Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes
Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes
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DOI：
10.1128/jvi.79.2.732-744.2005
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发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Nagata, K
中科院分区：
文献类型：
--
作者：
Kawaguchi, A;Naito, T;Nagata, K
流感病毒的RNA依赖性RNA聚合酶由PB 1、PB 2和PA三个亚基组成，合成vRNA、cRNA和mRNA三种病毒RNA。PB 1是催化亚基; PB 2识别帽结构以产生转录引物; PA被认为参与病毒RNA复制。然而，聚合酶复合物组装的过程和参与三种不同RNA种类合成的聚合酶复合物的确切性质尚不清楚。ts 53病毒是从A/WSN/33（A. Sugiura，M.上田，K. Tobita和C. Enomoto，Virology 65：363-373，1975）。我们证实，在非允许温度下，ts 53的mRNA合成水平不受影响，而vRNA的合成大大减少。编码ts 53 PA的基因的测序和重组病毒拯救实验揭示，氨基酸位置226处从Leu到Pro的氨基酸变化是温度敏感性的原因。通过甘油密度梯度分析的核提取物制备的野生型病毒感染的细胞，我们发现，聚合酶蛋白沉淀在三个馏分：一个（H馏分）由RNP复合物，另一个（M馏分）含有活性聚合酶，但不是病毒RNA，和其他（L馏分）含有非活性形式的聚合酶。脉冲追踪实验表明，L级分中的聚合酶转化为M级分中的聚合酶。在ts 53感染的细胞中，聚合酶在L组分中积累。这些结果有力地表明，PA是参与组装的功能性病毒RNA聚合酶复合物从他们的非活性中间体。
The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear. ts53 virus is a temperature-sensitive (ts) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology 65:363-373, 1975). We confirmed that the mRNA synthesis level of ts53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding ts53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In ts53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.