Envelope Glycoprotein Incorporation and Function

包膜糖蛋白的掺入和功能

• 批准号: 10486935
• 负责人: eric freed
• 金额: $ 61.65万
• 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/10486935
• 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; Lentivirus; 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; Rhesus; Role; SIV; Signal Transduction; Structure; Surface Plasmon Resonance; T-Lymphocyte; Tail; Terminator Codon; Titrations; Universities; Viral; Virion; Virus; Virus Replication; Work; antiretroviral therapy; cell type; experimental study; inhibitor/antagonist; insight; interest; mutant; novel; overexpression; particle; recruit; trafficking; ubiquitin-protein ligase

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. We have also demonstrated that the cellular E3 ubiquitin ligase, MARCH8, restricts a number of viral envelope glycoproteins including that of HIV-1. Our findings indicate that MARCH8-mediated antagonism of HIV-1 Env does not require the cytoplasmic tail of gp41.

我们一直积极致力于定义逆转录病毒Env糖蛋白在组装过程中掺入病毒颗粒的分子机制。由于缺乏关于病毒体中Gag的基质结构域（MA）和gp 41的胞质尾区（CT）的结构信息，在Env掺入中对gp 41 CT的要求的细胞类型特异性差异，HIV-1和猴免疫缺陷病毒的SIVmac株之间gp 41 CT在人与猴细胞中的作用的明显差异，以及存在于逆转录病毒Env蛋白的CT中的大量运输和信号基序。最近，我们已经取得了重大进展，从MA的角度了解Env纳入的结构要求，并将建立在这些进展，以阐明gp 41 CT在Env纳入的作用。_若干证据表明，HIV 1 Env糖蛋白通过Env和MA之间的直接相互作用被募集到病毒体中;例如，MA和gp 41 CT中的突变可以阻断HIV 1 Env掺入。我们最近的研究结果强烈支持的假设，MA结构域的三聚体在Env的招聘中起着重要的作用：在推定的MA三聚体界面的突变能够拯救Env掺入缺陷所施加的一个大面板的MA突变和一个小的缺失的gp 41 CT，和突变，破坏MA三聚体形成块Env掺入。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，首先关注HIV-1，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。_多年前，我们发现HIV-1 Env可能以细胞类型依赖性方式与促进Env掺入的宿主细胞因子相互作用。最近的研究表明，Env掺入是由MA和47 kDa的宿主因子尾相互作用蛋白（TIP 47）之间的相互作用介导的。作为我们正在进行的理解HIV-1 Env掺入所需的宿主细胞机制的努力的一部分，我们重新评估了TIP 47在这一过程中的作用。通过NMR光谱滴定实验和表面等离子体共振[在我们的合作者Michael Summers博士（马里兰州大学）和Simon Cocklin博士（德雷克塞尔大学）的实验室中进行]证实了MA和TIP 47之间的直接相互作用。然而，在HeLa细胞中，TIP 47过表达或RNAi介导的耗竭对HIV-1 Env掺入、病毒释放或颗粒感染性没有显著影响。类似地，Jurkat T细胞系中TIP 47的消耗不损害HIV-1 Env掺入、病毒释放、感染性或复制。因此，我们的结果不支持TIP 47在HIV-1 Env掺入或病毒体感染性中的作用。_最近，斯皮尔曼实验室证明了另一种宿主蛋白Rab 11-FIP 1c在Env运输和掺入病毒体中起着重要作用。还建议逆转录复合物在Env贩运中起作用。慢病毒Env掺入的细胞类型特异性性质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，gp 41 CT的截短阻断了HIV-1复制，但SIVmac在人类细胞中繁殖时获得了gp 41 CT终止密码子。当突变病毒在猴细胞中繁殖时，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 gp 41 CT在病毒复制中起积极作用，而SIVmac gp 41 CT在人细胞中起消极作用，但在猴细胞中起积极作用。了解这些观察的基础可能会为gp 41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gp 41中的决定因素。_尽管自1996年以来就已经存在三聚体MA晶体结构，但功能性MA三聚体的证据一直难以捉摸。同样，HIV-1 Env募集到病毒体中的机制也不清楚。我们鉴定了MA中的点突变（62 QR），其挽救了由广泛的MA和Env突变组施加的Env掺入缺陷。将突变映射到推定的MA三聚体上揭示了在gp 41 CT可以插入的MA晶格中的推定间隙的周边处，在三聚体的尖端处聚集有纯化缺陷的突变。相比之下，拯救突变位于三聚体界面，表明其通过MA三聚体相互作用的修饰赋予Env掺入的拯救。这些数据强烈支持MA三聚体在未成熟的Gag晶格中的存在，并表明Env掺入缺陷的拯救是由MA三聚体界面处的改性相互作用介导的。三聚体界面在拯救HIV-1 Env掺入中的重要性表明MA的三聚体排列在允许Env掺入Gag晶格中起关键作用。可以开发抑制剂，通过破坏MA三聚体中的这种基本结构元件来阻断HIV-1 Env掺入。未来的工作还可以通过破坏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断HIV-1 Env掺入。我们还证明了细胞E3泛素连接酶MARCH 8限制了许多病毒包膜糖蛋白，包括HIV-1。我们的研究结果表明，MARCH 8介导的HIV-1 Env拮抗作用不需要gp 41的胞质尾区。