Assembly and Release of HIV-1 and Other Retroviruses

我实验室的工作重点是了解HIV-1组装、释放和成熟的各个方面。主要研究领域包括HIV-1组装的亚细胞靶向，脂筏在HIV-1复制中的作用，HIV-1萌发中的病毒和宿主因素，以及HIV-1成熟抑制剂PA-457(Bevirimat)的作用机制。宿主因子Tsg101和Alix已被证明与HIV-1 Gag的p6区相互作用，我们先前证明Tsg101的p6相互作用区的过表达以显性-负向的方式抑制HIV-1的萌发。我们现在发现，Alix的Gag结合域的过表达也有效地抑制了HIV-1Gag的处理和病毒的释放。这些研究表明，逆转录病毒复制周期的萌芽阶段可以以一种后期特定区域的方式进行靶向。我们还研究了质膜胆固醇在HIV-1组装和释放中的作用，继我们发现病毒组装和萌发发生在富含胆固醇的脂筏微域中。我们观察到胆固醇耗竭破坏了GAG与质膜的结合和GAG的多聚体，表明质膜胆固醇，可能还有脂筏，有助于创建一个稳定的GAG膜结合、GAG多聚体和病毒释放的平台。我们还确定了胆固醇结合化合物AME的作用靶点和作用机制。我们与Panacos制药公司合作，继续表征抗HIV-1化合物PA-457的作用机制。我们的数据表明，PA-457通过干扰GAG处理级联中的一个特定步骤来阻止病毒复制：将衣壳间隔肽1(CA-SP1)处理中间到成熟的CA。GAG处理过程中的这一缺陷通过阻止HIV-1病毒粒子从药物处理细胞释放后成熟来消除病毒的感染性。我们试图确定能够赋予PA-457耐药性的具有复制能力的HIV-1变异体的全谱。分离到6个独立地赋予PA-457抗性的单一氨基酸替换。所有PA-457耐药突变均定位于GAG的CA-SP1连接。研究了PA-457抗性分离株对病毒复制、GAG加工动力学、病毒颗粒组装和释放以及病毒粒子形态的影响。结果表明，PA-457耐药的获得与多种病毒表型有关，提示了多种耐药机制。这项研究为出现对PA-457的耐药性提供了重要而新颖的见解。这里提出的结果将为患者对PA-457耐药性的分析和其他化合物的开发提供信息，这些化合物如PA-457，通过扰乱GAG处理途径中的特定步骤来阻止HIV-1的成熟。在过去的一年里，PA-457已经证明有能力减少感染患者的病毒载量，并已开始扩大该药物的临床试验。

The work in my laboratory is focused on understanding a variety of aspects of HIV-1 assembly, release, and maturation. The major areas of investigation include the subcellular targeting of HIV-1 assembly, the role of lipid rafts in HIV-1 replication, viral and host factors in HIV-1 budding, and mechanism of action of the HIV-1 maturation inhibitor PA-457 (Bevirimat). The host factors Tsg101 and Alix have been shown to interact with the p6 region of HIV-1 Gag, and we previously demonstrated that overexpression of the p6-interacting region of Tsg101 impairs HIV-1 budding in a dominant-negative manner. We now find that overexpression of the Gag-binding domain from Alix also potently inhibits HIV-1 Gag processing and virus release. These studies indicate that the budding step of the retroviral replication cycle can be targeted in a late-domain-specific manner. We have also investigated the role of plasma membrane cholesterol in HIV-1 assembly and release, following up on our finding that virus assembly and budding take place in cholesterol-enriched lipid raft microdomains. We observe that cholesterol depletion disrupts both binding of Gag to the plasma membrane and Gag multimerization, suggesting that plasma membrane cholesterol, and probably lipid rafts, help create a platform for stable Gag membrane binding, Gag multimerization, and virus release. We have also defined the target and mechanism of action of the cholesterol-binding compound AME.In collaboration with Panacos Pharmaceuticals, we have continued to characterize the mechanism of action of the anti-HIV-1 compound PA-457. Our data indicate that PA-457 blocks virus replication by interfering with a specific step in the Gag processing cascade: the conversion of the capsid-spacer peptide 1 (CA-SP1) processing intermediate to mature CA. This defect in Gag processing abolishes virus infectivity by preventing maturation of HIV-1 virions following release from drug-treated cells. We sought to identify the full spectrum of replication-competent HIV-1 variants capable of conferring PA-457 resistance. Six single amino acid substitutions, which independently confer PA-457-resistance, were isolated. All PA-457-resistance-conferring mutations mapped to the CA-SP1 junction of Gag. The PA-457-resistant isolates were characterized with respect to their effects on virus replication, the kinetics of Gag processing, virus particle assembly and release, and virion morphology. The results demonstrate that acquisition of PA-457-resistance is associated with a variety of viral phenotypes, suggesting multiple mechanisms of resistance. This study provides significant and novel insights into the emergence of resistance to PA-457. The results presented here will inform both the analysis of PA-457 resistance in patients and the development of additional compounds that, like PA-457, block HIV-1 maturation by disrupting specific steps in the Gag processing pathway. During the past year, PA-457 has demonstrated the ability to reduce viral loads in infected patients and expanded clinical trials of this drug have been initiated.