CAT/MOUSE MODELS TO EVALUATE PR-TARGETED ANTIVIRALS

FIV在家猫中引起艾滋病样综合征， 代表了研究慢病毒的最小自然动物系统 感染.该蛋白酶（PR）与以下蛋白酶的PR具有实质性的结构同源性： 但HIV-1具有独特的底物和抑制剂特性，其中一些是 与耐药HIV的PR相同。迄今为止的研究已经确定 FIV和HIV-1 PR的关键氨基酸残基，其负责 已经制备了观察到的底物/抑制剂特异性和突变体FJV 显示HIV样底物/抑制剂特异性。这些资料已经 用于开发一种广泛的抑制剂（称为TL-3）， 在体外抗FIV和HIV-1 PR以及在组织培养中抗病毒感染。 我们建议通过鉴定进一步定义底物/抑制剂相互作用， 耐药HIV-1和FIV逃逸的结构变化 突变体和建立蛋白酶突变性的限制。初始实验 已经鉴定出一种突变型HIV，其对TL-3的抗性增加了19倍。的 该分离株的PR以及野生型和其他耐药PR已被 亚克隆，在E. coli中，并纯化用于酶分析。 这些PR将与噬菌体展示文库结合使用， 选择耐药变体PR首选的底物， 将鉴定切割序列。获得的信息可用于 制备对耐药PR具有更高选择性的抑制剂。共识 将鉴定出代表最佳切割靶的序列， FIV和HIV-I PR的广谱性，其可用作制备 对多种FIV和HIV有效的广谱抑制剂 变体。将在组织培养物和体内进行试验， FIV（在猫中）和HIV-1（在NOD/SCID小鼠中），以监测抑制剂疗效， 测试相对病毒适合度和耐药性的发展。这些 研究将促进我们对药物结构基础的理解， 耐药性，并导致开发有效的抑制剂， 通过PR内的突变逃逸。

FlV causes an AIDS-like syndrome in the domestic cat and represents the smallest natural animal system for study of lentivirus infections. The protease (PR) shares substantial structural homology with PR of HIV-1 but has unique substrate and inhibitor properties, some of which are shared with PRs of drug-resistant HIVs. Studies to date have identified critical amino acid residues of FlV and HIV-1 PRs that are responsible for the observed substrate/inhibitor specificities and mutant FJVs have been prepared that show HIV-like substrate/inhibitor specificities. This information has been used to develop a broad-based inhibitor (termed TL-3) that is efficacious against both FlV and HIV-l PRs in vitro and virus infection in tissue culture. We propose to further define substrate/inhibitor interactions by identifying structural changes responsible for escape by drug-resistant HIV- I and FIV mutants and establishing the limits of protease mutability. Initial experiments have identified a mutant HIV with a 19-fold increase in resistance to TL-3. The PR of this isolate as well as wild-type and other drug resistant PRs have been sub-cloned, over-expressed in E. coli, and purified for enzymatic analyses. These PRs will be employed in conjunction with phage display libraries to select for substrates preferred by drug resistant variant PRs and optimal cleavage sequences will be identified. The information gained can be used to prepare inhibitors with greater selectivity for drug-resistant PRs. Consensus sequences will be identified that represent optimal cleavage targets for a broad spectrum of F1V and HIV-l PRs, which can be used as frameworks to prepare broad-based inhibitors efficacious against a wide range of FIV and HIV variants. Tests will be performed in tissue culture and in vivo against both FIV (in cats) and HIV-1 (in NOD/SCID mice) to monitor inhibitor efficacy and to test for relative viral fitness and development of drug resistance. These studies will advance our understanding of the structural basis for drug resistance and lead to development of effective inhibitors less susceptible to escape via mutations within PR.