Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy
Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy
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DOI：
10.1128/jvi.01533-09
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发表时间：
2010-02-01
影响因子：
5.4
通讯作者：
Ellenberg, Jan
中科院分区：
文献类型：
--
作者：
Huet, Sebastien;Avilov, Sergiy V.;Ellenberg, Jan
依赖于异源三聚体RNA聚合酶的亚基在细胞内的转运和组装构成了流感病毒复制周期的关键组成部分。最近的结果表明，高效的聚合酶组装是重组病毒生存能力的限制因素。PB1、PB2和PA三个聚合酶亚基的核导入和组装机制仍然存在争议，但这显然对理解具有大流行潜力的新毒株的出现具有重要意义。在这项研究中，我们利用荧光互相关光谱(FCCS)和定量共聚焦显微镜系统地研究了聚合酶亚基之间的相互作用及其在活细胞中的定位。我们可以证明，PB1和PA在细胞质中形成二聚体，并分别从PB2输入到细胞核中。一旦进入细胞核，PB1/PA二聚体与PB2结合形成三聚体聚合酶。光子计数直方图分析表明，三聚体聚合酶复合体可以在细胞核内形成更高阶的低聚物。我们进一步证明，通过突变PB2的核定位信号来损害其核输入会导致细胞质中三聚体聚合酶的异常形成。综上所述，我们的结果证明了前面讨论的流感病毒聚合酶运输模型中的哪一种在活细胞中起作用。我们的研究揭示了亚基的核进口和流感病毒聚合酶组装之间的相互作用，并为未来分析不同宿主范围突变的影响提供了一个方法学框架。
Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.