Enzymology of Influenza Virus RNA Replication

描述（由申请方提供）：拟定研究调查了流感病毒RNA复制的酶学;流感病毒RNA依赖性RNA聚合酶是一种多亚基酶，对病毒mRNA表达和病毒RNA复制至关重要，没有真核类似的细胞对应物。 目前没有针对这种酶的流感抗病毒药物。已经使用在不存在流感病毒感染的情况下表达的重组聚合酶复合物阐明了病毒mRNA中涉及的机制。这些聚合酶复合物不能有效地催化病毒RNA复制。为了阐明病毒RNA复制的机制并促进新的抗病毒疗法的创造，我们建议利用从流感感染细胞的细胞核中纯化的流感聚合酶复合物。具体目标是：1）使用来自流感病毒感染的细胞核提取物的纯化的流感病毒聚合酶复合物建立体外病毒RNA复制的要求。2)确定流感病毒RNA复制所必需的聚合酶亚基和宿主细胞因子的修饰。3)通过利用从流感感染细胞核提取物和外源模板RNA纯化的无模板聚合酶复合物组装在vRNA复制中起作用的起始复合物来确定vRNA复制的模板RNA需求。

DESCRIPTION (provided by applicant): The proposed research investigates the enzymology of influenza virus RNA replication; the influenza RNA-dependent RNA polymerase is a multi-subunit enzyme central to viral mRNA expression and viral RNA replication with no eukaryotic analogous cellular counterpart. Currently no influenza anti-viral drugs are aimed against this enzyme. Mechanisms involved in viral mRNA have been elucidated using reconstituted polymerase complexes expressed in the absence of influenza virus infection. These polymerase complexes do not efficiently catalyze viral RNA replication. To elucidate the mechanism of viral RNA replication and facilitate creation of new anti-viral therapies, we propose to utilize influenza polymerase complexes purified from the nuclei of influenza-infected cells. Specific aims are: 1) Establish requirements for virus RNA replication in vitro using purified influenza virus polymerase complexes from influenza virus infected cell nuclear extracts. 2) Identify modifications of polymerase subunits and host cellular factors that are essential for influenza virus RNA replication. 3) Determine template RNA requirements for vRNA replication by assembling initiation complexes that function in vRNA replication utilizing template-free polymerase complexes purified from influenza infected cell nuclear extracts and exogenous template RNAs.