Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication
Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication
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DOI：
10.1128/jvi.02293-07
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发表时间：
2009-01-01
影响因子：
5.4
通讯作者：
Krug, Robert M.
中科院分区：
文献类型：
--
作者：
Newcomb, Laura L.;Kuo, Rei-Lin;Krug, Robert M.
甲型流感病毒聚合酶转录并复制八个病毒粒子RNA （vRNA）片段。转录是由病毒聚合酶内在的内切酶从细胞前mrna中切除的带帽RNA引物开始的。病毒RNA的复制分两步进行：首先制造vRNA的全长拷贝，称为cRNA，然后复制该cRNA以产生vRNA。与病毒mRNA合成的起始不同，cRNAs和vRNAs的合成不需要引物，并且需要病毒核衣壳蛋白（NP）。未引物病毒RNA复制的机制尚不清楚。为了阐明这一机制，我们利用纯化的重组流感病毒聚合酶复合物和NP建立了一个体外系统，利用50个核苷酸长的RNA模板催化无引物合成cRNA和vRNA。纯化的病毒聚合酶和NP足以催化这种RNA合成而不需要引物，这表明不需要宿主细胞因子。我们使用这个纯化的体外复制系统来证明NP的rna结合活性不需要用于无引物合成cRNA和vRNA。这一结果排除了两种假设NP的RNA结合活性介导了从带帽RNA引物转录到无引物病毒RNA复制的转换的模型。由于我们发现缺乏rna结合活性的NP直接与病毒聚合酶结合，因此NP与病毒聚合酶之间的直接相互作用可能会导致聚合酶的修饰，从而有利于未引物起始。
The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.