Regulation of hepatitis C virion production via phosphorylation of the NS5A protein.
Regulation of hepatitis C virion production via phosphorylation of the NS5A protein.
复制标题
通过NS5A蛋白的磷酸化来调节丙型肝炎的产生。
DOI：
10.1371/journal.ppat.1000032
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发表时间：
2008-03-21
期刊：
影响因子：
6.7
通讯作者：
Foss, Katie L.
中科院分区：
文献类型：
--
作者：
Tellinghuisen, Timothy L.;Foss, Katie L.
丙型肝炎病毒 (HCV) 是一种重要的病原体，感染全世界约 1.7 亿人。持续的病毒感染常常导致肝硬化和肝癌。在受感染的细胞中，必须发生许多 RNA 指导的过程才能维持和传播感染。病毒基因组RNA不断复制，作为翻译模板，被包装成新的病毒颗粒；不能同时发生的过程。人们对这些事件的监管知之甚少。病毒 NS5A 磷蛋白多年来一直被提议作为 HCV 生命周期中事件的调节因子，但细节仍然是个谜。 NS5A 是一种三结构域蛋白，RNA 复制所需的结构域 I 和 II 已被充分记录。 NS5A 结构域 III 不是 RNA 复制所必需的，并且该区域在 HCV 生命周期中的功能尚不清楚。我们在结构域 III 中发现了一个小缺失，它会破坏感染性病毒颗粒的产生，但不会改变 HCV RNA 复制的效率。这种缺失会破坏病毒在组装的早期阶段的产生，因为不会产生细胞内病毒，也不会从细胞中释放病毒RNA和核衣壳蛋白。遗传图谱表明缺失中的单个丝氨酸残基导致了观察到的表型。该丝氨酸残基位于酪蛋白激酶 II 共有基序内，模拟磷酸化的突变表明该位置的磷酸化调节感染性病毒的产生。我们通过基因沉默和化学抑制实验表明，NS5A 需要酪蛋白激酶 II 在该位置磷酸化才能产生病毒粒子。该位置模拟磷酸化的突变对酪蛋白激酶 II 活性的这些操作不敏感。这些数据为 NS5A 结构域 III 的功能提供了第一个证据，并表明 NS5A 作为 HCV RNA 复制和病毒粒子组装的重要调节因子。如本文所述，将病毒产生与RNA复制解偶联的能力可能有助于理解HCV组装并且可能在治疗上很重要。丙型肝炎病毒 (HCV) 是一种危及生命的感染，困扰着全世界约 1.7 亿人，目前的抗病毒疗法对治疗这些患者的效果有限。显然，需要更有效的 HCV 抗病毒药物。对于这一过程来说，最重要的是了解 HCV 用于感染细胞、复制病毒基因组、组装子代病毒和退出细胞的详细机制。通过基因图谱，我们鉴定了 HCV NS5A 蛋白的单个氨基酸残基，该残基被宿主细胞激酶磷酸化，并且这种修饰调节新的感染性病毒颗粒的产生。 NS5A 的这种修饰导致一些病毒基因组从复制事件中释放出来，从而使该材料可用于子代病毒颗粒的生产。我们已经确定了调节这一事件的遗传和化学方法，从而使我们能够在实验室中控制传染性病毒颗粒的产生。分离病毒遗传物质的复制和新病毒的组装的能力使我们成为监测这一过程如何发生的宝贵工具，并有可能成为开发急需的抗病毒药物的新靶标。
Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important. Hepatitis C virus (HCV) is a life-threatening infection afflicting some 170 million people worldwide, and current antiviral therapies are only marginally effective in treating these patients. Clearly, more effective anti-viral drugs for HCV are needed. Of paramount importance to this process is understanding the detailed mechanisms HCV uses to infect cells, replicate the viral genome, assemble progeny virus, and exit the cell. Using genetic mapping, we have identified a single amino acid residue of the HCV NS5A protein that is phosphorylated by host cell kinase, and this modification regulates the production of new infectious virus particles. This modification of NS5A results in the release of some of the viral genome from replicative events, thereby making this material available for progeny virus particle production. We have identified genetic and chemical methods to modulate this event, resulting in our ability to control the production of infectious virus particles in the laboratory. The ability to separate the replication of the virus genetic material and the assembly of new viruses allows us a valuable tool to monitor how this process occurs and, potentially, a novel target for the development of much needed anti-viral drugs.
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DOI：
10.1006/bbrc.1999.0460
发表时间：
1999-04-21
影响因子：
3.1
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通讯作者：
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