The Cell Biology of HIV-1 Genome Trafficking

HIV-1 基因组贩运的细胞生物学

• 批准号: 8731031
• 负责人: Nathan M Sherer
• 金额: $ 33.05万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-15 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8731031
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Animal Model; Antiviral Agents; Binding; Cell Culture Techniques; Cells; Cellular biology; Cessation of life; Complex; Data; Development; Elements; Engineering; Environment; Evolution; Gagging; Gene Expression Regulation; Genes; Genome; Genomics; HIV-1; Human; In Vitro; Infection; Introns; Knowledge; Leucine; Life; Life Cycle Stages; Link; Maps; Messenger RNA; Monitor; Multi-Drug Resistance; Mus; Mutagenesis; Nuclear Export; Nuclear RNA; Pathway interactions; Peptides; Phase; Polyproteins; Production; Proteins; Recording of previous events; Recruitment Activity; Regulation; Response Elements; Rodent; Role; Signal Transduction; Staging; Structural Protein; System; Testing; Time; Translations; Treatment Cost; Tropism; Vaccines; Vertebrates; Viral; Viral Genome; Viral Proteins; Virion; Virus; base; cellular imaging; insight; interest; mutant; nucleocytoplasmic transport; public health relevance; receptor; rev Protein; trafficking

DESCRIPTION (provided by applicant): Human mmunodeficiency virus type 1 (HIV-1) causes the acquired immunodeficiency syndrome (AIDS). The lack of an HIV-1 vaccine, multi-drug resistance and complications from (and cost of) treatment emphasize a continuing need to identify new virus-host interfaces with the potential for targeting with antiviral strategies. We recently identified a species-specific attribute of the cellular CRM1 nuclear export receptor that suppresses the nucleocytoplasmic transport of HIV-1's intron-containing mRNAs (including gRNAs) in murine cells. CRM1 is remarkably well conserved among vertebrates and regulates the nuclear export of a broad range of cellular and viral proteins encoding hydrophobic peptides known as nuclear export signals (NESs). The HIV-1 Rev protein encodes a leucine-rich NES and recruits CRM1 to viral mRNAs that encode the cis- acting Rev response element (RRE) in order to facilitate their nuclear export. That CRM1's activities are species limited in the context of Rev leads to important questions regarding CRM1's protein evolution and cell- specific modes of action. First, what is human CRM1 (hCRM1) doing that murine CRM1 (mCRM1) is not? We have mapped the hCRM1 species-specific determinant that regulates Rev activity. In Aim 1 we will study the role of this determinant in Rev/gRNA complex formation and determine if the mCRM1 block to HIV-1 Rev function can be made manifest in human cells. Second, why is HIV-1 Rev adapted to preferentially exploit hCRM1? In Aim 2, we will characterize newly identified HIV-1 Rev and gRNA mutants that rescue virion production in murine cells in the absence of hCRM1, and test if hCRM1 or these viral mutants can provide for active HIV-1 replication in murine cell culture. Finally, in Aim 3 we will establish a systems- based live cell imaging strategy to study how perturbations affecting HIV-1 gRNA nuclear export can influence the downstream stages of gRNA trafficking, Gag translation, gRNA packaging and efficient virus particle assembly.

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）导致获得性免疫缺陷综合征（AIDS）。缺乏HIV-1疫苗、多重耐药性和治疗并发症（以及治疗费用）突出表明，继续需要确定新的病毒-宿主界面，并有可能用抗病毒战略进行靶向治疗。我们最近确定了细胞CRM 1核输出受体的种属特异性属性，该受体抑制小鼠细胞中含HIV-1内含子的mRNA（包括gRNA）的核质转运。CRM 1在脊椎动物中非常保守，并调节广泛的细胞和病毒蛋白质的核输出，这些蛋白质编码称为核输出信号（NES）的疏水肽。HIV-1 Rev蛋白编码富含亮氨酸的内斯，并将CRM 1募集到编码顺式作用Rev反应元件（RRE）的病毒mRNA中，以促进其核输出。CRM 1的活动是物种有限的， 对Rev的研究导致了关于CRM 1蛋白进化和细胞特异性作用模式的重要问题。首先，什么是人CRM 1（hCRM 1）做，鼠CRM 1（mCRM 1）不是？我们已经绘制了hCRM 1物种特异性决定因素，调节Rev活性。在目标1中，我们将研究这种决定因素在Rev/gRNA复合物形成中的作用，并确定mCRM 1对HIV-1 Rev功能的阻断是否可以在人类细胞中表现出来。第二，为什么HIV-1 Rev能够优先利用hCRM 1？在目标2中，我们将表征新鉴定的HIV-1 Rev和gRNA突变体，其在不存在hCRM 1的情况下拯救鼠细胞中的病毒体产生，并测试hCRM 1或这些病毒突变体是否可以在鼠细胞培养物中提供活跃的HIV-1复制。最后，在目标3中，我们将建立基于系统的活细胞成像策略，以研究影响HIV-1 gRNA核输出的扰动如何影响gRNA运输、Gag翻译、gRNA包装和有效病毒颗粒组装的下游阶段。