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，加工，然后运输出细胞核，翻译成蛋白质。在被HIV-1感染的细胞中，病毒RNA基因组必须不经加工就被输出出细胞核，这样它们才能被包装成新的病毒颗粒。要做到这一点，细胞必须从细胞核中的宿主RNA中结合自己的RNA基因组。这是通过识别病毒RNA中的Rev应答元件（RRE）的HIV-1 Rev蛋白实现的。一旦与RRE结合，Rev就会自我结合并结合其他宿主蛋白，形成从细胞核输出的多蛋白- rna复合物。我们目前的研究旨在描述这种复合物的分子细节。除了提供关于病毒复制机制的基本信息外，这些研究还可能突出易感性的点，这些易感性可能是包括Rev本身在内的治疗干预的合适靶点。