Enzymology of Influenza Virus RNA Replication

描述（由申请人提供）：拟议的研究旨在研究流感病毒RNA复制的酶学；流感RNA依赖的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，在真核生物中没有类似的细胞对应物。目前还没有针对这种酶的流感抗病毒药物。在没有流感病毒感染的情况下，利用重组的聚合酶复合物表达了病毒mRNA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建，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。具体目的是：1)利用流感病毒感染的细胞核提取物纯化的流感病毒聚合酶复合物，建立病毒RNA体外复制的要求。2)鉴定流感病毒RNA复制所必需的聚合酶亚基和宿主细胞因子的修饰。3)利用从流感感染的细胞核提取物中纯化的无模板聚合酶复合物和外源模板RNA组装起vRNA复制作用的起始复合物，确定vRNA复制所需的模板RNA。

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.