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Envelope Glycoprotein Incorporation and Function

包膜糖蛋白的掺入和功能

基本信息

批准号：
10926290
负责人：
eric freed
金额：
$ 87.04万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926290
关键词：
Antiretroviral drug resistance Biology Cell Line Cells Cellular biology Complex Cytoplasmic Tail Data Defect Development Drug resistance Elements Future Glycoproteins Goals HIV HIV-1 Hela Cells Human Integrase Integration Host Factors Lentivirus Maps 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 in vivo inhibitor insight interest mutant novel overexpression particle recruit trafficking ubiquitin-protein ligase

项目摘要

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. We have shown that mutations in Env can confer broad resistance to antiretroviral drugs by enhancing the efficiency of cell-cell transfer. This effect is particularly pronounced for integrase strand transfer inhibitors. Current efforts are aimed at deciperhing the mechanism by which Env mutations confer drug resistance, and determining whether this phenomenon occurs in vivo.

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

HIV-1 gag: an emerging target for antiretroviral therapy.

DOI：
10.1007/82_2015_436
发表时间：
2015
期刊：
Current topics in microbiology and immunology
影响因子：
0
作者：
Tedbury PR;Freed EO
通讯作者：
Freed EO

Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins.

DOI：
10.1128/mbio.00219-21
发表时间：
2021-03-16
期刊：
mBio
影响因子：
6.4
作者：
Lun CM;Waheed AA;Majadly A;Powell N;Freed EO
通讯作者：
Freed EO

Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle.

HIV-1包膜糖蛋白的突变可以广泛地挽救病毒复制周期多个步骤的阻断。

DOI：
10.1073/pnas.1820333116
发表时间：
2019
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
VanDuyne,Rachel;Kuo,LillianS;Pham,Phuong;Fujii,Ken;Freed,EricO
通讯作者：
Freed,EricO

"Expand and Click": A New Method for Labeling HIV-1 Envelope Glycoproteins.