Assembly dynamics and role of PI4P enriched replication organelles for enterovira

富含 PI4P 的复制细胞器的组装动力学和作用

• 批准号: 8115580
• 负责人: Nihal Altan-Bonnet
• 金额: $ 48.45万
• 依托单位: RUTGERS THE STATE UNIV OF NJ NEWARK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115580
• 关键词: 1-Phosphatidylinositol 4-Kinase; Address; Binding; Binding Sites; Biochemical; Biochemical Genetics; Capsid Proteins; Cells; Clathrin; Complex; Computing Methodologies; Coupling; DNA-Directed RNA Polymerase; Diffusion; Drug Delivery Systems; Elements; Enzymes; Event; Genetic; Goals; Image; Integration Host Factors; Intracellular Membranes; Light; Link; Lipid Binding; Lipids; Location; Membrane; Membrane Lipids; Methodology; Nuclear Magnetic Resonance; Organelles; Pathway interactions; Phosphatidylinositols; Phosphotransferases; Plant Roots; Polymerase; Process; Property; Proteins; RNA; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Reaction; Regulation; Resolution; Role; Site; Surface; Tail; Testing; Time; Translations; Viral; Viral Proteins; Virus; Virus Diseases; Work; base; combat; density; design; human disease; imaging modality; in vivo; insight; light microscopy; nanoscale; novel; novel therapeutics; phosphatidylinositol 4-phosphate; prevent; single molecule; spatiotemporal; spectroscopic imaging; viral RNA; virology

DESCRIPTION (provided by applicant): Cells infected with plus-strand RNA viruses undergo dramatic remodeling of their intracellular membranes into so-called replication organelles. The functional properties of the replication membranes that are required to support viral RNA replication for plus-strand viruses are unknown. Our recent work demonstrated that RNA viruses manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication which are distinct in protein and lipid composition from that of the host. We found that enteroviral 3A protein, hijacks host phosphatidylinositol-4-kinase III? (PI4KIII?) to membranes to generate organelles enriched in phosphatidylinositol-4-phosphate (PI4P) lipids. We discovered that the PI4P-rich lipid membrane microenvironment is essential for both enteroviral and flaviviral RNA synthesis, and PI4KIII? enzymes are critical for generating this lipid microenvironment. Furthermore we found that enteroviral RNA polymerases specifically and preferentially bind PI4P lipids. Our findings have brought forward a new paradigm to virology; revealing how viruses can selectively exploit specific elements of the host to form specialized organelles where host phosphoinositide lipids are key to the regulation of viral RNA replication. Based on these findings we hypothesize that "PI4P lipids are critical regulators of enteroviral RNA polymerase activity and link membrane reorganization with optimized viral RNA replication in vivo". Our specific aims are: to determine the mechanisms by which PI4P lipids regulate enteroviral RNA synthesis and to identify the PI4P lipid-binding domain on enteroviral RNA polymerases. Using biochemical, spectroscopic, genetic and computational methods we will determine the location of PI4P binding site on the RNA polymerase and whether PI4P binding can modulate the enzymatic activity of the polymerase and other viral proteins within the replication complex. This will further our understanding of all positive strand RNA viral infection which depend on membranes for RNA synthesis; to elucidate the mechanisms by which enteroviral 3A protein selectively promotes recruitment of host PI4KIII?. Using a variety of biochemical, genetic and imaging methods we will test whether PI4KIII? is hijacked directly by 3A or through intermediate host factors. This information will be important for designing and targeting drugs to block this critical event; to determine the nanoscale association of viral and host components with PI4P lipid enriched replication organelles using quantitative super-resolution light-based imaging. Using Photoactivated Light Microscopy (PALM), a single molecule super-resolution imaging method, we will investigate the membrane structure of PI4P-lipid enriched replication organelles and determine the spatial organization and density of replication complexes. This will provide high-resolution spatiotemporal information on the coupling between membrane dynamics and viral RNA replication. Collectively these studies will address an important new paradigm in virology, providing insight into how phosphoinositide lipids can regulate viral RNA replication. These findings will have far reaching implications for designing new therapeutics to combat viral infections. PUBLIC HEALTH RELEVANCE: Plus-strand RNA viruses are at the root of many human diseases. Upon infecting cells, they hijack specific cellular proteins to build novel membrane-bound organelles; the virus uses the surface of the membranes of the organelles for RNA replication. The hijacked proteins impart on these organelle membranes unique properties, which we found were necessary for replicating viral RNA. Our goals are to determine how these membrane properties regulate viral RNA replication and how specific cellular proteins are hijacked to impart these properties.

描述（由申请人提供）：感染正链RNA病毒的细胞经历其细胞内膜急剧重塑为所谓的复制细胞器。支持正链病毒RNA复制所需的复制膜的功能特性尚不清楚。我们最近的工作表明，RNA病毒操纵细胞分泌途径的多个组件，以产生专门用于复制的细胞器，这些细胞器在蛋白质和脂质组成上与宿主不同。我们发现肠道病毒3A蛋白，劫持宿主磷脂酰肌醇-4-激酶III？（PI4KIII？）膜以产生富含磷脂酰肌醇-4-磷酸（PI 4P）脂质的细胞器。我们发现，PI 4P丰富的脂质膜微环境是必不可少的肠道病毒和黄病毒RNA的合成，PI 4KIII？酶是产生这种脂质微环境的关键。此外，我们发现肠道病毒RNA聚合酶特异性和优先结合PI 4P脂质。我们的发现为病毒学提出了一个新的范式;揭示了病毒如何选择性地利用宿主的特定元素形成专门的细胞器，其中宿主磷酸肌醇脂质是调节病毒RNA复制的关键。基于这些发现，我们假设“PI 4P脂质是肠道病毒RNA聚合酶活性的关键调节剂，并将膜重组与体内优化的病毒RNA复制联系起来”。我们的具体目标是：确定PI 4P脂质调节肠道病毒RNA合成的机制，并鉴定肠道病毒RNA聚合酶上的PI 4P脂质结合结构域。使用生物化学，光谱学，遗传学和计算方法，我们将确定RNA聚合酶上的PI 4P结合位点的位置，以及PI 4P结合是否可以调节聚合酶和复制复合物内其他病毒蛋白的酶活性。这将进一步加深我们对依赖于RNA合成膜的所有正链RNA病毒感染的理解;阐明肠道病毒3A蛋白选择性促进宿主PI 4KIII？募集的机制。使用各种生化，遗传和成像方法，我们将测试是否PI 4KIII？被3A直接劫持或通过中间宿主因素劫持。这些信息对于设计和靶向药物以阻止这一关键事件将是重要的;使用定量超分辨率光基成像来确定病毒和宿主组分与PI 4P脂质富集复制细胞器的纳米级关联。使用光活化光显微镜（PALM），单分子超分辨率成像方法，我们将研究富含PI 4P-脂质的复制细胞器的膜结构，并确定复制复合物的空间组织和密度。这将提供高分辨率的时空信息膜动力学和病毒RNA复制之间的耦合。总的来说，这些研究将解决一个重要的病毒学新范式，提供洞察磷脂酰肌醇脂质如何调节病毒RNA复制。这些发现将对设计新的治疗方法来对抗病毒感染产生深远的影响。 公共卫生相关性：正链RNA病毒是许多人类疾病的根源。在感染细胞后，它们劫持特定的细胞蛋白来构建新的膜结合细胞器;病毒使用细胞器膜的表面进行RNA复制。被劫持的蛋白质赋予这些细胞器膜独特的特性，我们发现这是复制病毒RNA所必需的。我们的目标是确定这些膜特性如何调节病毒RNA复制，以及特定的细胞蛋白质如何被劫持以赋予这些特性。