Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants
Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants
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DOI：
10.1074/jbc.271.30.17979
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发表时间：
1996-07-26
影响因子：
4.8
通讯作者：
Livingston, DJ
中科院分区：
文献类型：
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作者：
Pazhanisamy, S;Stuver, CM;Livingston, DJ
人类免疫缺陷病毒1型(HTV-1)在羟乙氨基磺胺抑制剂VX-478或VB-11328浓度增加的情况下在T淋巴细胞系中的传代导致HIV1蛋白酶突变的顺序积累。我们鉴定了含有这些突变的重组HTV-1蛋白酶(L10F、M46I、I47V、I50V)或组合(双突变体L10F/I50V和三个突变体M46I/I47V/I50V)。对磺胺类抑制剂和其他类抑制剂的催化性能和亲和力进行了测定，对于I50V突变体，设计用于模拟HIV-1 Gag-Pol多肽中裂解连接的多肽的处理效率(k(Cat)/K-m)降低了25倍。对于含有Phe/Pro和Tyr/Pro裂解位点的多肽底物，三个突变体的处理效率是I50V的1倍，这表明M46I和I47V突变是代偿性的，突变对处理效率的影响与抑制常数(K-I)相结合来评估突变对病毒复制的优势。这些分析支持病毒学观察，即在I50V突变背景上添加M46I和I47V突变能够提高HIV-1病毒在VX-478存在下复制时的存活率，HTV-1蛋白酶突变体活性部位的晶体结构和分子模型表明，活性部位的变化可以选择性地影响抑制剂的结合能，而底物结合几乎没有相应的变化。
Passage of human immunodeficiency virus type-1 (HTV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV 1 protease, We have characterized recombinant HTV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined, For the I50V mutant, the efficiency (k(cat)/K-m) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a a-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory, The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (K-i) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478, Crystal structures and molecular models of the active site of the HTV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.