Induced Maturation of Human Immunodeficiency Virus
Induced Maturation of Human Immunodeficiency Virus
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DOI：
10.1128/jvi.02271-14
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发表时间：
2014-12-01
影响因子：
5.4
通讯作者：
Mueller, Barbara
中科院分区：
文献类型：
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作者：
Mattei, Simone;Anders, Maria;Mueller, Barbara
HIV-1在产生病毒的细胞的质膜上作为一个未成熟的、非感染性的颗粒聚集。病毒蛋白酶（PR）对Gag和Gag- pol多蛋白的加工激活了病毒酶，并导致病毒粒子内部剧烈的结构重排——称为成熟——这是传染性的先决条件。尽管它对病毒复制至关重要，但目前对蛋白质水解的调控以及成熟的动力学和结构中间体知之甚少。这主要是由于HIV-1的释放和成熟在单个细胞和单个细胞的颗粒释放水平上都是异步发生的。在这里，我们报告了一种基于纯化的未成熟病毒粒子的蛋白酶抑制剂（PI）冲洗的体外同步HIV-1蛋白水解的方法，从而暂时解耦病毒组装和成熟。药物洗脱导致蛋白质水解的诱导，其裂解效率与相应PR-PI复合物的关闭率相关。Gag的蛋白水解几乎完全，并产生了正确的产物，最佳半衰期（t(1/2)）接近5小时，但病毒感染性没有恢复。PI洗脱后无法获得感染性可能是由于观察到异常病毒衣壳的形成和/或与RT活性缺乏相关的逆转录酶（RT）异源二聚体加工的明显缺陷。基于我们的研究结果，我们假设多蛋白加工动力学以及未成熟颗粒组装和PR激活的紧密时间耦合对于正确的多蛋白加工和形态成熟以及因此对HIV-1感染至关重要。病毒蛋白酶将Gag和Gag- pol HIV-1多蛋白分解为其功能亚基，激活病毒酶并引起HIV-1感染所必需的主要结构重排。这种蛋白水解成熟与病毒释放同时发生，组织培养中的病毒群体在组装和成熟的所有阶段都含有颗粒，这一事实阻碍了对其动力学的研究。在这里，我们开发了一种抑制剂冲洗策略来同步激活野生型病毒中的蛋白酶。我们证明了在优化条件下完成了几乎完整的Gag处理和未成熟病毒结构的解析。然而，大多数颗粒表现出不规则的形态，Gag-Pol加工没有忠实地重建，传染性没有恢复。这些数据表明，HIV-1的成熟对加工过程的动态很敏感，而且正确的多蛋白加工需要病毒组装和PR激活之间的紧密时间联系。
HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion-termed maturation-that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of similar to 5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity.IMPORTANCECleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.