A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

APOBEC3 degradation is the primary function of HIV-1 Vif determining virion infectivity in the myeloid cell line THP-1.

HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+ T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity in A3F/A3G-null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.

DNA Deamination Is Required for Human APOBEC3A-Driven Hepatocellular Carcinoma In Vivo.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3's signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a "master driver" that fuels tumor formation through a DNA deamination-dependent mechanism.

An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations.

Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumor regression but ultimately fail to elicit cures. As a result, there is an intense interest in how to best combine targeted therapies with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumors with the host immune system are inadequate, in part due to the low tumor mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumor mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumor mutational burden and autochthonous tumors remained refractory to immunotherapy. However, establishing clonal cell lines from these tumors enabled the generation of an immunogenic syngeneic transplantation model of KRAS-mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, antitumor immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C -expressing tumors was markedly potentiated by the adaptive immune system, highlighting the importance of using immunocompetent models for evaluating targeted therapies. Overall, this model provides a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer. SIGNIFICANCE: This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.

Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro/3CLpro in Living Cells.

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 report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, 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. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

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.

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

Disease Expression and Familial Transmission of Fuchs Endothelial Corneal Dystrophy With and Without CTG18.1 Expansion.

Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD). Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed. Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (∼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD. Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.

Gain-of-function assay for SARS-CoV-2 M pro inhibition in living cells.

The main protease, M pro , 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 M pro 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.

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

PURPOSE: Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. EXPERIMENTAL DESIGNS: APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. RESULTS: One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. CONCLUSIONS: These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

The deaminase APOBEC3B triggers the death of cells lacking uracil DNA glycosylase.

Human cells express up to 9 active DNA cytosine deaminases with functions in adaptive and innate immunity. Many cancers manifest an APOBEC mutation signature and APOBEC3B (A3B) is likely the main enzyme responsible. Although significant numbers of APOBEC signature mutations accumulate in tumor genomes, the majority of APOBEC-catalyzed uracil lesions are probably counteracted in an error-free manner by the uracil base excision repair pathway. Here, we show that A3B-expressing cells can be selectively killed by inhibiting uracil DNA glycosylase 2 (UNG) and that this synthetic lethal phenotype requires functional mismatch repair (MMR) proteins and p53. UNG knockout human 293 and MCF10A cells elicit an A3B-dependent death. This synthetic lethal phenotype is dependent on A3B catalytic activity and reversible by UNG complementation. A3B expression in UNG-null cells causes a buildup of genomic uracil, and the ensuing lethality requires processing of uracil lesions (likely U/G mispairs) by MSH2 and MLH1 (likely noncanonical MMR). Cancer cells expressing high levels of endogenous A3B and functional p53 can also be killed by expressing an UNG inhibitor. Taken together, UNG-initiated base excision repair is a major mechanism counteracting genomic mutagenesis by A3B, and blocking UNG is a potential strategy for inducing the selective death of tumors.

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1.

Apolipoprotein B mRNA editing enzyme catalytic subunit-like protein 3B (APOBEC3B or A3B), as other APOBEC3 members, is a single-stranded (ss)DNA cytosine deaminase with antiviral activity. A3B is also overexpressed in multiple tumor types, such as carcinomas of the bladder, cervix, lung, head/neck, and breast. A3B generates both dispersed and clustered C-to-T and C-to-G mutations in intrinsically preferred trinucleotide motifs (TCA/TCG/TCT). A3B-catalyzed mutations are likely to promote tumor evolution and cancer progression and, as such, are associated with poor clinical outcomes. However, little is known about cellular processes that regulate A3B. Here, we used a proteomics approach involving affinity purification coupled to MS with human 293T cells to identify cellular proteins that interact with A3B. This approach revealed a specific interaction with cyclin-dependent kinase 4 (CDK4). We validated and mapped this interaction by co-immunoprecipitation experiments. Functional studies and immunofluorescence microscopy experiments in multiple cell lines revealed that A3B is not a substrate for CDK4-Cyclin D1 phosphorylation nor is its deaminase activity modulated. Instead, we found that A3B is capable of disrupting the CDK4-dependent nuclear import of Cyclin D1. We propose that this interaction may favor a more potent antiviral response and simultaneously facilitate cancer mutagenesis.

Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity.

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.

A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.

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.

APOBEC3B Nuclear Localization Requires Two Distinct N-Terminal Domain Surfaces.

The APOBEC3 family of cytosine deaminases catalyzes the conversion of cytosines-to-uracils in single-stranded DNA. Traditionally, these enzymes are associated with antiviral immunity and restriction of DNA-based pathogens. However, a role for these enzymes in tumor evolution and metastatic disease has also become evident. The primary APOBEC3 candidate in cancer mutagenesis is APOBEC3B (A3B) for three reasons: (1) A3B mRNA is upregulated in several different cancers, (2) A3B expression and mutational loads correlate with poor clinical outcomes, and (3) A3B is the only family member known to be constitutively nuclear. Previous studies have mapped non-canonical A3B nuclear localization determinants to a single surface-exposed patch within the N-terminal domain (NTD). Here, we show that A3B has an additional, distinct, surface-exposed NTD region that contributes to nuclear localization. Disruption of residues within the first 30 amino acids of A3B (import surface 1) or loop 5/α-helix 3 (import surface 2) completely abolish nuclear localization. These import determinants also graft into NTDs of related family members and mediate re-localization from cell-wide-to-nucleus or cytoplasm-to-nucleus. These findings demonstrate that both sets of residues are required for non-canonical A3B nuclear localization and describe unique surfaces that may serve as novel therapeutic targets.

Simian Immunodeficiency Virus Vif and Human APOBEC3B Interactions Resemble Those between HIV-1 Vif and Human APOBEC3G.

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 SIVmac239 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 SIVmac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIVmac239 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 SIVmac239 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 SIVmac239 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. SIVmac239 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 SIVmac239 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 SIVmac239 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.