Structural basis for drug resistance in HIV and FIV PRs

拟议的研究涉及使用FIV和HIV进行比较研究，以调查耐药性的发展和底物专一性，确定耐药性发展的时间进程和结构基础，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。编码嵌合蛋白酶(PR)的FIV将被产生，这些突变具有与HIV-1 PR相似的药物敏感性和底物特异性，用于针对一组特定的多肽底物和噬菌体文库进行分析，以及用于体外感染。将监测感染的进展，以研究在单一和多个药物选择下药物敏感性/耐药性发展的分子过程。嵌合PR对HIV多肽底物的切割将用于识别与HIV底物特异性相关的残基，先前的研究表明，这些残基不是导致当前药物耐药的主要残基。此外，还将评估在存在和不存在PI的情况下HIV PR的体外进化，所获得的信息将为FIV研究提供信息。其目的是：1)制备一组编码HIV PR分子的感染性FIV，其中FIV PR的多个残基已被HIV PR的等量残基取代。将对病毒后代进行表型、基因和结构分析，并使用体外GAG处理试验评估底物特异性变化；2)继续使用新型PR抑制剂产生一组PR耐药突变株。野生型和抗药性艾滋病毒之间的体外竞争实验将作为相对病毒适合性的衡量标准进行。反过来，这些HIV PR将确定在目标1中用于嵌合PR研究的FIV PR中需要改变的额外残基。最后，将对选定的FIV和HIV耐药PR进行结构评估，以评估完全耐药PR的蛋白酶抑制物(PI)效力的丧失和酶功能的改变。这些研究的结果将为了解PR在导致病毒抗药性和功能改变的残基变化方面的功能可塑性提供见解。 ?？ ?？ ?？ ?？ ARRA请求：1 R01 AI081585-01A2 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.