Production Of HIV And HIV Related Proteins For Structural Studies

用于结构研究的 HIV 和 HIV 相关蛋白的生产

• 批准号: 10018384
• 负责人: PAUL T WINGFIELD
• 金额: $ 92.68万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 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/10018384
• 关键词: 3-Dimensional; Affinity; Amino Acids; Antibodies; Antiviral Agents; Architecture; Bacteria; Binding; Binding Sites; Biological Assay; Biosynthetic Proteins; C-terminal; Cell Nucleus; Cells; Chromatin; Complex; Cryoelectron Microscopy; Crystallization; Cyclic Peptides; Cysteine; DNA; Data; Disease; Drug Design; Drug Screening; Drug Targeting; Electron Microscopy; Engineering; Filament; Fluorescent Probes; Genome; HIV; HIV-1; Histones; Human; Immunoglobulin Fragments; Immunoglobulin Variable Region; Label; Lead; Leg; Life Cycle Stages; Measures; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Molecular Weight; Monitor; Multidimensional NMR Techniques; N-terminal; NPM1 gene; Nuclear Export; Nuclear Magnetic Resonance; Nucleosomes; Oryctolagus cuniculus; Peptides; Phage Display; Positioning Attribute; Preparation; Process; Production; Proteins; RNA; RNA Conformation; RNA Folding; RNA Sequences; Recombinant DNA; Resolution; Response Elements; Roentgen Rays; Role; Shapes; Site; Structural Protein; Structure; System; Therapeutic Intervention; Time; Translating; Viral; Viral Proteins; Virus; Virus Replication; Work; X ray diffraction analysis; X-Ray Crystallography; antibody libraries; aptamer; arm; base; chimeric antibody; density; design; dimer; high throughput screening; improved; interest; novel; nucleophosmin; particle; polymerization; prevent; protein structure; rev Protein; stable isotope; targeted treatment; three dimensional structure; time use; trafficking; viral RNA

In uninfected cells, RNA is transcribed from DNA, processed, and then transported out of the nucleus and translated into protein. In cells infected with HIV-1, the viral RNA genomes must be exported out of the nucleus without being processed so they can be packaged into new viral particles. To do this the cell must bind its own RNA genome from among the host RNA in the nucleus. This is achieved using the HIV-1 Rev protein that recognizes a Rev response element (RRE) in the viral RNA. Once bound to RRE, Rev self-associates and binds other host proteins, forming a multiprotein-RNA complex that is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. In addition to contributing fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability that may be suitable targets for therapeutic intervention including Rev itself. A picture of how Rev binds to RRE has come from our previous structural studies of both Rev and the RRE. The structure of the N-terminal half of dimeric Rev (the region involved in RNA interaction) was solved for first time by using an antibody fragment (Fab) as a crystallization chaperone. The RRE RNA forms an A shape with one leg shorter than the other. The legs are about 55 Angstrom part and position the known binding sites for Rev on either arm of the A. The higher affinity binding site is on the lower part of the short arm and the lower affinity site is on the lower part of the longer arm, placing them about 55 Angstrom from each other. This spacing matches the interaction domains of the Rev dimer that are also about 55 Angstrom apart. Once bound to RRE, Rev oligomerizes forming a complex that engages with the host nuclear export machinery. The oligomerization of Rev on RRE is essential for formation of an active nuclear export complex. To study this protein association, filaments were generated from the soluble Rev dimers and their structure was determined by high resolution electron microscopy incorporating X-ray data from the N-terminal domain of Rev dimers. Our data revealed a third interface between Rev which offers an explanation for how the arrangement of Rev subunits adapts to the A-shaped architecture of the RRE in the export-active complexes. Also, the structures contained additional density indicating that C-terminal domains (CTD) become partially ordered in the context of filaments. This is the first time structural information on the Rev CTD has been acquired as this domain is disordered in the crystals used for X-ray determinations. Further studies are required to determine in more detail the structure of the export-competent ensemble to expand our understanding of HIV-1 Rev's key role in the nuclear export of viral mRNA. We have focused first on Rev RNA interactions. Using a shortened and non-polymerizing form of Rev that incorporates amino acid residues 1-93 (wild type Rev is 1-115) and is further stabilized with a single chain variable fragment (scFv) antibody, we have prepared complexes with various RNA preparations corresponding to regions of the RRE. Also we have used RNA aptamers, which are RNAs that fold into 3-dimensional conformations that bind to their targets (in this case Rev). Aptamers that bind with higher affinity than Rev-binding sites on RRE have potential anti-HIV activity. We have recently determined a high resolution structure by X-ray crystallography of Rev with a high affinity binding aptamer. In this structure, dimeric Rev bridges two discontinuous aptamers, suggesting when it binds to RRE the Rev dimer is binding two RNA sequences co-localized by the RNA conformation. Using the Rev 1-93 - scFv as a proven crystallization platform we are extending structural studies to solve interactions with other high affinity aptamers. We are also studying other Rev complexes including with the nucleosome assembly protein 1 (Nap-1) which is a major histone chaperone involved in chromatin formation. Nap-1 binds to Rev and appears to mediate nuclear export of Rev. The structure of Nap-1 in complex with Rev has been solved at low resolution by X-ray crystallography and shows that Nap-1 forms a tetramer of dimers. Attempts to increase the resolution are being made by cryo-EM analysis. In a similar manner, Rev interactions with B23/nucleophosmin are of great interest. B23 has diverse molecular functions including roles in nucleo-cytoplasmic trafficking. As B23 is also targeted by a number of other viral proteins, the structural analyses of its binding sites may lead to novel anti-viral drugs. We have made crystals of complexes of B23 with Rev that diffract weakly but at high resolution. Further structural analysis by cryo-EM is planned. The antibody fragment (Fab) used for stabilizing Rev for structural studies was derived from a phage display antibody library. This chimeric antibody (human framework and rabbit variable domains), expressed in bacteria, was humanized and was effective by binding to Rev with a very high affinity, thereby preventing its oligomerization. In previous work we showed that this antibody had anti-HIV-1 activity. We also showed that cyclic peptides (up to 12 amino acids long) from the antibody variable regions (CDRs) could bind to Rev but we have not yet shown whether they also have anti-HIV-1 activity. In addition, we are attempting to co-crystallize the peptides with Rev in order to obtain high-resolution structures of the complexes, which may help design or model low-molecular weight mimics with improved (stronger) binding to Rev. In a parallel approach to targeting Rev, we are using the fact that polymerization or self-association of Rev is required for function and hence is a target for drug screening. As a first step we are developing assays that can be used to measure Rev self-association and then applied to high-throughput screening where compounds that block Rev-Rev interactions can be rapidly identified. To develop robust assay we have engineered Rev to include site-specific cysteine residues for introducing site -specific fluorescent probes, which will allow sensitive monitoring.

在未感染的细胞中，RNA从DNA转录，处理，然后从细胞核中运出并转化为蛋白质。在感染HIV-1的细胞中，必须将病毒RNA基因组从细胞核中输出而不会被处理，以便可以将它们包装到新的病毒颗粒中。为此，细胞必须从核中的宿主RNA中结合其自己的RNA基因组。这是使用HIV-1 REV蛋白来实现的，该蛋白识别病毒RNA中的Rev Respent元件（RRE）。一旦与RRE结合，REV会自缔合并结合其他宿主蛋白，形成从细胞核中导出的多蛋白-RNA复合物。我们目前的研究旨在描述该复合物的分子细节。除了贡献有关病毒复制机制的基本信息外，这些研究还可能突出脆弱性点，这可能是包括Rev本身在内的治疗干预措施的合适靶标。 RE与RRE结合的图片来自我们先前对REV和RRE的结构研究。首次使用抗体片段（FAB）作为结晶伴侣，首次解决了Dimeric Rev（RNA相互作用所涉及的区域）的N末端一半的结构。 RRE RNA形成一个比另一个腿短的A形。腿约为55埃绳肌，并定位A上的Rev的已知结合位点。较高的亲和力结合位点位于短臂的下部，下部亲和力位点位于较长臂的下部，将它们彼此放置约55埃。该间距与Rev Dimer的相互作用域相匹配，Rev Dimer的相互作用域也大约是55盎司。一旦绑定到RRE，Rev Rev会形成与宿主核出口机械接合的复合物。 RER在RRE上的低聚对于形成活跃的核出口复合物至关重要。为了研究这种蛋白质的关联，从可溶的Rev Dimers产生了细丝，它们的结构是由高分辨率电子显微镜确定的，这些电子显微镜掺入了Rev Dimers的N末端域中的X射线数据。我们的数据揭示了REV之间的第三个接口，该接口提供了解释Rev亚基的布置如何适应RRE在出口活性复合物中的A形架构。同样，结构包含额外的密度，表明C末端域（CTD）在细丝的背景下被部分排序。这是Rev CTD的首次获得结构信息，因为该域在用于X射线测定的晶体中是无序的。需要进一步的研究更详细地确定出口能力集合的结构，以扩展我们对HIV-1 Rev在病毒mRNA核输出中的关键作用的理解。 我们首先关注RNA相互作用。使用缩短和非聚合形式的Rev形式，该形式结合了氨基酸残基1-93（野生型Rev为1-115），并用单个链可变片段（SCFV）抗体进一步稳定，我们已经准备了与RRE区域相对应的各种RNA制剂。另外，我们还使用了RNA适体，它们是折叠成3维构象的RNA（在这种情况下为Rev）。与RRE上的Rev-Binding位点结合的适体具有潜在的抗HIV活性。我们最近通过具有高亲和力结合适体的Rev的X射线晶体学确定了高分辨率结构。在这种结构中，Dimeric Rev桥接了两个不连续的适体，这表明它与RRE时，Rev Dimer何时结合了通过RNA构象共定位的两个RNA序列。使用REV 1-93 -SCFV作为一个经过验证的结晶平台，我们正在扩展结构研究，以解决与其他高亲和力适体的相互作用。 我们还研究了其他REV复合物，包括核小体组装蛋白1（NAP-1），该蛋白1（NAP-1）是参与染色质形成的主要组蛋白伴侣。 NAP-1与REV结合，并似乎介导了Rev的核出口。与Rev的NAP-1结构通过X射线晶体学以低分辨率解决了REV的结构，并表明NAP-1形成了二聚体的四聚体。通过冷冻EM分析进行提高分辨率的尝试。以类似的方式，与B23/核磷脂的Rev相互作用引起了极大的关注。 B23具有不同的分子功能，包括在核总质运输中的作用。由于B23也受许多其他病毒蛋白的靶向，因此其结合位点的结构分析可能导致新型的抗病毒药物。我们使B23复合物的晶体具有微弱衍射但高分辨率的Rev。计划通过冷冻EM进行进一步的结构分析。 用于稳定结构研究的REV的抗体片段（FAB）源自噬菌体显示抗体库。这种嵌合抗体（人类框架和兔可变结构域）在细菌中表达，是人性化的，并且通过具有很高亲和力的Rev结合而有效，从而防止其低聚。在先前的工作中，我们表明该抗体具有抗HIV-1活性。我们还表明，来自抗体可变区域（CDR）的环状肽（长达12个氨基酸长）可以与REV结合，但我们尚未证明它们是否也具有抗HIV-1活性。此外，我们正在尝试将肽与Rev共结合，以获得复合物的高分辨率结构，这可能有助于设计或模拟低分子量的模拟模拟物，并在靶向Rev的平行方法中改善（更强的）与Rev.的结合（更强），我们使用的事实是，对于功能和HECENENINIP的REV是必不可少的事实，是对型和nefforting of Forgation and Fornation和Heceenting sexenenting和Heceenting screening ancenenting ancenting ancenting ancenenting ancenenting ancenenting ancenenting。作为第一步，我们正在开发可用于测量Rev自我关联的测定方法，然后应用于高通量筛选，其中可以快速识别阻断Rev-Rev相互作用的化合物。为了开发强大的测定，我们已经设计了REV，以包括用于引入特定地点的特定荧光探针的位点特异性半胱氨酸残基，这将允许敏感监测。