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。下文简述了主要的调查领域。1. HIV-1组装的亚细胞靶向。逆转录病毒颗粒从感染细胞的产生是由Gag前体蛋白介导的。在胞质溶胶中合成后，Gag被快速特异性地转运到病毒组装位点。Gag被靶向到适当的亚细胞位置的分子机制仍然知之甚少。基于对突变HIV-1 Gag蛋白的分析，我们和其他人先前已经证明，Gag的基质（MA）结构域中的高度碱性补丁是Gag转运到质膜的主要决定因素。我们最近确定（Ono & Freed，J. Virol.，2004），在HeLa和T细胞中，在质膜靶向方面有缺陷的MA突变型Gag在CD 63阳性隔室中形成病毒颗粒，所述隔室被定义为晚期内体或多泡体（MVB）。有趣的是，我们发现，在原代人巨噬细胞中，野生型（WT）和MA突变型Gag蛋白都特异性靶向MVB。这些结果表明，Gag靶向MVB并在MVB中组装是巨噬细胞中HIV-1病毒颗粒产生的生理学重要步骤，并且这种细胞类型中的颗粒释放可能遵循外泌体途径。为了确定靶向MVB的Gag是否是p6 Gag中的晚期结构域与MVB分选机制之间相互作用的结果（例如，Tsg 101），我们检查了缺乏p6的Gag突变体的靶向和组装。值得注意的是，在不存在p6的情况下仍然观察到Gag的MVB定位，这表明Gag靶向MVB不需要Gag和Tsg 101之间的相互作用。这些数据与Gag靶向模型一致，该模型假定Gag有两种不同的细胞结合伴侣，一种在质膜上，另一种在MVB中。最近的实验室研究提供了特异性磷酸肌醇在HIV-1 Gag靶向中的作用的证据（Ono et al.，PNAS，2004）。2.质膜筏在HIV-1复制中的作用。我们和其他人已经观察到，逆转录病毒Gag蛋白不是均匀分布在质膜上，而是集中在细胞表面的离散区域。由于这些假定的组装中心可能富集在病毒组装、Env掺入和颗粒释放中发挥积极作用的宿主蛋白，因此确定这些区域的组成将非常重要。许多兴奋和争议已经产生的认识，质膜，而不是一个统一的海洋脂质，含有各种微区与特定的脂质和蛋白质组成。特别感兴趣的是富含胆固醇/鞘糖脂的“筏”。“我们已经证明（Ono & Freed，PNAS，2001）HIV-1 Gag与筏相关，并且用胆固醇消耗剂破坏这些脂质结构域显着并特异性地抑制病毒颗粒释放。我们还观察到，从胆固醇耗尽的细胞产生的病毒体显示受损的感染性。当务之急将是全面了解筏在整个病毒复制周期中所起的作用。3. HIV-1 Env糖蛋白掺入和Env/Gag相互作用。HIV-1组装的关键步骤包括将Env糖蛋白掺入出芽颗粒中。多年来，我们一直在研究这种合并的机制。我们证明了跨膜糖蛋白的长胞质尾区在Env掺入中的重要性，并描述了该功能的细胞类型依赖性（Murakami & Freed，PNAS，2000; Murakami & Freed，J. Virol.，2000年）。

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 are described briefly below. 1. The subcellular targeting of HIV-1 assembly. The production of retrovirus particles from infected cells is mediated by the Gag precursor protein. Following its synthesis in the cytosol, Gag is rapidly and specifically transported to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant HIV-1 Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. We recently determined (Ono & Freed, J. Virol., 2004) that, in HeLa and T cells, the MA-mutant Gags that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that, in primary human macrophages, both wild type (WT) and MA-mutant Gag proteins are targeted specifically to the MVB. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages, and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6Gag and MVB sorting machinery (e.g., Tsg101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and Tsg101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB. Recent studies in the lab have provided evidence of a role for specific phosphoinositides in HIV-1 Gag targeting (Ono et al., PNAS, 2004). 2. Role of plasma membrane rafts in HIV-1 replication. We and others have observed that retroviral Gag proteins, rather than being uniformly distributed at the plasma membrane, concentrate in discrete regions at the cell surface. Since these presumed centers of assembly are likely to be enriched for host proteins that play an active role in virus assembly, Env incorporation, and particle release, it will be very important to determine the composition of these regions. Much excitement and controversy have been generated by the realization that the plasma membrane, rather than being a uniform sea of lipid, contains a variety of microdomains with specific lipid and protein compositions. Of particular interest has been the cholesterol/glycosphingolipid-enriched "rafts." We have demonstrated (Ono & Freed, PNAS, 2001) that HIV-1 Gag associates with rafts, and that disruption of these lipid domains with cholesterol-depleting agents markedly and specifically suppresses virus particle release. We also observe that virions produced from cholesterol-depleted cells display impaired infectivity. A top priority will be to develop a full understanding of the role rafts play throughout the virus replication cycle. 3. HIV-1 Env glycoprotein incorporation and Env/Gag interactions. A critical step in HIV-1 assembly involves the incorporation of the Env glycoproteins into budding particles. For a number of years, we have been investigating the mechanism by which this incorporation takes place. We demonstrated the importance of the long cytoplasmic tail of the transmembrane glycoprotein in Env incorporation, and described the cell-type-dependent nature of this function (Murakami & Freed, PNAS, 2000; Murakami & Freed, J. Virol., 2000).