Alleviation of HIV-1 Rev Block in Astrocytes by an E3 Ubiquitin Ligase, NKLAM

E3 泛素连接酶 NKLAM 减轻星形胶质细胞中的 HIV-1 Rev 阻断

• 批准号: 8140739
• 负责人: RAGHAVENDAR R THIPPARTHI
• 金额: $ 22.8万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8140739
• 关键词: AIDS Dementia Complex; Acquired Immunodeficiency Syndrome; Asses; Astrocytes; Binding; Brain; Cell Nucleus; Data; Development; Environment; Enzymes; Epidemic; Gene Expression; Genetic Transcription; Goals; HIV; HIV-1; Highly Active Antiretroviral Therapy; Human; Infection; Intervention; Knowledge; Laboratories; Lead; Life Cycle Stages; Lytic; Mediating; Methodology; Microscopic; Mission; Molecular; Outcome; Pathway interactions; Patients; Post-Transcriptional Regulation; Prevention; Production; Proteins; Public Health; Publishing; RNA; Reporting; Research; Resistance; Role; SRC-associated p68 protein; Testing; Therapeutic; Time; Transactivation; Ubiquitination; Vaccines; Variant; Viral; Work; base; cellular targeting; combat; experience; innovation; insight; mRNA Export; mortality; novel strategies; novel therapeutics; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how HIV-1 maintains latency in astrocytes. This gap denotes a significant problem, because, unless this gap is filled, understanding the molecular mechanisms of HIV-1 latency leading to HIV-associated dementia (HAD) cannot be achieved. Furthermore, the knowledge required to develop novel therapeutic strategies for HAD patients will remain largely unknown. The long-term goal is to understand the molecular mechanisms that contribute to HIV-1 latency in the brain and develop a therapeutic strategy to combat HIV. The objective of this application is to determine the role of Natural Killer Lytic-Associated Molecule (NKLAM) in the post-transcriptional regulation of HIV-1 gene expression. The central hypothesis of this application is that NKLAM synergizes with Sam68 and alleviates a post-transcriptional block to HIV-1 production in astrocytes. The hypothesis has been formulated from the strong preliminary data, demonstrating that NKLAM interacts with Sam68, which in turn, regulates HIV-1 Rev function in astrocytes. The rationale for the proposed project is that, once the role of NKLAM in the HIV-1 life cycle is understood, its production can be modulated in such a way that impacts HIV-1 production in the prevention and treatment of AIDS, including HAD. Guided by the preliminary data, the central hypothesis will be tested to accomplish the objective of this application by pursuing the following two specific aims: 1) Determine the functional significance of NKLAM in HIV-1 replication. The working hypothesis is that NKLAM relieves a post-transcriptional block to HIV-1 production in astrocytes. Under this aim, stable NKLAM expressing astrocytes will be created and assessed for the alleviation of Rev block and HIV-1 production by examining viral mRNA export. 2) Investigate the mechanism of action of NKLAM in Sam68/RRE-mediated transactivation. The working hypothesis is that NKLAM must act in the nucleus to synergize with Sam68. Employing mutational, microscopic and ubiquitination analyses, the mechanism of action of NKLAM in Sam68/Rev/RRE function will be determined. The proposed work is innovative, because, so far, no E3 ubiquitin ligase(s), specifically NKLAM, has been implicated in the post-transcriptional regulation of HIV-1 gene expression. Most importantly, to date, there are no published reports implicating, any E3 ligase(s), particularly, NKLAM- mediated Sam68 ubiquitination in alleviating the Rev block in astrocytes. The proposed research is significant because it is expected to provide the knowledge needed to develop novel strategies to eliminate HIV-1 reservoirs and combat HAD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because, determining the role of NKLAM in HIV-1 replication is eventually expected to increase our understanding of the molecular basis of HIV-1 latency operating at the level of post-transcription in astrocytes. Therefore, the proposed research is relevant to the part of NIH's mission that relates to the knowledge needed to develop novel therapeutic strategies for HAD patients.

描述（由申请人提供）：我们对HIV-1如何在星形胶质细胞中保持潜伏期的理解存在根本性差距。这一差距意味着一个重要的问题，因为除非填补这一差距，否则无法理解HIV-1潜伏期导致HIV相关性痴呆（HAD）的分子机制。此外，为HAD患者开发新的治疗策略所需的知识在很大程度上仍是未知的。长期目标是了解导致HIV-1在大脑中潜伏的分子机制，并开发出对抗HIV的治疗策略。本申请的目的是确定自然杀伤细胞裂解相关分子（NKLAM）在HIV-1基因表达的转录后调节中的作用。本申请的中心假设是NKLAM与Sam 68协同作用，并在星形胶质细胞中对HIV-1的产生进行转录后阻断。该假设是根据强有力的初步数据制定的，表明NKLAM与Sam 68相互作用，而Sam 68反过来又调节星形胶质细胞中的HIV-1 Rev功能。拟议项目的理由是，一旦了解NKLAM在HIV-1生命周期中的作用，就可以调节其生产，从而影响艾滋病预防和治疗中的HIV-1生产，包括HAD。在初步数据的指导下，将通过追求以下两个具体目标来测试中心假设以实现本申请的目的：1）确定NKLAM在HIV-1复制中的功能意义。工作假设是NKLAM解除了星形胶质细胞中HIV-1产生的转录后阻断。在此目标下，将创建稳定的NKLAM表达星形胶质细胞，并通过检查病毒mRNA输出来评估Rev阻断和HIV-1产生的缓解。2)研究NKLAM在Sam 68/RRE介导的反式激活中的作用机制。工作假设是NKLAM必须在细胞核中起作用以与Sam 68协同作用。采用突变、显微镜和泛素化分析，将确定NKLAM在Sam 68/Rev/RRE功能中的作用机制。所提出的工作是创新的，因为到目前为止，没有E3泛素连接酶，特别是NKLAM，涉及HIV-1基因表达的转录后调节。最重要的是，迄今为止，没有公开的报道涉及任何E3连接酶，特别是NKLAM介导的Sam 68泛素化在减轻星形胶质细胞中的Rev阻断。这项拟议中的研究意义重大，因为它有望提供开发新策略所需的知识，以消除HIV-1储存库和对抗HAD。 公共卫生关系：拟议的研究与公共卫生有关，因为确定NKLAM在HIV-1复制中的作用最终有望增加我们对HIV-1潜伏期在星形胶质细胞转录后水平上运作的分子基础的理解。因此，拟议的研究与NIH的使命的一部分有关，该使命涉及为HAD患者开发新的治疗策略所需的知识。