HIV Protease Activation and Viral Replication

描述（由申请人提供）：激活HIV-1蛋白酶是病毒复制的重要步骤。与所有逆转录病毒蛋白酶一样，酶的活性需要形成蛋白酶二聚体。阻断二聚化的突变会干扰蛋白酶的功能；编码无功能酶的病毒变异是异常地组装和非传染性的。在体外对抑制酶二聚化的化合物的研究也产生了类似的结果。尽管蛋白酶二聚化在所有逆转录病毒系统中具有不变的性质，但重要的生物学和结构问题仍未得到解答。首先，虽然结构研究已经确定了参与二聚体界面的残基，但特定氨基酸在促进和维持二聚体形成中的作用尚未得到检验。其次，大量的结构和生物学信息表明，在有序的蛋白酶介导的前体加工、颗粒组装和感染性之间存在着密切的联系。然而，蛋白酶二聚化和随后的酶活化对颗粒组装和感染性的确切影响尚不清楚。最后，尽管临床可用的基于底物的HIV蛋白酶抑制剂对疾病进展产生了巨大影响，但在使用这些活性位点导向化合物治疗的患者中经常出现耐药变异。迫切需要新的抑制剂设计方法来开发其他有效的治疗药物。在下面的研究中，我们将定义对维持HIV蛋白酶二聚体至关重要的相互作用，以及最终结构在病毒复制中所起的作用。此外，我们将探索通过破坏二聚体形成来抑制病毒蛋白酶的新策略。总的来说，我们的研究将为蛋白酶结构、酶功能和逆转录病毒生物学提供重要的见解。具体而言，我们将：

DESCRIPTION (provided by applicant): Activation of the HIV-1 protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activity requires the formation of protease homodimers. Mutations that block dimerization interfere with the protease function; viral variants encoding non-functional enzymes are aberrantly assembled and non-infectious. In vitro studies of compounds that inhibit the enzyme from dimerizing produce similar results. Despite the invariant nature of protease dimerization across all retroviral systems, important biological and structural questions remain unanswered. First, although structural studies have identified the residues involved in the dimer interface, the role of specific amino acids in promoting and maintaining dimer formation has not been examined. Second, a wealth of structural and biological information suggests that there is a close association between ordered, protease-mediated precursor processing, particle assembly and infectivity. However, the precise effect of protease dimerization and subsequent enzyme activation on particle assembly and infectivity is unclear. Finally, although clinically available substrate-based inhibitors of the HIV protease have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. Novel approaches to inhibitor design are urgently needed to develop additional effective therapeutic agents. In the studies described below, we will define the interactions critical for maintenance of the HIV protease dimer and the role that the final structure plays in viral replication. Further, we will explore novel strategies for inhibition of the viral protease through disrupting dimer formation. Overall, our studies will provide important insights into protease structure, enzyme function and retroviral biology. Specifically, we will: I. Define the role of individual amino acids in dimer formation. II. Characterize the effect of substitutions in the F-IIV protease dimer interface on viral replication. IlI. Characterize HIV protease activation within GagPol as a potential target for inhibition.