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: 8559288
• 负责人: PAUL T WINGFIELD
• 金额: $ 60.16万
• 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/8559288
• 关键词: 3-Dimensional; Affinity; Anti-HIV Agents; Antibodies; Bacteria; Binding; Biological; Cell Nucleus; Cells; Clinical; Complex; Crystallization; Cyclic Peptides; Drug Delivery Systems; Drug Design; Drug resistance; Fab domain; HIV; HIV Protease; HIV-1; Immunologic Deficiency Syndromes; Intervention; Label; Lead; Length; Life Cycle Stages; Link; Mediating; Messenger RNA; Methods; Molecular Structure; Multi-Drug Resistance; Organized by Structure Protein; Parents; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Production; Proteins; RNA; Recombinant DNA; Refractory; Role; Structural Protein; Structure; Testing; Therapeutic; Transcript; Viral; Virus; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; antigen antibody binding; base; genetic regulatory protein; inhibitor/antagonist; novel; prevent; protein structure; rev Protein; small molecule; stable isotope

Rev is a key regulatory protein of HV-1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA thereby allowing the production of structural proteins. The structure was only recently determined due to the tendency of the protein to oligomerize and aggregate. Antibody antigen binding domains (Fab and scFv) can mediate co-crystallization of refractory proteins. Protein antibody complexes were produced which generated crystals from which the structure of Rev was determined by X-ray crystallography. The high affinity anti-Rev antibody used in these structural studies is also being tested for its anti HIV-1 potential as it prevents the Rev self association required for biological activity. Anti-Rev Fab antibody when internalized into infected cells showed high anti-HIV activity. The crystal structure of the antibody bound to Rev is also being used to guide the selection of small peptide inhibitors of Rev oligomerization. Cyclic peptides (12- 18 residues in length), where the N-terminus and C-terminus are linked mimicking the loop structure in the parent antibody protein, have been shown to bind to Rev. These studies may lead small molecules targeting the HIV-1 Rev protein and hence a valuable alternative therapeutic anti-HIV treatment. Also, the functional interactions of Rev with RNA and accessory proteins are being studied with the aim of gathering structural information which may be useful for targeted anti-HIV intervention. HIV protease, a homodimeric protein is essential in the viral life cycle and a major anti-HIV drug target. We have expressed and purified a number of drug resistant forms of the protease based on multi-drug-resistant clinical HIV-1 isolates. Novel drugs which bind to these isolates are being studied by co-crystallization and the crystal structures used to rationalize and optimize drug binding.

Rev是HV-1的关键调节蛋白。其功能是结合病毒转录物，并从细胞核输出未剪接的mRNA，从而产生结构蛋白。由于蛋白质具有寡聚化和聚集的趋势，其结构最近才被确定。抗体抗原结合结构域（Fab和scFv）可以介导难治蛋白的共结晶。产生蛋白质抗体复合物，其产生晶体，通过X射线晶体学确定Rev的结构。这些结构研究中使用的高亲和力抗Rev抗体也正在测试其抗HIV-1的潜力，因为它阻止了生物活性所需的Rev自缔合。抗Rev Fab抗体在内化到感染细胞中时显示出高的抗HIV活性。与Rev结合的抗体的晶体结构也被用于指导Rev寡聚化的小肽抑制剂的选择。环肽（长度为12- 18个残基），其中N-末端和C-末端连接模拟亲本抗体蛋白中的环结构，已被证明与Rev结合。这些研究可能导致靶向HIV-1 Rev蛋白的小分子，因此是一种有价值的替代治疗性抗HIV治疗。此外，Rev与RNA和辅助蛋白的功能相互作用正在研究中，目的是收集可能对靶向抗HIV干预有用的结构信息。 HIV蛋白酶是一种同源二聚体蛋白，在病毒生命周期中是必需的，也是抗HIV药物的主要靶点。我们已经表达和纯化了许多耐药形式的蛋白酶的基础上，多药耐药的临床HIV-1分离株。正在通过共结晶和用于合理化和优化药物结合的晶体结构来研究与这些分离物结合的新型药物。