核糖核酸酶
复制(统计)
酶
病毒复制
辅因子
冠状病毒
2019年冠状病毒病(COVID-19)
生物
细胞生物学
病毒
病毒学
遗传学
生物化学
基因
核糖核酸
医学
疾病
病理
传染病(医学专业)
核糖核酸酶P
作者
Samantha Grimes,Brook E. Heaton,Mackenzie L. Anderson,Kaitlyn N. Burke,Laura J. Stevens,Xiaotao Lu,Nicholas S. Heaton,Jordan Anderson-Daniels,Jordan Anderson-Daniels
标识
DOI:10.1101/2024.09.26.615217
摘要
Coronaviruses (CoVs) encode nonstructural proteins (nsps) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3′-5′ exoribonuclease (ExoN) in nsp14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro 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 nsp14-10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, hadimpairments intermediate between WT-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-10 interface alanine substitutions impaired in vitro 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 nsp14-10 interface for viral inhibition and attenuation.
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