Production Of HIV And HIV Related Proteins For Structural Studies

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。尽管其重要性，但由于蛋白质聚集的趋势，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。最近的更高分辨率的Rev结构被用于模拟蛋白质-蛋白质相互作用或自缔合，这对Rev作用至关重要。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。来自HIV-1蛋白酶二聚体界面的N末端和C末端区域的肽通过阻止二聚化（单体蛋白无活性）来抑制蛋白酶活性。在先前的工作中，已经表明通过连接HIV-Tat的转导结构域，肽的溶解性和细胞渗透性得到增强。产生了一种遗传构建体，其可用于研究HIV-1蛋白酶Gag-Pro-Pol加工级联中的第一步，并显示半胱氨酸的可逆氧化可在该初始步骤控制HIV-1蛋白酶的活性。该测定系统还具有筛选潜在二聚化抑制剂的潜力。

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. Despite its importance, the structure has only been recently determined due to the tendency of the protein to 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 more recent higher resolution structure of Rev is being used to model the protein-protein interactions or self-associations which are essential for Rev action. 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. Peptides derived from the N- and C-terminal regions of the HIV-1 protease dimer interface inhibit protease activity by preventing dimerization (monomeric protein is inactive). In previous work, it was shown that the solubility and cell permeability of the peptides was enhanced by linking the transduction domain of HIV-Tat. A genetic construct was generated that can be used to study the first step in the cascade of HIV-1 protease Gag-Pro-Pol processing and show that reversible oxidation of cysteines can control the activity of the HIV-1 protease at this initial step. This assay system also has the potential for the screening of potential dimerization inhibitors.