Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35
Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35
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DOI：
10.1128/jvi.02100-19
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发表时间：
2020-08-01
影响因子：
5.4
通讯作者：
Engel, Daniel A.
中科院分区：
文献类型：
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作者：
Miyake, Tsuyoshi;Farley, Charlotte M.;Engel, Daniel A.
埃博拉病毒（EBOV）包涵体（IBs）是核衣壳形成和RNA复制的细胞质位点，包含了病毒生命周期中值得进一步研究的关键步骤。在感染期间，IBs显示有关其大小和位置的动态属性。在进一步的病毒成熟、组装和释放之前，IB的内容也必须转变，这意味着IB功能的其他步骤。有趣的是，仅表达病毒核蛋白（NP）就足以产生IB，这表明它在感染过程中IB的形成中起重要作用。除了NP外，核衣壳的其他成分也定位于IBs，包括VP35、VP24、VP30和RNA聚合酶l。我们之前定义并解决了NP的c端结构域（NP- ct）的晶体结构，但其在病毒复制中的作用尚不清楚。在这里，我们发现当NP单独表达时，NP- ct是IB形成所必需的。有趣的是，我们发现感染性病毒样颗粒（VLPs）的产生也需要NP-Ct，并且具有NP-Ct缺失的缺陷VLPs的病毒RNA含量显着降低。此外，核衣壳组分VP35的共表达克服了NP-Ct在触发IB形成中的缺失，证明了两种蛋白之间的功能相互作用。在所有EBOV蛋白中，只有VP35能够克服NP-Ct缺失导致的IB形成缺陷。这种作用是由VP35和NP之间的一种新的蛋白质-蛋白质相互作用介导的，这种相互作用控制着IB形成和RNA复制本身的调节，并由一个新发现的NP功能域（中心结构域）介导。包涵体（IBs）是包括埃博拉病毒在内的多种负义RNA病毒的RNA合成细胞质位点。除了容纳病毒生命周期中的重要步骤外，IBs还保护新病毒RNA免受先天免疫攻击，并含有功能正在研究中的特定宿主蛋白。埃博拉病毒IB形成的一个关键病毒因子是核蛋白NP，它在RNA封装和合成中也很重要。在这项研究中，我们已经确定了两个控制包涵体形成的NP域。其中一个中心结构域（CD）与病毒蛋白VP35相互作用，控制包涵体的形成和RNA的合成。另一个是NP c端结构域（NP- ct），其功能以前没有报道过。这些发现有助于建立一个模型，其中NP及其与VP35的相互作用将IBs的建立与病毒RNA的合成联系起来。
Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain.IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.