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）： 1 型人类免疫缺陷病毒 (HIV-1) 会导致获得性免疫缺陷综合症 (AIDS)。 HIV-1 疫苗的缺乏、多重耐药性和治疗并发症（以及治疗费用）强调了持续需要确定新的病毒-宿主界面，并具有抗病毒策略的靶向潜力。我们最近发现了细胞 CRM1 核输出受体的物种特异性属性，它抑制小鼠细胞中 HIV-1 含有内含子的 mRNA（包括 gRNA）的核质转运。 CRM1 在脊椎动物中非常保守，可调节多种编码疏水性肽（称为核输出信号 (NES)）的细胞和病毒蛋白的核输出。 HIV-1 Rev蛋白编码富含亮氨酸的NES，并将CRM1招募到编码顺式作用Rev反应元件（RRE）的病毒mRNA上，以促进其核输出。 CRM1 的活动受到物种限制 Rev 的研究引发了有关 CRM1 蛋白质进化和细胞特异性作用模式的重要问题。首先，人类 CRM1 (hCRM1) 能做什么而小鼠 CRM1 (mCRM1) 不能做什么？我们已经绘制了调节 Rev 活性的 hCRM1 物种特异性决定因素。在目标 1 中，我们将研究该决定因素在 Rev/gRNA 复合物形成中的作用，并确定 mCRM1 对 HIV-1 Rev 功能的阻断是否可以在人类细胞中显现出来。其次，为什么 HIV-1 Rev 适合优先利用 hCRM1？在目标 2 中，我们将表征新发现的 HIV-1 Rev 和 gRNA 突变体，它们在缺乏 hCRM1 的情况下挽救小鼠细胞中的病毒颗粒产生，并测试 hCRM1 或这些病毒突变体是否可以在小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 包装和高效病毒颗粒组装的下游阶段。