Structure and Function of Vif and APOBEC3 (A3) Proteins

Vif 和 APOBEC3 (A3) 蛋白的结构和功能

基本信息

批准号：
10702378
负责人：
VINAY K. PATHAK
金额：
$ 176.93万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10702378
关键词：
APOCEC3G gene Acquired Immunodeficiency Syndrome Affinity Alternative Splicing Antiviral Therapy Architecture Binding Biological Biomedical Research C-terminal CD34 gene Catalytic Domain Cell Line Cells Collaborations Complex Crystallization Cytidine DNA Binding DNA Integration DNA biosynthesis Deamination Direct Repeats Epidemic Evolution Exons Frequencies Genes Genetic Genetic Recombination Goals HIV HIV Infections HIV-1 Hematopoietic stem cells Host Defense Human Infection Innate Immune Response Integration Host Factors Knowledge Laboratories Lentivirus Vector Malignant Neoplasms Mediating Molecular Mutagenesis Mutation Mutation Analysis N-terminal Nucleotides Pharmaceutical Preparations Production Proteins RNA Splicing Reagent Research Resistance Reverse Transcription Reverse Transcription Inhibition Single-Stranded DNA Somatic Mutation Specificity Structure T-Lymphocyte Universities Variant Vertebral column Viral Genome Virion Virus Virus Replication antiviral drug development base cancer type cellular transduction comparative design gene therapy homologous recombination in vivo inhibitor insight member mutant novel novel therapeutic intervention pathogen preference prevent reconstitution translational genomics ubiquitin ligase vector viral DNA

项目摘要

APOBEC3 (A3) proteins are members of an innate immune response that provide a defense against HIV-1 and other pathogens. In the absence of the HIV-1 protein Vif, the A3 proteins are incorporated into virions in the virus producer cells and inhibit viral replication by deaminating cytidines in the minus-strand of viral DNA during reverse transcription in the target cells, resulting in extensive G-to-A hypermutation of the viral genome. In addition to inactivating most of the viral genomes through lethal hypermutation, we and others have shown that A3G and A3F also inhibit viral DNA synthesis and integration. To overcome these host defenses, Vif binds to the A3 proteins and targets them for proteasomal degradation, preventing their incorporation into virions. Defining the interactions of Vif with A3G and A3F at the molecular level could provide two potential targets for the development of antiviral drugs to suppress A3G and A3F degradation. Our goal is to understand the structure and function of Vif and A3 proteins. We will gain insights into the structures of Vif:A3 complexes through mutational and comparative analyses and generate reagents for structural studies. ___Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of A3G, we developed novel self-activating lentiviral vectors that efficiently deliver an HIV-1 Vif-resistant A3G-D128K mutant to target cells. To circumvent A3G expression in virus-producing cells, which diminishes virus production, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, homologous recombination between the direct repeats reconstituted an active A3G-D128K in 88-98% of transduced cells. Feasibility of human gene therapy was supported by 30% transduction of CD34+ hematopoietic stem and progenitor cells. A3G-D128K expression in T-cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of HIV-1 subtypes by C-to-U deamination, leading to lethal G-to-A hypermutation and inhibition of reverse transcription. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for 3.5 months without emergence of detectable resistant virus, suggesting a high genetic barrier for evolution of A3G-D128K resistance. These studies provide a proof-of-principle that A3G-D128K gene therapy is potentially a viable strategy to achieve a functional cure for HIV-1. ___Recent studies have shown that HIV-1 Vif interacts with host factor CBFbeta and that this interaction is critical for Vif-mediated degradation of A3 proteins. It was thought that the Vif-CBFbeta interaction increases the stability of Vif, which facilitates its interactions with cullin5-RBX2-ubiquitin ligase complex that are needed for inducing degradation of A3 proteins. In collaboration with Yong Xiong (Yale University), we determined the structure of a complex of Vif, CBFbeta, and A3F C-terminal domain, and unexpectedly found that A3F directly interacts with CBFbeta. The in vivo significance of the A3F-CBFbeta interaction was established by showing that mutations in CBFbeta prevent Vif-mediated degradation of A3F and that compensatory mutations that restore the interactions also restore A3F degradation. ___In addition to HIV-1 group M, the primary HIV-1 group responsible for the AIDS epidemic, HIV-1 groups N, O, and P have been shown to infect humans on rare occasions. We have characterized the Vifs from these four groups and found that some groups can induce degradation of A3G mutant D128K, which is resistant to degradation by group M Vif. Through mutational analyses, we have identified mutations in the N-terminal region of group M Vif that confer partial ability to induce degradation of the D128K mutant of A3G. These studies have helped to define the interactions between Vif and A3G that are critical for Vif's ability to overcome the A3G restriction. ___In collaboration with Hiroshi Matsuo (Leidos Biomedical Research, Inc., Frederick National Laboratory), we determined the structure of the C-terminal catalytic domain (CTD) of A3G in complex with a single-strand DNA (ssDNA) substrate. To overcome weak DNA-binding affinity between A3G and the CTD, we generated a catalytically active variant of A3G-CTD that binds ssDNA stronger than wild type. This A3G-CTD variant was co-crystallized with a 9-nucleotide ssDNA containing a 5'-TCCCA target sequence with all 9 nucleotides well resolved in the structure. The nucleotides within the 5'-TCCCA target sequence show numerous interactions with A3G-CTD, explaining the nucleotide specificity preferences. Furthermore, the backbone architecture of the protein changed upon ssDNA binding, enabling the target sequence to fit. These results provide fundamental insights into the mechanisms by which A3 proteins recognize their specific substrate sequences. ___Somatic mutations generated by A3B are common in many human cancers, but their burden varies within and between cancer types. In collaboration with Ludmila Prokunina-Olsson (Laboratory of Translational Genomics, NCI), we showed that alternative splicing of A3B (A3B) results in reduced expression of mutagenic A3B, leading to decreased A3B signature mutations. Importantly, we showed that A3B exon 5 splicing can be modulated by SF3B1 pladienolide B inhibitor leading to reduced mutagenic A3B1 protein levels. We propose that pladienolide B-based drugs may hold promise to modulate A3-mediated mutagenesis in human cancers.

APOBEC3（A3）蛋白质是先天免疫反应的成员，可防御HIV-1和其他病原体。在缺乏HIV-1蛋白VIF的情况下，将A3蛋白掺入病毒生产细胞中的病毒体中，并通过在靶细胞中逆转录过程中逆转录病毒DNA中的细胞丁基脱氨酸来抑制病毒复制，从而导致广泛的G-TO-A-TO-a-a to-a to-a-a蛋白。除了通过致命的过度突击灭活大多数病毒基因组外，我们和其他人还表明，A3G和A3F还抑制病毒DNA的合成和整合。为了克服这些宿主防御，VIF与A3蛋白结合并将其靶向蛋白酶体降解，从而阻止其掺入病毒体中。在分子水平上定义VIF与A3G和A3F的相互作用可以为开发抗病毒药物抑制A3G和A3F降解提供两个潜在的靶标。我们的目标是了解VIF和A3蛋白的结构和功能。我们将通过突变和比较分析来了解VIF：A3复合物的结构，并为结构研究生成试剂。需要___ ___策略来控制HIV-1复制而无需抗病毒疗法以实现功能治疗。为了利用A3G的先天抗病毒功能，我们开发了新型的自动慢病毒载体，这些载体有效地将HIV-1 VIF VIF A3G-D128K突变体提供给靶细胞。为了绕过降低病毒产生的病毒细胞中的A3G表达，设计了一个含有A3G-D128K的两个重叠片段的载体，该载体被设计为在病毒产生细胞中以不活跃的形式保持基因。但是，在靶细胞转导期间，直接重复之间的同源重组在88-98％的转导细胞中重新组装了活性A3G-D128K。 CD34+造血茎和祖细胞的30％转导支持人类基因治疗的可行性。 T细胞线CEM，CEMS和PM1中的A3G-D128K表达通过C-TO-U脱氨基有效地抑制HIV-1亚型的扩散感染，从而导致致命的G-TO-A TO-A超偏变和逆转录的抑制。 CEM细胞中的A3G-D128K表达有效抑制了3.5个月的HIV-1复制，而不会出现可检测的抗性病毒，这表明A3G-D128K抗性的进化具有很高的遗传障碍。这些研究提供了原则上的证明，即A3G-D128K基因治疗可能是实现HIV-1功能治疗的可行策略。 _____的研究表明，HIV-1 VIF与宿主因子CBFBETA相互作用，并且这种相互作用对于VIF介导的A3蛋白的降解至关重要。人们认为，VIF-CBFBETA相互作用提高了VIF的稳定性，这有助于其与Cullin5-RBX2-泛素蛋白连接酶复合物的相互作用，这是诱导A3蛋白降解所需的。与Yong Xiong（耶鲁大学）合作，我们确定了VIF，CBFBETA和A3F C末端域的复合体的结构，并且出乎意料地发现A3F直接与CBFBETA相互作用。通过表明CBFBETA中的突变可以防止VIF介导的A3F降解，并且恢复相互作用的补偿性突变还可以恢复A3F降解，从而确定了A3F-CBFBETA相互作用的体内意义。 ___添加HIV-1组M，是负责AIDS流行病，HIV-1组N，O和P的主要HIV-1组，在极少数情况下会感染人类。我们已经表征了这四个组的VIF，发现某些组可以诱导A3G突变体D128K的降解，这是M VIF组抗性的降解。通过突变分析，我们已经确定了M VIF组的N末端区域中的突变，从而赋予了诱导A3G D128K突变体降解的部分能力。这些研究有助于定义VIF和A3G之间的相互作用，这对于VIF克服A3G限制的能力至关重要。 ___在与弗雷德里克国家实验室（Frederick National Laboratory）的Hiroshi Matsuo（Leidos Biomedical Research，Inc。）合作，我们确定了与单链DNA（SSDNA）底物在复合体中A3G的C末端催化域（CTD）的结构。为了克服A3G和CTD之间的弱DNA结合亲和力，我们生成了A3G-CTD的催化活性变体，该变体结合了比野生型强的ssDNA。该A3G-CTD变体与9-核苷酸ssDNA共结晶，该核苷酸ssDNA包含5'-TCCCA靶序序，所有9个核苷酸都在结构中很好地分辨出来。 5'-TCCCA目标序列中的核苷酸与A3G-CTD相互作用，这解释了核苷酸特异性偏好。此外，该蛋白质的骨干结构在ssDNA结合后发生了变化，使靶序序拟合。这些结果提供了对A3蛋白识别其特定底物序列的机制的基本见解。 ___Somatic mutations generated by A3B are common in many human cancers, but their burden varies within and between cancer types.与Ludmila Prokunina-Olsson（转化基因组学实验室，NCI）合作，我们表明A3B（A3B）的替代剪接导致诱变A3B的表达降低，从而导致A3B签名突变减少。重要的是，我们表明A3B外显子5剪接可以由SF3B1 pladienolide B抑制剂调节，从而导致诱变A3B1蛋白水平降低。我们建议基于Pladienolide B的药物可能有望调节人类癌症中A3介导的诱变。