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APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3'-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G's cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes., (© 2024. The Author(s).)

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. Whilst recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV's counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction., (© 2023. The Author(s).)

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles 1-4 . The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene., (© 2023. The Author(s).)

APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3's catalytic activity as therapeutics as well as tools to study A3's biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2'-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-H 2 O mimics the transition state by coordinating the active site Zn 2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3's transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future., (© 2022. The Author(s).)

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes., Competing Interests: Conflict of interest N. J. K. has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, and Interline Therapeutics. He is a shareholder in Tenaya Therapeutics, Maze Therapeutics, and Interline Therapeutics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn 2+ , yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3' to 5' directional preference in processive deamination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 American Society for Microbiology.)

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: R.S.H. is a co-founder, shareholder and consultant of ApoGen Biotechnologies.

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer's and Parkinson's diseases. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting that interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind the on-surface aggregation process remain unclear. Here, we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This physical-chemical theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic α-synuclein protein and non-amyloidogenic cytosine deaminase APOBEC3G.

APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunodeficiency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD's search function. These processes explain A3G's ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process., Competing Interests: MM, MN, YF, LC, IR, MW No competing interests declared, (© 2019, Morse et al.)

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human APOBEC3G (A3G) inhibits the replication of human immunodeficiency virus-1 by deaminating cytidine at the 3'-end in the target motif 5'-CCC-3' in viral cDNA during reverse transcription. It in vitro deaminates two consecutive cytidines in a 3'->5' order. Although a crystal structure of the A3G catalytic domain (A3G-CD2) with DNA was reported, it is unknown why residues involved in enzymatic reaction are distributed widely. Here, we introduced an iodine atom into the C-5 position of cytidine (dC 6 I ) in DNA 5'-ATTC 4 C 5 C 6 I A 7 ATT-3' (TCCC 6 I ). It switches the deamination sequence preference from CCC to TCC, although small dC 6 I deamination was observed. Solution structures of A3G-CD2 in complexes with products DNA TCUC 6 I and TCUU 6 I indicate that the substrate DNA binds A3G-CD2 in TCC and CCC modes. The dC 6 deamination correlates with the 4 th base type. The CCC mode favours dC 6 deamination, while the TCC mode results in dC 5 deamination. These studies present an extensive basis to design inhibitors to impede viral evolvability., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

APOBEC3G (A3G) is a cellular protein that inhibits HIV-1 infection through virion incorporation. The interaction of the A3G N-terminal domain (NTD) with RNA is essential for A3G incorporation in the HIV-1 virion. The interaction between A3G-NTD and RNA is not completely understood. The A3G-NTD is also recognized by HIV-1 Viral infectivity factor (Vif) and A3G-Vif binding leads to A3G degradation. Therefore, the A3G-Vif interaction is a target for the development of antiviral therapies that block HIV-1 replication. However, targeting the A3G-Vif interactions could disrupt the A3G-RNA interactions that are required for A3G's antiviral activity. To better understand A3G-RNA binding, we generated in silic o docking models to simulate the RNA-binding propensity of A3G-NTD. We simulated the A3G-NTD residues with high RNA-binding propensity, experimentally validated our prediction by testing A3G-NTD mutations, and identified structural determinants of A3G-RNA binding. In addition, we found a novel amino acid residue, I26 responsible for RNA interaction. The new structural insights provided here will facilitate the design of pharmaceuticals that inhibit A3G-Vif interactions without negatively impacting A3G-RNA interactions.

The APOBEC3 enzyme family are host restriction factors that induce mutagenesis of HIV-1 proviral genomes through the deamination of cytosine to form uracil in nascent single-stranded (-)DNA. HIV-1 suppresses APOBEC3 activity through the HIV-1 protein Vif that induces APOBEC3 degradation. Here we compared two common polymorphisms of APOBEC3F. We found that although both polymorphisms have HIV-1 restriction activity, APOBEC3F 108 A/231V can restrict HIV-1 ΔVif up to 4-fold more than APOBEC3F 108 S/231I and is partially protected from Vif-mediated degradation. This resulted from higher levels of steady state expression of APOBEC3F 108 A/231 V. Individuals are commonly heterozygous for the APOBEC3F polymorphisms and these polymorphisms formed in cells, independent of RNA, hetero-oligomers between each other and with APOBEC3G. Hetero-oligomerization with APOBEC3F 108 A/231V resulted in partial stabilization of APOBEC3F 108 S/231I and APOBEC3G in the presence of Vif. These data demonstrate functional outcomes of APOBEC3 polymorphisms and hetero-oligomerization that affect HIV-1 restriction., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

APOBEC3G (A3G) belongs to the family of cytosine deaminases that play an important role in the innate immune response. Similar to other, two-domain members of the APOBEC family, A3G is prone to concentration-dependent oligomerization, which is an integral for its function in the cell. It is shown that oligomerization of A3G is related to the packing mechanism into virus particle and, is critical for the so-called roadblock model during reverse transcription of proviral ssDNA. The role of oligomerization for deaminase activity of A3G is widely discussed in the literature; however, its relevance to deaminase activity for different oligomeric forms of A3G remains unclear. Here, using Atomic Force Microscopy, we directly visualized A3G-ssDNA complexes, determined their yield and stoichiometry and in parallel, using PCR assay, measured the deaminase activity of these complexes. Our data demonstrate a direct correlation between the total yield of A3G-ssDNA complexes and their total deaminase activity. Using these data, we calculated the relative deaminase activity for each individual oligomeric state of A3G in the complex. Our results show not only similar deaminase activity for monomer, dimer and tetramer of A3G in the complex, but indicate that larger oligomers of A3G retain their deaminase activity.

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The human APOBEC3G protein is a cytidine deaminase that generates cytidine to deoxy-uridine mutations in single-stranded DNA (ssDNA), and capable of restricting replication of HIV-1 by generating mutations in viral genome. The mechanism by which APOBEC3G specifically deaminates 5'-CC motifs has remained elusive since structural studies have been hampered due to apparently weak ssDNA binding of the catalytic domain of APOBEC3G. We overcame the problem by generating a highly active variant with higher ssDNA affinity. Here, we present the crystal structure of this variant complexed with a ssDNA substrate at 1.86 Å resolution. This structure reveals atomic-level interactions by which APOBEC3G recognizes a functionally-relevant 5'-TCCCA sequence. This complex also reveals a key role of W211 in substrate recognition, implicating a similar recognition in activation-induced cytidine deaminase (AID) with a conserved tryptophan.

Human APOBEC3G (hA3G) is a restriction factor that inhibits human immunodeficiency 1 virus (HIV-1) replication. The virally encoded protein Vif binds to hA3G and induces its degradation, thereby counteracting the antiviral activity of hA3G. Vif-mediated hA3G degradation clearly represents a potential target for anti-HIV drug development. Herein, we have performed virtual screening to discover small molecule inhibitors that target the binding interface of the Vif/hA3G complex. Subsequent biochemical studies have led to the identification of a small molecule inhibitor, IMB-301 that binds to hA3G, interrupts the hA3G-Vif interaction and inhibits Vif-mediated degradation of hA3G. As a result, IMB-301 strongly inhibits HIV-1 replication in a hA3G-dependent manner. Our study further demonstrates the feasibility of inhibiting HIV replication by abrogating the Vif-hA3G interaction with small molecules.

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis-for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antiviral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production and cytidine-to-uridine-driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3'-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires the specific interaction of A3G with reverse transcriptase itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between reverse transcriptase, A3G and cellular DNA repair machinery, and identify the suppression of HIV-1 reverse transcriptase by a directly interacting host protein as a new cell-mediated antiviral mechanism.

APOBEC3G (A3G), an anti-human immunodeficiency virus 1 factor, deaminates cytidines. We examined deamination of two cytidines located separately on substrate ssDNA by the C-terminal domain (CTD) of A3G using real-time NMR monitoring. The deamination preference between the two cytidines was lost when either the substrate or non-substrate ssDNA concentration increased. When the non-substrate ssDNA concentration increased, the deamination activity first increased, but then decreased. This indicates that even a single domain, A3G-CTD, undergoes intersegmental transfer for a target search.

The present article describes techniques for classical simulations of proteins and protein-nucleic acid complexes, revealing their dynamics and protein-substrate binding energies. The approach is based on classical atomistic molecular dynamics (MD) simulations of the experimentally determined structures of the complexes. MD simulations can provide dynamics of complexes in realistic solvents on microsecond timescales, and the free energy methods are able to provide Gibbs free energies of binding of substrates, such as nucleic acids, to proteins. The chapter describes methodologies for the preparation of computer models of biomolecular complexes and free energy perturbation methodology for evaluating Gibbs free energies of binding. The applications are illustrated with examples of snapshots of proteins and their complexes with nucleic acids, as well as the precise Gibbs free energies of binding.

Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17- N -allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4 + T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart., Importance: The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif., (Copyright © 2017 American Society for Microbiology.)

APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

APOBEC3G (A3G) is a potent restriction factor of HIV-1. The N-terminal domain of A3G (A3G-CD1) is responsible for oligomerization and nucleic acid binding, both of which are essential for anti-HIV activity. As a countermeasure, HIV-1 viral infectivity factor (Vif) binds A3G-CD1 to mediate A3G degradation. The structural basis for the functions of A3G-CD1 remains elusive. Here, we report the crystal structures of a primate A3G-CD1 (rA3G-CD1) alone and in complex with single-stranded DNA (ssDNA). rA3G-CD1 shares a conserved core structure with the previously determined catalytic APOBECs, but displays unique features for surface charge, dimerization and nucleic acid binding. Its co-crystal structure with ssDNA reveals how the conformations of loops and residues surrounding the Zn-coordinated centre (Zn-centre) change upon DNA binding. The dimerization interface of rA3G-CD1 is important for oligomerization, nucleic acid binding and Vif-mediated degradation. These findings elucidate the molecular basis of antiviral mechanism and HIV-Vif targeting of A3G.

Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)