Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle
Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle
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DOI：
10.1128/jvi.01925-19
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发表时间：
2020-02-01
影响因子：
5.4
通讯作者：
Reggiori, Fulvio
中科院分区：
文献类型：
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作者：
Cong, Yingying;Ulasli, Mustafa;Reggiori, Fulvio
冠状病毒（CoV）核衣壳（N）蛋白是将基因组RNA整合到子代病毒颗粒中的关键。在感染的细胞中，N蛋白存在于复制-转录复合物（RTC），即CoV RNA合成的位点。研究表明，N蛋白对病毒复制很重要，并且小鼠肝炎病毒（MHV）（一种常用的CoV模型）的N蛋白与RTCs的非结构蛋白3（nsp 3）相互作用。然而，CoV生命周期的这两个方面并没有联系。通过使用系统性酵母双杂交方法，我们发现MHV N蛋白仅与nsp 3结合，而不与其他RTC成分结合，并且我们确定了N蛋白中冗余介导这种相互作用的两个不同区域。在这两个区域携带点突变的选择性N蛋白变体在体外不能结合nsp 3，导致其在体内向RTC募集的抑制。此外，与野生型N蛋白相反，该N蛋白变体损害体内和体外基因组RNA和病毒mRNA转录的刺激，这反过来导致MHV复制和后代产生的损害。总而言之，我们的研究结果表明，N蛋白通过与nsp 3结合募集到RTC是CoV生命周期中的重要步骤，因为它对最佳病毒RNA合成至关重要。重要性CoV长期以来一直被认为是人类相对无害的病原体。然而，严重急性呼吸综合征冠状病毒和中东呼吸综合征冠状病毒引起的严重呼吸道感染暴发在人类中造成了高致病性和死亡率。这些疫情突出了能够控制CoV感染的相关性。我们使用模型CoV，MHV，来研究募集N蛋白（CoV病毒体的核心组分）到细胞内平台的重要性，在细胞内平台上CoV复制、转录和翻译其基因组。通过鉴定这些细胞内平台的主要结合伴侣并产生特定的突变体，我们发现N蛋白募集到这些位置对于促进病毒RNA合成至关重要。此外，阻断这种募集强烈抑制病毒感染。因此，我们的研究结果解释了CoV生命周期的一个重要方面，并揭示了可以在抗病毒治疗中靶向的病毒蛋白的相互作用。
Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo. Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.