Beck Z Q, Lin Y C, Elder J H
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Virol. 2001 Oct;75(19):9458-69. doi: 10.1128/JVI.75.19.9458-9469.2001.
We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.
我们使用随机六聚体噬菌体文库来描绘1型人类免疫缺陷病毒（HIV-1）和猫免疫缺陷病毒（FIV）蛋白酶（PR）底物特异性之间的异同。鉴定出了每种蛋白酶特异性切割的肽序列，以及两种酶切割效果相同的序列。基于底物P3-P3'区域内似乎与这些切割特异性相关的氨基酸差异，我们在HIV-1和FIV PR切割效率基本相同的肽序列框架内制备了一系列合成肽，Ac-KSGVF/VVNGLVK-NH₂（箭头表示切割位点）。我们使用所得的肽组来评估特定氨基酸取代对两种蛋白酶切割特性的影响。研究结果表明，当P2位置的Val被Asn取代时，HIV-1 PR切割底物的速率比FIV PR快得多。同样，P2'位置的Val被Glu或Gln取代也会产生对HIV-1 PR特别敏感的肽。相反，当P1'位置的Val被Ser取代时，FIV PR切割底物的速率比HIV-1 PR高得多。此外，P1位置的Asn或Gln与合适的P3氨基酸Arg组合，也强烈有利于FIV PR而非HIV PR的切割。结构分析确定了几个可能决定观察到的特异性差异的蛋白酶残基。有趣的是，影响S2和S2'亚位点特异性的HIV PR Asp30（FIV PR中的Ile-35），以及影响S1和S1'亚位点特异性的HIV-1 PR Pro-81和Val-82（FIV PR中的Ile-98和Gln-99），是HIV-1蛋白酶耐药性发展中经常涉及的残基。两种PR都能切割的肽底物KSGVF/VVNGK被用作设计还原酰胺抑制剂Ac-GSGVF Psi(CH₂NH)VVNGL-NH₂的模板。该化合物对FIV和HIV-1 PR的抑制效率大致相同。这些发现为人和猫慢病毒PR底物特异性差异建立了分子基础，并为开发高效的广谱抑制剂提供了框架。
