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冠状病毒nsp14外切核糖核酸酶与辅因子nsp10的界面对于有效的病毒复制和酶活性至关重要。

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

作者信息

Grimes Samantha L, Heaton Brook E, Anderson Mackenzie L, Burke Katie, Stevens Laura, Lu Xiaotao, Heaton Nicholas S, Denison Mark R, Anderson-Daniels Jordan

机构信息

Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA.

出版信息

J Virol. 2025 Feb 25;99(2):e0170824. doi: 10.1128/jvi.01708-24. Epub 2025 Jan 10.

Abstract

Coronaviruses (CoVs) encode non-structural proteins (nsp's) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3'-5' exoribonuclease in non-structural protein 14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The enzymatic activity of nsp14-ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-nsp10 interface and recovered mutant viruses with a range of impairments in replication and biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between wild type-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-nsp10 interface alanine substitutions impaired exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-nsp10 interface for viral inhibition and attenuation.IMPORTANCECoronavirus replication requires assembly of a replication transcription complex composed of nsp's, including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity requires the non-enzymatic cofactor nsp10, but the determinants and importance of the nsp14-nsp10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-nsp10 interface are essential for efficient viral replication and exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non-active-site targets for virus inhibition and attenuation.

摘要

冠状病毒(CoV)编码非结构蛋白(nsp)1 - 16,这些蛋白组装形成在病毒RNA合成中起作用的复制转录复合体。所有冠状病毒在非结构蛋白14(nsp14 - ExoN)中编码一种校对3' - 5'外切核糖核酸酶,该酶介导校对和高保真复制,并且在复制和发病机制中的其他作用至关重要。在辅因子nsp10存在的情况下,nsp14 - ExoN的酶活性会增强。我们在鼠肝炎病毒(MHV)的nsp14的nsp14 - nsp10界面引入丙氨酸替代,并获得了一系列在复制和生化外切核酸酶活性方面受损的突变病毒。其中两个替代,nsp14 K7A和D8A,其损伤程度介于野生型MHV nsp14和已知的ExoN( - )D89A / E91A nsp14催化失活突变体之间。所有引入的nsp14 - nsp10界面丙氨酸替代均损害了外切核酸酶活性。K7A和D8A突变病毒传代后,在nsp14中选择了与改善的突变病毒复制和外切核酸酶活性相关的第二位点非同义突变。这些结果证实了nsp14 - nsp10相互作用对于有效酶活性和病毒复制的重要作用,确定了nsp14 - nsp10相互作用的近端和远距离决定因素,并支持将nsp14 - nsp10界面作为病毒抑制和减毒的靶点。

重要性

冠状病毒复制需要由nsp组成的复制转录复合体的组装,这些nsp包括聚合酶、解旋酶、外切核酸酶、加帽酶和非酶辅因子。冠状病毒nsp14外切核糖核酸酶在病毒生命周期中介导多种功能,包括基因组和亚基因组RNA合成、RNA重组、RNA校对和高保真复制,以及对许多核苷类似物的天然抗性。nsp - 14外切核酸酶活性需要非酶辅因子nsp10,但病毒复制过程中nsp14 - nsp10相互作用的决定因素和重要性尚未明确。在这里,我们表明对于冠状病毒鼠肝炎病毒,nsp14 - nsp10界面处的nsp14残基对于有效的病毒复制和外切核酸酶活性至关重要。这些结果为冠状病毒复制转录复合体内蛋白质相互作用的要求提供了新的见解,并且它们可能揭示用于病毒抑制和减毒的新的非活性位点靶点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e717/11852845/ea86c3069039/jvi.01708-24.f001.jpg

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