Envelope Glycoprotein Incorporation

包膜糖蛋白掺入

• 批准号: 9344061
• 负责人: eric freed
• 金额: $ 48.54万
• 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/9344061
• 关键词: Antiviral Agents; Biology; Cell Line; Cells; Cellular biology; Complex; Cytoplasmic Tail; Data; Defect; Development; Future; Glycoproteins; Goals; HIV; HIV-1; Hela Cells; Human; Indium; Integration Host Factors; Macaca mulatta; Maps; Maryland; Mediating; Modification; Molecular; Monkeys; Mutation; NMR Spectroscopy; Natural Immunity; Nature; Pathway interactions; Play; Point Mutation; Primates; Process; Proteins; RNA Interference; Recruitment Activity; Reporting; Research; Retroviridae; Role; SIV; Signal Transduction; Site Visit; Staging; Structure; Subfamily lentivirinae; Surface Plasmon Resonance; T-Lymphocyte; Tail; Terminator Codon; Titrations; Universities; Virion; Virus; Virus Replication; Work; antiretroviral therapy; base; cell type; env Gene Products; env Glycoproteins; inhibitor/antagonist; insight; interest; mutant; novel; overexpression; particle; programs; rab11 protein; research study; 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._____[Corresponds to Freed Project 2 in the July 2016 site visit report of the HIV Dynamics and Replication Program]

我们一直在积极参与定义分子机制，通过该机制将逆转录病毒ENV糖蛋白掺入组装过程中的病毒颗粒中。 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.猴子细胞，以及逆转录病毒Env蛋白CT中存在的大量运输和信号基序。最近，我们从MA的角度理解设想的结构要求方面取得了重大进展，并将基于这些进步，以阐明GP41 CT在Invimation中的作用。 _____Several lines of evidence suggest that HIV 1 Env glycoproteins are recruited into virions via direct interactions between Env and MA;例如，MA和GP41 CT中的突变都可以阻止HIV 1 Envionation。我们最近的发现强烈支持以下假设：MA领域的修剪化在Env招聘中起着重要作用：假定的MA Trimer界面的突变能够营救由MA突变的大型MA突变施加的Env-Incoberation缺陷，并且在GP41 CT中的一个小型删除以及GP41 CT中的小型删除，并破坏MA TrimerTrimerTrimerTrimer形成Envitions Invienty block Invormation block Invimation block Invimation intrimation block Invormation Incompation Incompation Incompation Incompation。在该项目中，我们旨在进一步阐明Embionation的结构要求，首先关注HIV-1，然后将分析扩展到其他慢病毒，更广泛地将其扩展到其他逆转录病毒。 _____我们几年前表明，HIV-1 Env可能与细胞类型的方式相互作用，并与促进Envistion的宿主细胞因子相互作用。最近的研究表明，Embionation是由MA与47 kDa的宿主因子尾部相互作用蛋白之间的相互作用介导的（TIP47）。作为我们为了解HIV-1设想所需的宿主细胞机械努力的一部分，我们重新评估了TIP47在此过程中的作用。 NMR光谱滴定实验和表面等离子体共振证实了MA和TIP47之间的直接相互作用[在我们的协作者DRS的实验室中进行。迈克尔·萨默斯（Michael Summers）（马里兰大学）和西蒙·科克林（Simon Cocklin）（德雷克塞尔大学）]。然而，在HeLa细胞中，TIP47过表达或RNAi介导的耗竭对HIV-1环境掺入，病毒释放或颗粒感染性没有显着影响。同样，在Jurkat T细胞系中TIP47的耗竭也不会损害HIV-1设想的成立，病毒释放，感染力或复制。因此，我们的结果不支持tip47在HIV-1环境中或病毒感染感染中的作用。还建议逆转录综合体在ENV运输中发挥作用。慢病毒设想的细胞类型特异性性质的一个有趣的方面是，尽管在大多数相关的人类细胞类型中，GP41 CT的截断是HIV-1的复制，但Sivmac在人类细胞中传播时会获得GP41 CT停止密码子。当突变病毒在猴子细胞中传播时，这些终止密码子恢复为野生型序列（例如，恒河猴PBMC）。因此，HIV-1 GP41 CT在病毒复制中起积极的作用，而SIVMAC GP41 CT在人类细胞中起负作用，但在猴子细胞中起积极作用。了解这些观察结果的基础可能会为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动物慢病毒ENV糖蛋白掺入中的作用，以及Embionation所需的MA和GP41中的决定因素。__________________，尽管自1996年以来就可以使用三聚体MA晶体结构，但功能性MA三聚体的证据是难以捉摸的。 HIV-1 Env募集到病毒体的机制也尚不清楚。我们确定了MA（62QR）中的一个点突变，该突变挽救了由大量MA和Envistions造成的Env-Instruberation缺陷。将突变映射到推定的MA三聚体上表明，在三聚体的尖端，在MA晶格中gp41 CT可以插入的MA晶格的周长处，掺入缺陷的突变簇。相比之下，救援突变位于三聚体界面上，表明它通过修改MA三聚体相互作用来赋予Enviue Incompination。这些数据强烈支持未成熟的插科打晶格中MA三聚体的存在，并证明了拯救Env-Oberation缺陷是由MA Trimer界面的修改相互作用介导的。三聚体界面在拯救HIV-1环境中的重要性表明，MA的三聚体排列在允许将ENV纳入GAG晶格中起着至关重要的作用。可以开发抑制剂来阻止HIV-1环化合物通过破坏MA修剪中的这一基本结构元素。未来的工作还可以通过破坏宿主因素的功能或主机因素与ENV或GAG之间的相互作用来产生阻止HIV-1环境的策略。