Assembly dynamics and role of PI4P enriched replication organelles for enterovira

描述（由申请人提供）：被正链RNA病毒感染的细胞经历胞内膜的戏剧性重塑，形成所谓的复制细胞器。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种成分来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的细胞器不同。我们发现肠病毒3A蛋白劫持宿主磷脂酰肌醇-4-激酶III？（PI4KIII?）到膜上生成富含磷酸磷脂酰肌醇（PI4P）脂质的细胞器。我们发现富含pi4p的脂质膜微环境对肠病毒和黄病毒RNA合成至关重要，而PI4KIII？酶是产生这种脂质微环境的关键。此外，我们发现肠病毒RNA聚合酶特异性和优先结合PI4P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来形成专门的细胞器，其中宿主磷酸肌醇脂是调节病毒RNA复制的关键。基于这些发现，我们假设“PI4P脂质是肠病毒RNA聚合酶活性的关键调节剂，并将膜重组与优化的病毒RNA在体内复制联系起来”。我们的具体目标是：确定PI4P脂质调节肠病毒RNA合成的机制，并确定肠病毒RNA聚合酶上的PI4P脂质结合结构域。我们将使用生化、光谱学、遗传学和计算方法确定PI4P结合位点在RNA聚合酶上的位置，以及PI4P结合是否可以调节复制复合体内聚合酶和其他病毒蛋白的酶活性。这将进一步加深我们对所有依赖膜进行RNA合成的正链RNA病毒感染的理解；阐明肠病毒3A蛋白选择性促进宿主PI4KIII募集的机制。我们将使用多种生化、遗传学和影像学方法检测PI4KIII？直接被3A或通过中间宿主因子劫持。这一信息对于设计和靶向药物以阻断这一关键事件将是重要的；利用定量超分辨率光成像技术确定病毒和宿主成分与PI4P脂质富集复制细胞器的纳米级关联。利用光活化光显微镜（PALM），一种单分子超分辨率成像方法，我们将研究pi4p -脂富集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。这将提供膜动力学和病毒RNA复制之间耦合的高分辨率时空信息。总的来说，这些研究将解决病毒学中一个重要的新范式，为磷脂肌苷脂如何调节病毒RNA复制提供见解。这些发现将对设计对抗病毒感染的新疗法产生深远的影响。

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.