Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.


  • 影响因子:4.02
  • DOI:10.1128/JVI.01925-19
  • 作者列表:"Cong Y","Ulasli M","Schepers H","Mauthe M","V'kovski P","Kriegenburg F","Thiel V","de Haan CAM","Reggiori F
  • 发表时间:2020-01-31

: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.


: 冠状病毒 (CoV) 核衣壳蛋白 (N) 是将基因组 RNA 掺入子代病毒颗粒的关键。在感染的细胞中,N 蛋白存在于复制-转录复合物 (rtc),CoV RNA 合成的位点。研究表明,N 蛋白对病毒复制很重要,小鼠肝炎病毒 (MHV) 是一种常用的模型 CoV,与非结构蛋白 3 (nsp3) 相互作用, RTCs 的一个组件。然而,CoV 生命周期的这两个方面并没有联系起来。我们发现 MHV N 蛋白通过使用系统酵母双杂交方法专门与 nsp3 而不是其他 RTC 成分结合, 我们在 N 蛋白中发现了两个不同的区域,冗余地介导了这种相互作用。在这两个区域携带点突变的选择性 N 蛋白变体在体外未能结合 nsp3,导致其在体内招募到 RTCs 的抑制。与野生型 N 蛋白相比,该 N 蛋白变体在体内和体外损害基因组 RNA 和病毒 mRNA 转录的刺激,这反过来又导致 MHV 复制和子代生产受损。总之,我们的结果表明,N 蛋白通过与 nsp3 结合招募到 RTCs,是 CoV 生命周期中必不可少的一步,因为它对最佳病毒 RNA 合成至关重要。重要性 CoVs 长期以来被认为是对人类相对无害的病原体。然而,由严重急性呼吸综合征 (CoV) 和中东呼吸综合征 (CoV) 引起的严重呼吸道感染在人类中引起高致病性和高死亡率。这些暴发突出了能够控制 CoV 感染的相关性。我们使用了一个模型 CoV MHV,来研究 CoV 病毒粒子的中心成分 N 蛋白招募到胞内平台的重要性,在胞内平台上 CoV 复制、转录和翻译它们的基因组。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,阻断这种招募强烈抑制病毒感染。因此,我们的结果解释了 CoV 生命周期的一个重要方面,并揭示了病毒蛋白的相互作用,可以靶向抗病毒治疗。



作者列表:["Cong Z","Li D","Tao Y","Lv X","Zhu X"]

METHODS::Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α2 -adrenoceptor (α2 -AR) could attenuate lung injury induced by endotoxin in rats. α2A -adrenoceptor (α2A -AR), a subtype of α2 -AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α2A -AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α2A -AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA.

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