Conserved cysteines of the human immunodeficiency virus type 1 protease are involved in regulation of polyprotein processing and viral maturation of immature virions
Conserved cysteines of the human immunodeficiency virus type 1 protease are involved in regulation of polyprotein processing and viral maturation of immature virions
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DOI：
10.1128/jvi.73.2.1156-1164.1999
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发表时间：
1999-02-01
影响因子：
5.4
通讯作者：
Yarchoan, R
中科院分区：
文献类型：
--
作者：
Davis, DA;Yusa, K;Yarchoan, R
我们研究了人类免疫缺陷病毒1型（HIV-1）蛋白酶的两个高度保守的半胱氨酸残基，半胱氨酸67和95，在病毒成熟过程中调节蛋白酶活性的作用。为此，我们产生了四个HIV-1分子克隆：野生型，含有两个半胱氨酸残基;蛋白酶突变体，其中67位的半胱氨酸被丙氨酸（C67 A）取代; C95 A蛋白酶突变体;和双突变体（C67 A C95 A）。当在HIV-1蛋白酶抑制剂KNI-272的存在下产生不成熟病毒体，并且随后去除抑制剂时，在20小时的时间内观察到野生型病毒体制备物的有限的多蛋白加工。用还原剂二硫苏糖醇处理不成熟野生型病毒体显著改善了Gag加工的速率和程度，表明蛋白酶部分地，通过半胱氨酸残基的氧化可逆失活。为了支持这一点，在不存在还原剂的情况下，C67 A和C95 A病毒体处理Gag的速度比野生型病毒体快5倍。此外，氧化剂，如H2 O2和二酰胺，抑制Gag加工的野生型病毒体，这种效果是依赖于半胱氨酸95的存在。电子显微镜检查显示，更大比例的双突变体病毒体比野生型病毒体开发一个成熟样形态的抑制剂去除。这些研究提供的证据表明，在正常培养条件下，HIV-1蛋白酶的半胱氨酸在病毒成熟过程中易受氧化，从而阻止未成熟病毒体在释放后进行完全加工。这与半胱氨酸参与氧化应激下细胞中病毒成熟的调节是一致的。
We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period, Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H2O2 and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.