Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery.
Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery.
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DOI：
10.7554/elife.26910
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发表时间：
2017-07-31
期刊：
影响因子：
7.7
通讯作者：
Mehle A
中科院分区：
文献类型：
--
作者：
Mondal A;Dawson AR;Potts GK;Freiberger EC;Baker SF;Moser LA;Bernard KA;Coon JJ;Mehle A
流感病毒在感染早期表达转录物，并在稍后的时间点向基因组复制转变。该过程需要病毒复制机制、由病毒聚合酶、基因组RNA和寡聚核蛋白（NP）组成的大核糖核蛋白复合物（RNP）的从头组装。尽管RNP在感染过程中起着核心作用，但决定它们何时何地聚集的因素却知之甚少。在这里，我们表明，人类蛋白激酶C（PKC）家族成员调节RNP组装。活化的PKCδ与聚合酶亚基PB2相互作用，并在感染期间磷酸化调节NP寡聚化和RNP组装。与其在调节RNP组装中的作用一致，PKCδ的敲除通过选择性地破坏基因组复制来削弱病毒感染。然而，通过感染颗粒沉积的预形成的RNP的初级转录不受影响。因此，流感病毒利用宿主PKC来调节RNP组装，这是在感染周期期间从初级转录过渡到基因组复制所需的步骤。为了能够繁殖，流感病毒需要进入宿主的细胞，欺骗它们复制病毒的遗传信息并组装新的病毒颗粒。病毒的遗传信息存储在核糖核酸（RNA）分子中，并编码参与制造新病毒颗粒的几种病毒蛋白。这些蛋白质包括一种称为病毒聚合酶的酶和一种“核蛋白”。病毒聚合酶复制RNA，然后核蛋白与新的RNA结合以保护它，直到它被包装成新的病毒颗粒。许多核蛋白单元组装成长链，覆盖整个RNA，但目前还不清楚这一过程是如何控制的。在细胞中，其他被称为激酶的酶能够通过磷酸化修饰蛋白质的结构来改变许多蛋白质的活性。流感病毒核蛋白以前被证明是磷酸化的。因此，流感病毒可能利用磷酸化作用来控制核蛋白沿着RNA沿着组装成链。然而，病毒的RNA本身不编码任何激酶，因此它必须依赖于宿主细胞的激酶。人类细胞产生许多激酶，这些激酶可以分为几个不同的蛋白质家族。Mondal等人研究了蛋白激酶C家族在制造新病毒颗粒中的作用。实验表明，修饰该蛋白质家族的成员使其具有永久活性会导致病毒核蛋白在蛋白质上的两个特定位点磷酸化。这调节核蛋白在RNA上组装成长链，并最终促进新病毒颗粒的产生。更仔细的检查显示，这种作用主要归因于一种称为PKCδ的特定激酶。这种病毒在缺少PKCδ的人肺细胞中繁殖的能力较低，这是因为核蛋白链的形成不再受到调节，这些细胞产生的病毒蛋白质数量较低。综上所述，这些发现表明宿主细胞产生的激酶可以通过修饰病毒核蛋白来控制病毒复制的能力。未来，有可能开发出针对PKCδ和病毒需要的其他细胞因子的新药，以帮助治疗流感感染。
Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood. Here we demonstrate that human protein kinase C (PKC) family members regulate RNP assembly. Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection. Consistent with its role in regulating RNP assembly, knockout of PKCδ impairs virus infection by selectively disrupting genome replication. However, primary transcription from pre-formed RNPs deposited by infecting particles is unaffected. Thus, influenza virus exploits host PKCs to regulate RNP assembly, a step required for the transition from primary transcription to genome replication during the infectious cycle. To be able to multiply, the influenza virus needs to enter the cells of its host and trick them into copying the virus’ genetic information and assembling new virus particles. The genetic information of the virus is stored in molecules of ribonucleic acid (RNA) and encodes several viral proteins that are involved in making the new virus particles. These proteins include an enzyme known as the viral polymerase and a “nucleoprotein”. The viral polymerase copies the RNA and then the nucleoprotein binds to the new RNA to protect it until it is packaged into new virus particles. Many nucleoprotein units assemble into long chains that coat the whole length of the RNA, but it is not yet known exactly how this process is controlled. In cells, other enzymes known as kinases are able to alter the activities of many proteins by modifying the structures of proteins by a process called phosphorylation. Influenza nucleoprotein was previously shown to be phosphorylated. It is therefore possible that the influenza virus may use phosphorylation to control the assembly of nucleoproteins into chains along the RNA. However, the virus’ RNA does not encode any kinase enzymes of its own, so it must rely on kinases from its host cell. Human cells produce many kinase enzymes that can be grouped into several different protein families. Mondal et al. studied the role of the protein kinase C family in making new virus particles. The experiments show that modifying the members of this protein family to be permanently active causes the viral nucleoprotein to be phosphorylated at two specific sites on the protein. This regulates the assembly of the nucleoproteins into long chains on the RNA, and ultimately promotes the production of new virus particles. Closer examination revealed that this effect was primarily down to one specific kinase known as PKCδ. The virus was less able to multiply in human lung cells that were missing PKCδ – specifcially because the formation of nucleoprotein chains was no longer regulated – and these cells produced lower quantities of virus proteins. Taken together, these findings show that kinases produced by host cells can control the ability of viruses to replicate by modifying the viral nucleoproteins. In the future, it may be possible to develop new drugs that target PKCδ and other cellular factors the virus needs to help treat influenza infections.