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本并影响未剪接 mRNA 从细胞核的输出，从而产生结构蛋白。由于蛋白质有寡聚和聚集的趋势，该结构最近才被确定。抗体抗原结合结构域（Fab 和 scFv）可以介导难熔蛋白的共结晶。产生的蛋白质抗体复合物产生晶体，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 潜力，因为它可以阻止生物活性所需的 Rev 自缔合。 Anti-Rev Fab 抗体内化到受感染细胞中后表现出高抗 HIV 活性。与 Rev 结合的抗体的晶体结构也被用来指导 Rev 寡聚化小肽抑制剂的选择。环肽（长度为 12-18 个残基），其中 N 末端和 C 末端连接起来，模仿亲本抗体蛋白中的环结构，已被证明可以与 Rev 结合。这些研究可能会导致针对 HIV- 1 Rev 蛋白因此是一种有价值的抗 HIV 替代疗法。此外，Rev 与 RNA 和辅助蛋白的功能相互作用正在研究中，目的是收集可能有助于有针对性的抗 HIV 干预的结构信息。 HIV 蛋白酶是一种同型二聚体蛋白，在病毒生命周期中至关重要，也是主要的抗 HIV 药物靶点。我们基于多重耐药的临床 HIV-1 分离株表达并纯化了多种耐药形式的蛋白酶。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药物。