Enzymology of Influenza Virus RNA Replication

描述(申请人提供)：拟议的研究调查流感病毒RNA复制的酶学；流感RNA依赖的RNA聚合酶是一种多亚单位酶，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表达的重组聚合酶复合体已经阐明了涉及病毒mRNA的机制。这些聚合酶复合体不能有效地催化病毒的RNA复制。为了阐明病毒RNA复制的机制和促进新的抗病毒疗法的创造，我们提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体。具体目标是：1)建立使用从流感病毒感染细胞核提取液中纯化的流感病毒聚合酶复合体进行病毒RNA体外复制的要求。2)确定对流感病毒RNA复制至关重要的聚合酶亚单位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液中纯化的无模板聚合酶复合体和外源模板RNA组装在vRNA复制中起作用的起始复合体，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.