Envelope Glycoprotein Incorporation and Function

包膜糖蛋白的掺入和功能

• 批准号: 10702637
• 负责人: eric freed
• 金额: $ 82.37万
• 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/10702637
• 关键词: 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; antagonist; antiretroviral therapy; cell type; experimental study; inhibitor; 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确定逆转录病毒Env糖蛋白在组装过程中被纳入病毒颗粒的分子机制。由于缺乏Gag （MA）的基质结构域和病毒粒子中gp41的细胞质尾（CT）的结构信息，gp41 CT在Env结合中需求的细胞类型特异性差异，HIV-1和猴免疫缺陷病毒SIVmac株之间gp41 CT在人与猴细胞中的作用的明显差异，阻碍了对这一过程的完整理解。以及在逆转录病毒Env蛋白的ct中存在过多的转运和信号基序。最近，我们在从MA的角度理解Env结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结合中的作用。一些证据表明，HIV - 1 Env糖蛋白通过Env和MA之间的直接相互作用被招募到病毒粒子中；例如，MA和gp41 CT的突变都可以阻止HIV - 1 Env的结合。我们最近的研究结果有力地支持了MA结构域的三聚化在Env募集中起重要作用的假设：在假定的MA三聚体界面上的突变能够挽救由大量MA突变和gp41 CT中的小缺失造成的Env整合缺陷，而破坏MA三聚体形成的突变会阻止Env的整合。在这个项目中，我们的目标是进一步阐明Env结合的结构要求，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。我们在几年前就表明HIV-1 Env可能以细胞类型依赖的方式与促进Env结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与宿主因子尾部相互作用蛋白47 kDa （TIP47）之间的相互作用介导的。作为我们持续努力了解HIV-1 Env结合所需的宿主细胞机制的一部分，我们重新评估了TIP47在这一过程中的作用。MA和TIP47之间的直接相互作用被核磁共振光谱滴定实验和表面等离子体共振证实[在我们的合作者博士的实验室进行。Michael Summers（马里兰大学）和Simon Cocklin（德雷塞尔大学）]。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，在Jurkat t细胞系中，TIP47的缺失不会损害HIV-1 Env的结合、病毒释放、感染性或复制。因此，我们的结果不支持TIP47在HIV-1 Env结合或病毒粒子感染中的作用。最近，斯皮尔曼实验室证明了另一种宿主蛋白Rab11-FIP1c在Env运输和整合到病毒粒子中起着重要作用。逆转录复合体也被认为在环境贩运中起作用。慢病毒Env结合的细胞类型特异性的一个有趣方面是，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在人类细胞中繁殖时获得gp41 CT停止密码子。当突变病毒在猴细胞（如恒河猴PBMCs）中繁殖时，这些终止密码子恢复到野生型序列。因此，HIV-1 gp41 CT在病毒复制中起积极作用，而SIVmac gp41 CT在人类细胞中起消极作用，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。我们将评估宿主因子在灵长类慢病毒Env糖蛋白整合中的作用，以及Env整合所需的MA和gp41的决定因素。虽然自1996年以来，三聚体MA晶体结构已经可用，但功能性MA三聚体的证据一直难以捉摸。HIV-1 Env募集到病毒粒子的机制也同样不清楚。我们在MA （62QR）中发现了一个点突变，它挽救了大量MA和Env突变所造成的Env结合缺陷。将突变映射到假定的MA三聚体上显示，合并缺陷突变聚集在三聚体的尖端，位于gp41 CT可以插入的MA晶格假定间隙的周长。相比之下，拯救突变位于三聚体界面，表明它通过修饰MA三聚体相互作用来拯救Env的结合。这些数据有力地支持了未成熟Gag晶格中MA三聚体的存在，并证明了环境结合缺陷的修复是由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-1 Env结合中的重要性表明，MA的三聚体排列在允许Env结合到Gag晶格中起关键作用。抑制剂可以通过破坏MA三聚化中的基本结构元件来阻断HIV-1 Env的结合。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结合的策略。我们还证明了细胞E3泛素连接酶MARCH8限制了包括HIV-1在内的许多病毒包膜糖蛋白。我们的研究结果表明，march8介导的HIV-1 Env的拮抗不需要gp41的细胞质尾部。