Structural basis for drug resistance in HIV and FIV PRs

拟议的研究涉及使用FIV和HIV的比较研究，以调查耐药性发展和底物特异性，定义耐药性发展的时间过程和结构基础，最终目标是提供与开发广泛基础的抑制剂相关的结构信息。将产生编码嵌合蛋白酶（PR）的FIV，所述嵌合蛋白酶（PR）具有赋予与HIV-1 PR类似的药物敏感性和底物特异性的突变，用于针对一组特异性肽底物和噬菌体文库的分析以及用于离体感染。将监测感染进展，以检查在单一和多种药物选择下药物敏感性/耐药性发展的分子过程。通过嵌合PR切割HIV的肽底物将用于鉴定与HIV底物特异性相关的残基，先前的研究表明这些残基不是参与对当前药物耐药性的主要残基。此外，将评估存在和不存在PI时HIV PR的离体演变，获得的信息将为FIV研究提供信息。其目的是：1）制备一组感染性FIV编码“HIV化”PR分子，其中FIV PR的多个残基已被HIV PR的等效残基取代。将对病毒子代进行表型、基因型和结构分析，并使用离体Gag加工测定评估底物特异性变化; 2）使用新型PR抑制剂继续产生一组PR抗性突变体。将进行野生型和耐药HIV之间的离体竞争实验，作为相对病毒适应性的量度。反过来，这些HIV PR将鉴定FIV PR中要改变的其他残基，用于目的1中的嵌合PR研究。最后，将对选定的FIV和HIV耐药PR进行结构评价，以评估完全耐药PR的蛋白酶抑制剂（PI）效力损失和酶功能改变。这些研究的结果将提供洞察PR的功能可塑性方面的残基变化，导致耐药性和病毒功能的改变。 ?? ?? ?? ?? ARRA请求：1 R 01 AI 081585 - 01 A2 Elder，John H.

The proposed research involves comparative studies using both FIV and HIV to investigate drug resistance development and substrate specificity, defining the time-course and structural basis for resistance development with the ultimate goal of providing structural information relevant to developing broad-based inhibitors. FIVs encoding chimeric protease (PR) with mutations that impart drug sensitivities and substrate specificities similar to HIV-1 PR will be generated for analysis against a panel of specific peptide substrates and phage libraries and for ex vivo infections. Progress of infections will be monitored to examine the molecular course of drug sensitivity/resistance development under single and multiple drug selections. Cleavage of peptide substrates for HIV by chimeric PRs will be used to identify residues associated with HIV substrate specificity, which previous studies have shown are not the major residues involved in resistance to current drugs. In addition, the ex vivo evolution of HIV PR in the presence and absence of PI will be assessed and information gained will inform the FIV studies. The Aims are to: 1) Prepare a battery of infectious FIV encoding "HIVinized" PR molecules in which multiple residues of FIV PR have been substituted with the equivalent residue of HIV PR. Phenotypic, genotypic, and structural analyses will be performed on virus progeny and substrate specificity changes using an ex vivo Gag processing assay will be assessed; 2) Continue to generate a panel of PR resistance mutants using novel PR inhibitors. Ex vivo competition experiments between wild type and drug-resistant HIVs will be carried out as a measure of relative viral fitness. In turn, these HIV PRs will identify additional residues to alter in FIV PR for chimeric PR studies in Aim 1. Lastly, structural evaluation of selected FIV and HIV drug-resistant PRs will be undertaken in order to assess loss of protease inhibitor (PI) potency and altered enzyme function of fully resistant PRs. Findings from these studies will provide insights into the functional plasticity of PR in regard to residue changes leading to resistance and alteration of viral function. ?? ?? ?? ?? ARRA Request: 1 R01 AI081585-01A2 Elder, John H.