Envelope Glycoprotein Incorporation

包膜糖蛋白掺入

• 批准号: 10014786
• 负责人: eric freed
• 金额: $ 67.9万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014786
• 关键词: Antiviral Agents; Biology; Cell Line; Cells; Cellular biology; Complex; Crystallization; Cytoplasmic Tail; Data; Defect; Development; Elements; Future; Glycoproteins; Goals; HIV; HIV-1; Hela Cells; Human; Integration Host Factors; Macaca mulatta; Maryland; Mediating; Modification; Molecular; Monkeys; Mutation; NMR Spectroscopy; Natural Immunity; Nature; Pathway interactions; Peripheral Blood Mononuclear Cell; Play; Point Mutation; Primates; Process; Proteins; RNA Interference; Research; Retroviridae; Role; SIV; Signal Transduction; Structure; Subfamily lentivirinae; Surface Plasmon Resonance; T-Lymphocyte; Tail; Terminator Codon; Titrations; Universities; Virion; Virus; Virus Replication; Work; antiretroviral therapy; cell type; experimental study; inhibitor/antagonist; insight; interest; mutant; novel; overexpression; particle; recruit; trafficking

We have been actively engaged in defining the molecular mechanism by which retroviral Env glycoproteins are incorporated into virus particles during the assembly process. A complete understanding of this process has been stymied by a lack of structural information about the matrix domain of Gag (MA) and the cytoplasmic tail (CT) of gp41 in virions, cell-type-specific differences in the requirement for the gp41 CT in Env incorporation, clear differences in the roles of the gp41 CT between HIV-1 and the SIVmac strain of simian immunodeficiency virus in human vs. monkey cells, and the plethora of trafficking and signaling motifs present in the CTs of retroviral Env proteins. Recently, we have made significant progress in understanding the structural requirements for Env incorporation from the perspective of MA, and will build on these advances to elucidate the role of the gp41 CT in Env incorporation. ____Several lines of evidence suggest that HIV 1 Env glycoproteins are recruited into virions via direct interactions between Env and MA; for example, mutations in both MA and the gp41 CT can block HIV 1 Env incorporation. Our recent findings strongly support the hypothesis that trimerization of the MA domain plays an important role in Env recruitment: a mutation at the putative MA trimer interface is able to rescue the Env-incorporation defect imposed by a large panel of MA mutations and a small deletion in the gp41 CT, and mutations that disrupt MA trimer formation block Env incorporation. In this project, we aim to further elucidate the structural requirements for Env incorporation, focusing first on HIV-1 and then extending our analysis to other lentiviruses and, more broadly, other retroviruses. ____We showed a number of years ago that HIV-1 Env is likely to interact, in a cell-type-dependent manner, with host cell factors that promote Env incorporation. More recent studies suggested that Env incorporation is mediated by interactions between MA and the host factor tail-interacting protein of 47 kDa (TIP47). As part of our ongoing efforts to understand the host cell machinery required for HIV-1 Env incorporation, we reevaluated the role of TIP47 in this process. A direct interaction between MA and TIP47 was confirmed by NMR spectroscopy titration experiments and surface plasmon resonance [performed in the labs of our collaborators Drs. Michael Summers (University of Maryland) and Simon Cocklin (Drexel University)]. However, in HeLa cells, TIP47 overexpression or RNAi-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in the Jurkat T-cell line did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.____More recently, the Spearman lab demonstrated that another host protein, Rab11-FIP1c, plays an important role in Env trafficking and incorporation into virions. The retromer complex was also suggested to function in Env trafficking. An intriguing aspect of the cell-type-specific nature of lentiviral Env incorporation is that while in most relevant human cell types truncation of the gp41 CT blocks HIV-1 replication, SIVmac acquires gp41 CT stop codons when propagated in human cells. These stop codons revert to the wild-type sequence when the mutant viruses are propagated in monkey cells (e.g., rhesus PBMCs). Thus, the HIV-1 gp41 CT plays a positive role in virus replication, whereas the SIVmac gp41 CT plays a negative role in human cells but a positive role in monkey cells. Understanding the basis for these observations is likely to provide novel insights into the role of gp41 in lentiviral biology. We will evaluate the role of host factors in primate lentiviral Env glycoprotein incorporation and the determinants in MA and gp41 required for Env incorporation.___Although a trimeric MA crystal structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise has been unclear. We identified a point mutation in MA (62QR) that rescues the Env-incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, at the perimeter of a putative gap in the MA lattice into which the gp41 CT could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it confers rescue of Env incorporation via modification of MA trimer interactions. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env-incorporation defects is mediated by modified interactions at the MA trimer interface. The importance of the trimer interface in rescuing HIV-1 Env incorporation suggests that the trimeric arrangement of MA plays a critical role in permitting incorporation of Env into the Gag lattice. Inhibitors could be developed to block HIV-1 Env incorporation by disrupting this essential structural element in MA trimerization. Future work could also yield strategies to block HIV-1 Env incorporation by disrupting the function of host factors, or the interactions between host factors and either Env or Gag.

我们一直积极参与确定逆转录病毒包膜糖蛋白在组装过程中被整合到病毒颗粒中的分子机制。由于缺乏关于病毒粒子中gp41的基质结构域(MA)和gp41的细胞质尾部(CT)的结构信息，在Env掺入中对gp41 CT的要求存在细胞类型的差异，HIV-1株和SIVmac株的gp41 CT在人和猴子细胞中的作用存在明显差异，以及逆转录病毒Env蛋白的CT中存在过多的运输和信号基序，阻碍了对这一过程的完全了解。最近，我们在从MA的角度理解Env整合的结构要求方面取得了重大进展，并将在这些进展的基础上阐明gp41 CT在Env整合中的作用。_我们最近的发现有力地支持了这样的假设，即MA结构域的三聚化在Env的募集中起着重要的作用：在可能的MA三聚体界面上的突变能够修复由大量MA突变和gp41 CT的一小部分缺失以及扰乱MA三聚体形成的突变阻止Env的并入所造成的Env并入缺陷。在这个项目中，我们的目标是进一步阐明Env掺入的结构要求，首先集中在HIV-1上，然后将我们的分析扩展到其他慢病毒，更广泛地说，其他逆转录病毒。_几年前，我们证明了HIV-1Env很可能以一种依赖于细胞类型的方式与促进Env整合的宿主细胞因子相互作用。最近的研究表明，Env的掺入是由MA与宿主因子47 kDa的尾部相互作用蛋白(TIP47)相互作用所介导的。作为了解HIV-1Env整合所需宿主细胞机制的持续努力的一部分，我们重新评估了TIP47在这一过程中的作用。核磁共振光谱滴定实验和表面等离子体共振实验证实了MA和TIP47之间的直接相互作用[在我们的合作者Michael Summers博士(马里兰大学)和Simon Cocklin博士(Drexel大学)的实验室进行]。然而，在HeLa细胞中，TIP47过表达或RNAi介导的缺失对HIV-1 Env的掺入、病毒释放或颗粒感染性没有显著影响。同样，Jurkat T细胞系中TIP47的缺失不会损害HIV-1环境病毒的掺入、病毒释放、传染性或复制。因此，我们的结果不支持TIP47在HIV-1Env掺入或病毒粒子感染性中的作用。_最近，Spearman实验室证明了另一种宿主蛋白Rab11-FIP1c在环境病毒转运和病毒粒子掺入中发挥重要作用。回溯复合体也被认为在环境病毒贩运中发挥作用。慢病毒Env掺入的细胞类型特异性的一个有趣的方面是，虽然在大多数相关的人类细胞类型中，gp41 CT的截断阻止了HIV-1的复制，但SIVmac在人类细胞中传播时获得了gp41 CT终止密码子。当突变病毒在猴细胞(例如恒河猴外周血单核细胞)中繁殖时，这些终止密码子恢复到野生型序列。因此，HIV-1gp41CT在病毒复制中起积极作用，而SIVmac gp41CT在人细胞中起负面作用，而在猴细胞中起积极作用。了解这些观察的基础可能会为gp41在慢病毒生物学中的作用提供新的见解。我们将评估宿主因素在灵长类慢病毒Env糖蛋白掺入中的作用以及Env掺入所需的MA和gp41中的决定因素。尽管自1996年以来已经有了三聚体MA晶体结构，但关于MA三聚体功能的证据一直很难找到。同样，HIV-1Env募集到病毒粒子中的机制也不清楚。我们确定了MA(62QR)中的一个点突变，它可以挽救由一系列MA和Env突变造成的Env-Inc.缺陷。将突变映射到假定的MA三聚体上显示，整合缺陷突变聚集在三聚体的末端，位于gp41 CT可以插入的MA晶格中假定间隙的周长。相反，补救突变位于三聚体界面，这表明它通过修改MA三聚体相互作用来挽救Env的整合。这些数据有力地支持了MA三聚体在未成熟的Gag晶格中的存在，并证明了环境掺入缺陷的挽救是通过MA三聚体界面上的修饰相互作用介导的。三聚体界面在挽救HIV-1Env掺入中的重要性表明MA的三聚体排列在允许Env掺入Gag晶格中起着关键作用。通过破坏MA三聚化中的这一基本结构元素，可以开发出抑制HIV-1Env掺入的抑制剂。未来的工作还可能产生通过扰乱宿主因子的功能或宿主因子与Env或Gag之间的相互作用来阻止HIV-1Env整合的策略。