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Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.

Abstract Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Abstract Background RNA editing has been described as promoting genetic heterogeneity, leading to the development of multiple disorders, including cancer. The cytosine deaminase APOBEC3B is implicated in tumor evolution through DNA mutation, but whether it also functions as an RNA editing enzyme has not been studied. Results Here, we engineer a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B lead to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, RNA-sequencing of mouse tissues expressing high levels of APOBEC3B identifies frequent UCC-to-UUC RNA editing events that are not evident in the corresponding genomic DNA. Conclusions This work identifies, for the first time, a new deaminase-dependent function for APOBEC3B in RNA editing and presents a preclinical tool to help understand the emerging role of APOBEC3B as a driver of carcinogenesis.

Abstract The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2′-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Abstract The development of SARS-CoV-2 main protease (Mpro) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate Mpro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of Mpro. An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using Mpro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of Mpro with low nanomolar K i values. Furthermore, these compounds demonstrate efficacy against mutant forms of Mpro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.

APOBEC’s are a family of cytidine deaminases that induce mutations in viruses to inhibit their replication and maintain cell integrity. Here, Manjunath et al show that APOBEC3B also inhibits viral replication by stimulating the innate immune sensor protein kinase R causing translational shutdown and stress granule formation independently of its cytidine deaminase activity.

ABSTRACT 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.

ABSTRACT APOBEC3B is an innate immune effector enzyme capable of introducing mutations in viral genomes through DNA cytosine-to-uracil editing. Recent studies have shown that gamma-herpesviruses, such as Epstein-Barr virus (EBV), have evolved a potent APOBEC3B neutralization mechanism to protect lytic viral DNA replication intermediates in the nuclear compartment. APOBEC3B is additionally unique as the only human DNA deaminase family member that is constitutively nuclear. Nuclear localization has therefore been inferred to be essential for innate antiviral function. Here, we combine evolutionary, molecular, and cell biology approaches to address whether nuclear localization is a conserved feature of APOBEC3B in primates. Despite the relatively recent emergence of APOBEC3B approximately 30 to 40 million years ago (MYA) in Old World primates by genetic recombination (after the split from the New World monkey lineage 40 to 50 MYA), we find that the hallmark nuclear localization of APOBEC3B shows variability. For instance, although human and several nonhuman primate APOBEC3B enzymes are predominantly nuclear, rhesus macaque and other Old World primate APOBEC3B proteins are clearly cytoplasmic or cell wide. A series of human/rhesus macaque chimeras and mutants combined to map localization determinants to the N-terminal half of the protein with residues 15, 19, and 24 proving critical. Ancestral APOBEC3B reconstructed from present-day primate species also shows strong nuclear localization. Together, these results indicate that the ancestral nuclear localization of APOBEC3B is maintained in present-day human and ape proteins, but nuclear localization is not conserved in all Old World monkey species despite a need for antiviral functions in the nuclear compartment. IMPORTANCE APOBEC3 enzymes are single-stranded DNA cytosine-to-uracil deaminases with beneficial roles in antiviral immunity and detrimental roles in cancer mutagenesis. Regarding viral infection, all seven human APOBEC3 enzymes have overlapping roles in restricting virus types that require DNA for replication, including EBV, HIV, human papillomavirus (HPV), and human T-cell leukemia virus (HTLV). Regarding cancer, at least two APOBEC3 enzymes, APOBEC3B and APOBEC3A, are prominent sources of mutation capable of influencing clinical outcomes. Here, we combine evolutionary, molecular, and cell biology approaches to characterize primate APOBEC3B enzymes. We show that nuclear localization is an ancestral property of APOBEC3B that is maintained in present-day human and ape enzymes, but not conserved in other nonhuman primates. This partial mechanistic conservation indicates that APOBEC3B is important for limiting the replication of DNA-based viruses in the nuclear compartment. Understanding these pathogen-host interactions may contribute to the development of future antiviral and antitumor therapies.

ABSTRACT 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.

APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G2/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G0/early G1, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G0. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G2/M phase of the cell cycle.

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses such as the human T-cell leukemia virus type-1 (HTLV-1). Here the authors describe the structural role of the host co-factor protein phosphatase 2A B56γ subunit in supporting integration as part of the HTLV-1 intasome.

Abstract The single‐stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3‐family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high‐resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2‐pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

Abstract 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

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.

Human APOBEC3H has several haplotypes and splice variants with distinct anti-HIV-1 activities, but the genetics underlying the expression of these variants are unclear. Here, the authors identify an intronic deletion in A3H haplotype II resulting in production of the most active splice variant, which is counteracted by HIV-1 protease.

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. : 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. Keywords: cell cycle checkpoint, HIV-1, host-pathogen interaction, phosphatase regulation, Vif

ABSTRACT APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B. Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5′-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

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.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

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.

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamic simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a wobble model to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, how pathogens may evolve to escape innate host defenses.

HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

ABSTRACT 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.

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.

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.

ABSTRACT Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other tissues. A3B is upregulated in these tumor types relative to normal tissues, but the mechanism is unclear. Because A3B also has antiviral activity in multiple systems and is a member of the broader innate immune response, we tested the hypothesis that human papillomavirus (HPV) infection causes A3B upregulation. We found that A3B mRNA expression and enzymatic activity were upregulated following transfection of a high-risk HPV genome and that this effect was abrogated by inactivation of E6. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause A3B upregulation, and a panel of high-risk E6 proteins triggered higher A3B levels than did a panel of low-risk or noncancer E6 proteins. Knockdown experiments in HPV-positive cell lines showed that endogenous E6 is required for A3B upregulation. Analyses of publicly available head/neck cancer data further support this relationship, as A3B levels are higher in HPV-positive cancers than in HPV-negative cancers. Taken together with the established role for high-risk E6 in functional inactivation of TP53 and published positive correlations in breast cancer between A3B upregulation and genetic inactivation of TP53, our studies suggest a model in which high-risk HPV E6, possibly through functional inactivation of TP53, causes derepression of A3B gene transcription. This would lead to a mutator phenotype that explains the observed cytosine mutation biases in HPV-positive head/neck and cervical cancers. IMPORTANCE The innate immune DNA cytosine deaminase APOBEC3B (A3B) accounts for a large proportion of somatic mutations in cervical and head/neck cancers, but nothing is known about the mechanism responsible for its upregulation in these tumor types. Almost all cervical carcinomas and large proportions of head/neck tumors are caused by human papillomavirus (HPV) infection. Here, we establish a mechanistic link between HPV infection and A3B upregulation. The E6 oncoprotein of high-risk, but not low-risk, HPV types triggers A3B upregulation, supporting a model in which TP53 inactivation causes a derepression of A3B gene transcription and elevated A3B enzyme levels. This virus-induced mutator phenotype provides a mechanistic explanation for A3B signature mutations observed in HPV-positive head/neck and cervical carcinomas and may also help to account for the preferential cancer predisposition caused by high-risk HPV isolates.

APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.