RESUMEN
Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity.
Asunto(s)
Aminohidrolasas/metabolismo , Dimerización , VIH-1/crecimiento & desarrollo , Ensamble de Virus/genética , Aminohidrolasas/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Citosina Desaminasa/metabolismo , Células HEK293 , Células HeLa , Humanos , Estructura Secundaria de Proteína , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasa Pancreática/metabolismoRESUMEN
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.
Asunto(s)
Muerte Celular , Citidina Desaminasa/genética , ADN Glicosilasas/genética , Desaminasas APOBEC , Línea Celular Tumoral , ADN Glicosilasas/antagonistas & inhibidores , Reparación de la Incompatibilidad de ADN , Reparación del ADN , Técnicas de Inactivación de Genes , Humanos , Modelos Moleculares , UbiquitinaciónRESUMEN
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.
Asunto(s)
Desaminasa APOBEC-3G/genética , Citidina Desaminasa/genética , Inmunidad Innata/inmunología , Antígenos de Histocompatibilidad Menor/genética , Virus de la Inmunodeficiencia de los Simios/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Línea Celular , Células HEK293 , VIH-1/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genéticaRESUMEN
The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.
Asunto(s)
Aminohidrolasas/genética , Infecciones por VIH/genética , VIH-1/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Línea Celular , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/patogenicidad , Haplotipos , Humanos , Mutación , Polimorfismo Genético , Replicación Viral/genéticaRESUMEN
HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus-human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the α3 and α4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.
Asunto(s)
Citosina Desaminasa/metabolismo , VIH-1/inmunología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Sustitución de Aminoácidos , Animales , Citosina Desaminasa/genética , Análisis Mutacional de ADN , Humanos , Macaca mulatta , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Viruses have evolved diverse mechanisms to antagonize host immunity such as direct inhibition and relocalization of cellular APOBEC3B (A3B) by the ribonucleotide reductase (RNR) of Epstein-Barr virus. Here, we investigate the mechanistic conservation and evolutionary origin of this innate immune counteraction strategy. First, we find that human gamma-herpesvirus RNRs engage A3B via largely distinct surfaces. Second, we show that RNR-mediated enzymatic inhibition and relocalization of A3B depend upon binding to different regions of the catalytic domain. Third, we show that the capability of viral RNRs to antagonize A3B is conserved among gamma-herpesviruses that infect humans and Old World monkeys that encode this enzyme but absent in homologous viruses that infect New World monkeys that naturally lack the A3B gene. Finally, we reconstruct the ancestral primate A3B protein and demonstrate that it is active and similarly engaged by the RNRs from viruses that infect humans and Old World monkeys but not by the RNRs from viruses that infect New World monkeys. These results combine to indicate that the birth of A3B at a critical branchpoint in primate evolution may have been a driving force in selecting for an ancestral gamma-herpesvirus with an expanded RNR functionality through counteraction of this antiviral enzyme.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Ribonucleótido Reductasas , Virus , Humanos , Animales , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Herpesvirus Humano 4 , Inmunidad Innata , Platirrinos/metabolismo , Cercopithecidae/metabolismo , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismoRESUMEN
Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å2 binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.
RESUMEN
The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.
Asunto(s)
Replicación del ADN , ADN/química , ARN/química , Ribonucleasa H/química , Animales , Autoinmunidad/genética , Secuencia de Bases , Dominio Catalítico/genética , Cristalografía por Rayos X , ADN/metabolismo , Humanos , Ratones , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Especificidad por SustratoRESUMEN
The 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 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 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 host-pathogen 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.
Asunto(s)
Desaminasas APOBEC/genética , Herpesviridae/genética , Animales , Replicación del ADN/genética , Virus ADN/genética , ADN Viral/genética , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Interacciones Huésped-Patógeno/genética , Humanos , Replicación Viral/genéticaRESUMEN
Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.
Asunto(s)
Encefalopatías Metabólicas Innatas/enzimología , Mutación Missense , Ribonucleasa H/metabolismo , Sustitución de Aminoácidos , Encefalopatías Metabólicas Innatas/genética , Dominio Catalítico/genética , Humanos , Estructura Cuaternaria de Proteína/genética , Ribonucleasa H/genética , SíndromeRESUMEN
As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species. RNA binding is required for localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles and ultimately for effective virus restriction activity. Here we compared wild-type human A3H and RNA binding-defective mutants to ask whether RNA may be a factor in the functional interaction with HIV-1 Vif. We used structural modeling, immunoblotting, live cell imaging, and split green fluorescence protein (GFP) reconstitution approaches to assess the capability of HIV-1 Vif to promote the degradation of wild-type A3H in comparison to RNA binding-defective mutants. The results combined to show that RNA is not strictly required for Vif-mediated degradation of A3H, and that RNA and Vif are likely to bind this single-domain DNA cytosine deaminase on physically distinct surfaces. However, a subset of the results also indicated that the A3H degradation process may be affected by A3H protein structure, subcellular localization, and differences in the constellation of A3H interaction partners, suggesting additional factors may also influence the fate and functionality of this host-pathogen interaction.
Asunto(s)
Aminohidrolasas/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Aminohidrolasas/química , Genes Reporteros , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Multimerización de Proteína , Proteolisis , ARN Viral/química , ARN Viral/genética , Relación Estructura-Actividad , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
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.
Asunto(s)
Citidina Desaminasa/antagonistas & inhibidores , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas Virales/metabolismo , Sistemas CRISPR-Cas , Dominio Catalítico/genética , Línea Celular , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Antígenos de Histocompatibilidad Menor , Interferencia de ARN , ARN Interferente Pequeño/genética , Ribonucleótido Reductasas/genética , Proteínas Virales/genéticaRESUMEN
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.
Asunto(s)
Aminohidrolasas/metabolismo , VIH-1/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Carcinogénesis/metabolismo , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citidina Desaminasa/metabolismo , Citoplasma/metabolismo , Células HEK293 , Células HeLa , HumanosRESUMEN
Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I-VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a counter-defense mechanism against A3H in >80% of sub-Saharan African populations.
Asunto(s)
Empalme Alternativo/inmunología , Aminohidrolasas/inmunología , Proteasa del VIH/inmunología , VIH-1/inmunología , Haplotipos/inmunología , Empalme Alternativo/genética , Secuencia de Aminoácidos , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Secuencia de Bases , Células HEK293 , Proteasa del VIH/metabolismo , VIH-1/metabolismo , Haplotipos/genética , Humanos , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Polimorfismo de Nucleótido Simple/genética , Polimorfismo de Nucleótido Simple/inmunología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Replicación Viral/inmunología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring -1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The -1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5'-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.
Asunto(s)
Citidina Desaminasa/química , Antígenos de Histocompatibilidad Menor/química , Proteínas/química , Aminación , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Secuencia de Consenso , Cristalografía por Rayos X , Citidina Desaminasa/fisiología , Citosina , ADN de Cadena Simple/química , Humanos , Enlace de Hidrógeno , Cinética , Antígenos de Histocompatibilidad Menor/fisiología , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas/fisiología , Especificidad por SustratoRESUMEN
The APOBEC3 family of DNA cytosine deaminases is capable of restricting the replication of HIV-1 and other pathogens. Here, we report a 1.92 Å resolution crystal structure of the Vif-binding and catalytic domain of APOBEC3F (A3F). This structure is distinct from the previously published APOBEC and phylogenetically related deaminase structures, as it is the first without zinc in the active site. We determined an additional structure containing zinc in the same crystal form that allows direct comparison with the zinc-free structure. In the absence of zinc, the conserved active site residues that normally participate in zinc coordination show unique conformations, including a 90 degree rotation of His249 and disulfide bond formation between Cys280 and Cys283. We found that zinc coordination is influenced by pH, and treating the protein at low pH in crystallization buffer is sufficient to remove zinc. Zinc coordination and catalytic activity are reconstituted with the addition of zinc only in a reduced environment likely due to the two active site cysteines readily forming a disulfide bond when not coordinating zinc. We show that the enzyme is active in the presence of zinc and cobalt but not with other divalent metals. These results unexpectedly demonstrate that zinc is not required for the structural integrity of A3F and suggest that metal coordination may be a strategy for regulating the activity of A3F and related deaminases.
Asunto(s)
Citosina Desaminasa/química , Citosina Desaminasa/metabolismo , Zinc/metabolismo , Dominio Catalítico , Cisteína/metabolismo , VIH-1/metabolismo , Unión Proteica/fisiología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
RNA-DNA hybrids play essential roles in a variety of biological processes, including DNA replication, transcription, and viral integration. Ribonucleotides incorporated within DNA are hydrolyzed by RNase H enzymes in a removal process that is necessary for maintaining genomic stability. In order to understand the structural determinants involved in recognition of a hybrid substrate by RNase H we have determined the crystal structure of a dodecameric non-polypurine/polypyrimidine tract RNA-DNA duplex. A comparison to the same sequence bound to RNase H, reveals structural changes to the duplex that include widening of the major groove to 12.5 Å from 4.2 Å and decreasing the degree of bending along the axis which may play a crucial role in the ribonucleotide recognition and cleavage mechanism within RNase H. This structure allows a direct comparison to be made about the conformational changes induced in RNA-DNA hybrids upon binding to RNase H and may provide insight into how dysfunction in the endonuclease causes disease.
Asunto(s)
ADN/química , Modelos Moleculares , ARN/química , Ribonucleasa H/metabolismo , Ribonucleótidos/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/genética , ARN/metabolismoRESUMEN
Viruses possess elaborate defensive mechanisms to evade host innate immune responses. In this issue of Cell Host & Microbe, Stavrou et al. (2015) reveal how the murine leukemia virus uses a sugar-protein shield to protect from inevitable destruction by cellular innate immune factors including the APOBEC3 DNA mutating enzyme.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN Viral/metabolismo , Interferón beta/metabolismo , Virus de la Leucemia Murina/inmunología , Macrófagos/inmunología , AnimalesRESUMEN
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-ß 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.
Asunto(s)
Citosina Desaminasa/metabolismo , VIH-1/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Citosina Desaminasa/química , Citosina Desaminasa/genética , VIH-1/genética , Humanos , Inmunidad Innata , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Electricidad Estática , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5'-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5'-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5'-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5'-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5'-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained human immunodeficiency virus type 1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as human immunodeficiency virus type 1 cDNA.