the mechanisms that govern its interactions and define its antiviral activity, remains unknown. Here, we built a computer model of a full-length A3G using docking approaches and molecular dynamics simulations, based on the available X-ray and NMR structural data for the two protein domains. The model revealed a large-scale dynamics of the A3G monomer, as the two A3G domains can assume compact forms or extended dumbbell type forms with domains visibly separated from each other. To validate the A3G model, we performed time-lapse high-speed atomic force microscopy (HS-AFM) experiments enabling us to get images of a fully hydrated A3G and to directly visualize its dynamics. HS-AFM confirmed that A3G exists in two forms, a globular form (∼84% of the time) and a dumbbell form (∼16% of the time), and can dynamically switch from one form to the other. The obtained HS-AFM results are in line with the computer modeling, which demonstrates a similar distribution between two forms. Furthermore, our simulations capture the complete process of A3G switching from the DNA-bound state to the closed state. The revealed dynamic nature of monomeric A3G could aid in target recognition including scanning for cytosine locations along the DNA strand and in interactions with viral RNA during packaging into HIV-1 particles., APOBEC3G structure and dynamics are revealed in simulations and validated by high-speed AFM experiments: it exists in and switches between two forms, compact globular and extended dumbbell forms.

APOBEC3s (A3s) are single-stranded DNA cytosine deaminases that provide innate immune defences against retroviruses and mobile elements. A3s are specific to eutherian mammals because no direct homologs exist at the syntenic genomic locus in metatherian (marsupial) or prototherian (monotreme) mammals. However, the A3s in these species have the likely evolutionary precursors, the antibody gene deaminase AID and the RNA/DNA editing enzyme APOBEC1 (A1). Here, we used cell culture-based assays to determine whether opossum A1 restricts the infectivity of retroviruses including human immunodeficiency virus type 1 (HIV-1) and the mobility of LTR/non-LTR retrotransposons. Opossum A1 partially inhibited HIV-1, as well as simian immunodeficiency virus (SIV), murine leukemia virus (MLV), and the retrotransposon MusD. The mechanism of inhibition required catalytic activity, except for human LINE1 (L1) restriction, which was deamination-independent. These results indicate that opossum A1 functions as an innate barrier to infection by retroviruses such as HIV-1, and controls LTR/non-LTR retrotransposition in marsupials.

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of the HIV-1 virus in the absence of viral infectivity factor (Vif). The molecular mechanism of A3G antiviral activity is primarily attributed to deamination of single-stranded DNA (ssDNA); however, the nondeamination mechanism also contributes to HIV-1 restriction. The interaction of A3G with ssDNA and RNA is required for its antiviral activity. Here we used atomic force microscopy to directly visualize A3G-RNA and A3G-ssDNA complexes and compare them to each other. Our results showed that A3G in A3G-RNA complexes exists primarily in monomeric-dimeric states, similar to its stoichiometry in complexes with ssDNA. New A3G-RNA complexes in which A3G binds to two RNA molecules were identified. These data suggest the existence of two separate RNA binding sites on A3G. Such complexes were not observed with ssDNA substrates. Time-lapse high-speed atomic force microscopy was applied to characterize the dynamics of the complexes. The data revealed that the two RNA binding sites have different affinities for A3G. On the basis of the obtained results, a model for the interaction of A3G with RNA is proposed.

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations., IMPORTANCE A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors.

HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus-human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the α3 and α4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including human immunodeficiency virus type 1 (HIV-1). Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

SUMMARY HIV-1 Vif hijacks a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes and PP2A phosphatase regulators (PPP2R5A–E). APOBEC3 counteraction is essential for viral pathogenesis. However, Vif also functions through an unknown mechanism to induce G2 cell cycle arrest. Here, deep mutagenesis is used to define the Vif surface required for PPP2R5 degradation and isolate a panel of separation-of-function mutants (PPP2R5 degradation-deficient and APOBEC3G degradation-proficient). Functional studies with Vif and PPP2R5 mutants were combined to demonstrate that PPP2R5 is, in fact, the target Vif degrades to induce G2 arrest. Pharmacologic and genetic approaches show that direct modulation of PP2A function or depletion of specific PPP2R5 proteins causes an indistinguishable arrest phenotype. Vif function in the cell cycle checkpoint is present in common HIV-1 subtypes worldwide and likely advantageous for viral pathogenesis., In Brief Salamango et al. discovered that the HIV-1 accessory protein Vif degrades several PP2A phospho-regulators to induce G2 cell cycle arrest. This activity is prevalent among diverse HIV-1 subtypes and global viral populations, suggesting that virus-induced G2 arrest is advantageous for pathogenesis., Graphical Abstract

Tumor cells are able to evade cytotoxic therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor involved in the restriction of vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFNβ-dependent manner, which was responsible for the evolution of virus-resistanT-cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. ShRNA knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro, and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV in murine melanoma and glioblastoma lines, as well as in a human melanoma cell line in vitro and in vivo. Finally, we demonstrate that progeny virus obtained after passage through APOBEC3B overexpressing cells contained more defective interfering particles, thereby indicating that APOBEC3B directly affected the fitness of VSV, an RNA virus that has not previously been identified to be restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of oncolytic viruses as well as broader nucleic acid-based therapeutic platforms. Citation Format: Amanda L. Huff, Phonphimon Wongthida, Timothy Kottke, Jill Thompson, Christopher B. Driscoll, Matthew Schuelke, Kevin G. Shim, Reuben S. Harris, Amy Molan, Jose S. Pulido, Peter J. Selby, Kevin J. Harrington, Alan Melcher, Laura Evgin, Richard Vile. APOBEC3 confers resistance to oncolytic VSV therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A133.

Intrinsic restriction factors and viral nucleic-acid sensors are important for the anti-viral response. Here, we show how upstream sensing of retroviral reverse transcripts integrates with the downstream effector APOBEC3, an IFN-induced cytidine deaminase that introduces lethal mutations during retroviral reverse transcription. Using a Murine Leukemia Virus (MLV) variant with an unstable capsid that induces a strong IFNβ antiviral response, we identify three sensors, IFI203, DDX41 and cGAS, required for MLV nucleic-acid recognition. These sensors then signal using the adaptor STING, leading to increased production of IFNβ and other targets downstream of the transcription factor IRF3. Using knockout and mutant mice, we show that APOBEC3 limits the levels of reverse transcripts that trigger cytosolic sensing, and that nucleic-acid sensing in vivo increases expression of IFN-regulated restriction factors like APOBEC3 that in turn reduce viral load. These studies underscore the importance of the multiple layers of protection afforded by host factors.

The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.

SummaryHuman APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.

APOBEC3F and APOBEC3G are DNA cytosine deaminases that potently restrict Human Immunodeficiency Virus-type 1 replication when the virus is deprived of its accessory protein Vif. Vif counteracts these restriction factors by recruiting APOBEC3F and APOBEC3G to an E3 ubiquitin ligase complex that mediates their polyubiquitination and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream ubiquitin acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in APOBEC3G and proposed an antiparallel model of APOBEC3G interaction with the Vif-E3 ubiquitin ligase complex wherein Vif binding at one terminus of APOBEC3G orients the opposite terminus for polyubiquitination [Iwatani Y, et al. (2009) PNAS 106(46):19539–19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in APOBEC3F and used a complementary, unbiased proteomic approach to identify ubiquitin acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant ubiquitin acceptor sites in both APOBEC3F and APOBEC3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyubiquitination of APOBEC3F and APOBEC3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 ubiquitin ligase complex and diminish enthusiasm for the amenability of APOBEC3 ubiquitin acceptor sites to therapeutic intervention.

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.

Human immunodeficiency virus type 1 (HIV-1) requires the cellular transcription factor core binding factor subunit β (CBFβ) to stabilize its viral infectivity factor (Vif) protein and neutralize the APOBEC3 restriction factors. CBFβ normally heterodimerizes with the RUNX family of transcription factors, enhancing their stability and DNA-binding affinity. To test the hypothesis that Vif may act as a RUNX mimic to bind CBFβ, we generated a series of CBFβ mutants at the RUNX/CBFβ interface and tested their ability to stabilize Vif and impact transcription at a RUNX-dependent promoter. While several CBFβ amino acid substitutions disrupted promoter activity, none of these impacted the ability of CBFβ to stabilize Vif or enhance degradation of APOBEC3G. A mutagenesis screen of CBFβ surface residues identified a single amino acid change, F68D, that disrupted Vif binding and its ability to degrade APOBEC3G. This mutant still bound RUNX and stimulated RUNX-dependent transcription. These separation-of-function mutants demonstrate that HIV-1 Vif and the RUNX transcription factors interact with cellular CBFβ on genetically distinct surfaces.

The human APOBEC3G (A3G) enzyme restricts HIV-1 in the absence of the viral accessory protein viral infectivity factor (Vif) by deaminating viral cDNA cytosines to uracils. These uracil lesions base-pair with adenines during the completion of reverse transcription and result in A3G signature G-to-A mutations in the viral genome. Vif protects HIV-1 from A3G-mediated restriction by forming an E3-ubiquitin ligase complex to polyubiquitinate A3G and trigger its degradation. Prior studies indicated that Vif may also directly block the enzymatic activity of A3G and, provocatively, that Vif-derived peptides, Vif 25-39 and Vif 105-119, are similarly inhibitory. Here, we show that Vif 25-39 does not inhibit A3G enzymatic activity and that the inhibitory effect of Vif 105-119 and that of a shorter derivative Vif 107-115, although recapitulated, are non-specific. We also elaborate a simple method for assaying DNA cytosine deaminase activity that eliminates potential polymerase chain reaction-induced biases. Our results show that these Vif-derived peptides are unlikely to be useful as tools to study A3G function or as leads for the development of future therapeutics.

The APOBEC3 family of DNA cytosine deaminases has important roles in innate immunity and cancer. It is unclear how DNA tumor viruses regulate these enzymes and how these interactions, in turn, impact the integrity of both the viral and cellular genomes. Polyomavirus (PyVs) are small DNA pathogens that contain oncogenic potentials. In this study, we examined the effects of PyV infection on APOBEC3 expression and activity. We demonstrate that APOBEC3B is specifically upregulated by BK polyomavirus (BKPyV) infection in primary kidney cells and that the upregulated enzyme is active. We further show that the BKPyV large T antigen, as well as large T antigens from related polyomaviruses, is alone capable of upregulating APOBEC3B expression and activity. Furthermore, we assessed the impact of A3B on productive BKPyV infection and viral genome evolution. Although the specific knockdown of APOBEC3B has little short-term effect on productive BKPyV infection, our informatics analyses indicate that the preferred target sequences of APOBEC3B are depleted in BKPyV genomes and that this motif underrepresentation is enriched on the nontranscribed stand of the viral genome, which is also the lagging strand during viral DNA replication. Our results suggest that PyV infection upregulates APOBEC3B activity to influence virus sequence composition over longer evolutionary periods. These findings also imply that the increased activity of APOBEC3B may contribute to PyV-mediated tumorigenesis. IMPORTANCE Polyomaviruses (PyVs) are a group of emerging pathogens that can cause severe diseases, including cancers in immunosuppressed individuals. Here we describe the finding that PyV infection specifically induces the innate immune DNA cytosine deaminase APOBEC3B. The induced APOBEC3B enzyme is fully functional and therefore may exert mutational effects on both viral and host cell DNA. We provide bioinformatic evidence that, consistent with this idea, BK polyomavirus genomes are depleted of APOBEC3B-preferred target motifs and enriched for the corresponding predicted reaction products. These data imply that the interplay between PyV infection and APOBEC proteins may have significant impact on both viral evolution and virus-induced tumorigenesis.

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G.

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 double-strand 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.

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4 + T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4 + T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins—APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H—in HIV-1 restriction.

Tandem stop mutations K26X and H27X in human immunodeficiency virus type 1 (HIV-1) vif compromise virus replication in human T-cell lines that stably express APOBEC3F (A3F) or APOBEC3G (A3G). We previously reported that partial resistance to A3G could develop in these Vif-deficient viruses through a nucleotide A200-to-T/C transversion and a vpr null mutation, but these isolates were still susceptible to restriction by A3F. Here, long-term selection experiments were done to determine how these A3G-selected isolates might evolve to spread in the presence of A3F. We found that A3F, like A3G, is capable of potent, long-term restriction that eventually selects for heritable resistance. In all 7 instances, the selected isolates had restored Vif function to cope with A3F activity. In two isolates, Vif Q26-Q27 and Y26-Q27, the resistance phenotype recapitulated in molecular clones, but when the selected vif alleles were analyzed in the context of an otherwise wild-type viral background, a different outcome emerged. Although HIV-1 clones with Vif Q26-Q27 or Y26-Q27 were fully capable of overcoming A3F, they were now susceptible to restriction by A3G. Concordant with prior studies, a lysine at position 26 proved essential for A3G neutralization. These data combine to indicate that A3F and A3G exert at least partly distinct selective pressures and that Vif function may be essential for the virus to replicate in the presence of A3F.

BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.

The human APOBEC3 proteins are DNA cytidine deaminases that impede the replication of many different transposons and viruses. The genes that encode APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H were generated through relatively recent recombination events. The resulting high degree of inter-relatedness has complicated the development of specific quantitative PCR assays for these genes despite considerable interest in understanding their expression profiles. Here, we describe a set of quantitative PCR assays that specifically measures the mRNA levels of each APOBEC3 gene. The specificity and sensitivity of each assay was validated using a full matrix of APOBEC3 cDNA templates. The assays were used to quantify the APOBEC3 repertoire in multiple human T-cell lines, bulk leukocytes and leukocyte subsets, and 20 different human tissues. The data demonstrate that multiple APOBEC3 genes are expressed constitutively in most types of cells and tissues, and that distinct APOBEC3 genes are induced upon T-cell activation and interferon treatment. These data help define the APOBEC3 repertoire relevant to HIV-1 restriction in T cells, and they suggest a general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction.

Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of human immunodeficiency virus-1, other retroviruses, and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an alpha1-helix (residues 201-206) that was not apparent in the shorter protein, and it contributes to catalytic activity through interactions with hydrophobic core structures (beta1, beta3, alpha5, and alpha6). Both A3G catalytic domain solution structures have a discontinuous beta2 region that is clearly different from the continuous beta2 strand of another family member, APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain, including the interdomain linker and some of the last alpha-helix. These structured residues (residues 191-196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal beta2 regions are too distant from each other to participate in this interaction.