Structure and Function of Vif and APOBEC3 (A3) Proteins

APOBEC3 （A3）蛋白是先天免疫反应的成员，提供对HIV-1和其他病原体的防御。在缺乏HIV-1蛋白Vif的情况下，A3蛋白被整合到病毒产生细胞中的病毒粒子中，并在靶细胞的逆转录过程中通过脱氨病毒DNA负链中的胞苷抑制病毒复制，导致病毒基因组广泛的G-to-A高突变。除了通过致死性超突变使大多数病毒基因组失活外，我们和其他人已经证明A3G和A3F也抑制病毒DNA的合成和整合。为了克服这些宿主防御，Vif与A3蛋白结合，并将它们作为蛋白酶体降解的目标，阻止它们与病毒粒子结合。在分子水平上确定Vif与A3G和A3F的相互作用，可以为开发抑制A3G和A3F降解的抗病毒药物提供两个潜在靶点。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配合物的结构，并生成用于结构研究的试剂。需要在没有抗病毒治疗的情况下控制HIV-1复制的策略来实现功能性治愈。为了利用A3G的先天抗病毒功能，我们开发了一种新的自激活慢病毒载体，有效地将HIV-1抗vif的A3G- d128k突变体传递到靶细胞。为了避免A3G在病毒产生细胞中的表达，从而减少病毒的产生，设计了一个包含两个重叠的A3G- d128k片段的载体，使该基因在病毒产生细胞中保持无活性形式。然而，在靶细胞转导过程中，直接重复序列之间的同源重组在88-98%的转导细胞中重建了一个活性的A3G-D128K。30%的CD34+造血干细胞和祖细胞转导支持了人类基因治疗的可行性。A3G-D128K在t细胞系CEM、CEMSS和PM1中的表达可通过C-to-U脱胺抑制HIV-1亚型的传播感染，导致致命的G-to-A高突变和逆转录抑制。在CEM细胞中表达A3G-D128K可有效抑制HIV-1复制3.5个月而未出现可检测到的耐药病毒，这表明A3G-D128K耐药进化具有很高的遗传屏障。这些研究提供了一个原理证明，A3G-D128K基因治疗是实现HIV-1功能性治愈的潜在可行策略。最近的研究表明HIV-1 Vif与宿主因子cbfβ相互作用，这种相互作用对Vif介导的A3蛋白降解至关重要。人们认为Vif- cbfβ相互作用增加了Vif的稳定性，从而促进了Vif与cullin5- rbx2 -泛素连接酶复合物的相互作用，而cullin5- rbx2 -泛素连接酶复合物是诱导A3蛋白降解所必需的。我们与熊勇（耶鲁大学）合作，确定了Vif、cbf β和A3F c端结构域的复合物结构，意外地发现A3F直接与cbf β相互作用。A3F- cbf - β相互作用的体内意义是通过cbf - β突变阻止vif介导的A3F降解和恢复相互作用的代偿性突变也恢复A3F降解而建立的。除了导致艾滋病流行的主要HIV-1组M外，HIV-1组N、O和P在极少数情况下也会感染人类。我们对这四组Vif进行了表征，发现一些组可以诱导A3G突变体D128K的降解，该突变体对M组Vif的降解具有抗性。通过突变分析，我们已经确定了M Vif组n端区域的突变，这些突变赋予了诱导A3G的D128K突变体降解的部分能力。这些研究有助于确定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。我们与Hiroshi Matsuo （Leidos Biomedical Research, Inc.， Frederick国家实验室）合作，确定了A3G与单链DNA （ssDNA）底物配合物中c端催化结构域（CTD）的结构。为了克服A3G和CTD之间较弱的dna结合亲和力，我们产生了一种催化活性的A3G-CTD变体，其结合ssDNA的能力比野生型强。该A3G-CTD变体与含有5'-TCCCA靶序列的9核苷酸ssDNA共结晶，所有9个核苷酸在结构中都被很好地分解。5'-TCCCA靶序列内的核苷酸与A3G-CTD表现出许多相互作用，解释了核苷酸特异性偏好。此外，在ssDNA结合后，蛋白质的骨干结构发生了变化，使目标序列能够匹配。这些结果为A3蛋白识别其特定底物序列的机制提供了基本的见解。由A3B产生的体细胞突变在许多人类癌症中很常见，但是它们的负担在癌症类型内部和不同类型之间是不同的。与NCI翻译基因组学实验室的Ludmila Prokunina-Olsson合作，我们发现A3B （A3B）的选择性剪接导致诱变A3B的表达减少，导致A3B特征突变减少。重要的是，我们发现A3B外显子5剪接可以通过SF3B1铂烯内酯B抑制剂调节，从而降低致突变的A3B1蛋白水平。我们提出以铂烯内酯b为基础的药物可能有望调节人类癌症中a3介导的突变。

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.