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。它的功能是与病毒转录本结合，并从未贴花mRNA的核中导出，从而允许产生结构蛋白。该结构仅由于蛋白质降低和聚集的趋势而被确定。抗体抗原结合结构域（FAB和SCFV）可以介导难治性蛋白的共结晶。产生了蛋白抗体复合物，产生了由X射线晶体学确定Rev结构的晶体。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潜力，因为它可以防止生物活性所需的Rev自我关联。抗Rev Fab抗体内部化为感染细胞时显示出较高的抗HIV活性。与Rev结合的抗体的晶体结构也用于指导小肽抑制剂的选择。循环肽（长度为12-18个残基），其中N-末端和C末端与模仿父抗体蛋白中的环结构相关联。这些研究可能会导致针对HIV-1 REV蛋白的小分子，因此靶向HIV-1 REV蛋白，因此具有宝贵的替代治疗抗HIV治疗。同样，正在研究REV与RNA和附件蛋白的功能相互作用，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，一种同二聚体蛋白在病毒生命周期和主要抗HIV药物靶标中至关重要。我们已经根据多药耐药的临床HIV-1分离株表达并纯化了许多蛋白酶的抗药性形式。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株结合的新型药物。