Production Of HIV And HIV Related Proteins For Structural Studies

Rev是HV-1的关键调控蛋白。它的功能是与病毒转录物结合，并影响未剪接mRNA的细胞核输出，从而允许结构蛋白的产生。这种结构是最近才确定的，因为这种蛋白质倾向于寡聚和聚集。抗体抗原结合域（Fab和scFv）可以介导难熔蛋白的共结晶。制备了蛋白质抗体复合物，产生晶体，并用x射线晶体学测定了Rev的结构。在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，因为它可以阻止生物活性所需的Rev自我结合。Anti-Rev Fab抗体内化到感染细胞后显示出较高的抗hiv活性。结合Rev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。环状肽（长度为12- 18个残基），其中n端和c端连接，模仿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抗hiv治疗替代方法。此外，Rev与RNA和辅助蛋白的功能相互作用也正在研究中，目的是收集结构信息，这可能对靶向抗hiv干预有用。

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.