Role of HIV-1 p6gag and Ubiquitin in Virus Release

逆转录病毒Gag多蛋白具有特定的区域，称为晚期组装(L)结构域，促进组装的病毒粒子最终从细胞表面和彼此分离。HIV-1的L结构域位于C端的p6gag结构域，包含一个必需的P(T/S)APP基序，而癌逆转录病毒的L结构域在Gag中具有更多的N端位置和保守的PPXY核心基序。L结构域可以在HIV-1和无关逆转录病毒之间进行功能交换，表明它们代表了细胞相互作用伙伴的对接位点。与这一观点一致，我们最近观察到，HIV-1和其他病毒的L结构域诱导最小的HIV-1 Gag结构的泛素化，而取消L结构域活性的点突变阻止了Gag泛素化。我们的结果表明，细胞泛素化机制的招募在L结构域功能中起着至关重要的作用。我们的结果还表明，参与泛素化系统的HIV-1 Gag决定因素比肿瘤逆转录病毒中的决定因素更复杂。基于我们的初步结果，我们建议在HIV-1Gag中定位这些决定因素，以阐明Gag泛素化和病毒释放之间的关系，并确定参与这些事件的宿主因子的潜在相互作用位点。我们还建议研究泛素中特定决定因素的要求以及特定泛素连接酶的作用。此外，我们计划检查GAG本身是否需要泛素化，以及是否存在L结构域诱导的泛素化的细胞靶点。由于我们的初步结果显示，在Gag免疫沉淀物和病毒样颗粒中，一种42 kDa的蛋白质依赖于L结构域的浓缩，因此我们还打算研究肌动蛋白细胞骨架在HIV-1 L结构域功能中的作用。了解HIV-1 L结构域促进病毒释放的机制可能最终为尝试阻断HIV-1复制周期的这一阶段提供基础。

Retroviral Gag polyproteins have specific regions, known as late assembly (L) domains, which promote the final separation of assembled virions from the cell surface and from each other. The L domain of HIV-1 is in the C-terminal p6gag domain and contains an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses have a more N-terminal location within Gag and a conserved PPXY core motif. L domains can be functionally exchanged between HIV-1 and unrelated retroviruses, indicating that they represent docking sites for cellular interaction partners. Consistent with this view, we recently observed that the L domains of HIV-1 and other viruses induce the ubiquitination of minimal HIV-1 Gag constructs, and that point mutations which abolish L domain activity prevent Gag ubiquitination. Our results imply that the recruitment of the cellular ubiquitination machinery plays a crucial role in L domain function. Our results also indicate that the HIV-1 Gag determinants which engage the ubiquitination system are more complex than those in oncoretroviruses. Based on our preliminary results, we propose to map these determinants in HIV-1 Gag in order to clarify the relationship between Gag ubiquitination and virus release, and to define potential interaction sites for host factors involved in these events. We also propose to investigate the requirement for specific determinants in ubiquitin and the role of specific ubiquitin ligases. Furthermore, we plan to examine whether Gag itself needs to become ubiquitinated, and whether there is a cellular target for L domain-induced ubiquitination. Because our preliminary results show an L domain-dependent enrichment of a 42 kDa protein in Gag immunoprecipitates and in virus-like particles, we also intend to investigate the role of the actin cytoskeleton in HIV-1 L domain function. An understanding of the mechanism by which the HIV-1 L domain promotes virus release may ultimately provide a basis for attempts to block this stage of the HIV-1 replication cycle.