Reconstitution and functional characterization of SARS-CoV-2 proofreading complex.
Protein Expr Purif
; 185: 105894, 2021 09.
Article
in English
| MEDLINE | ID: covidwho-1209890
ABSTRACT
The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has led to a world-wild pandemic. The replication of SARS-CoV-2 RNA genome involves the core replication-transcription complex (RTC, nsp12-nsp7-nsp8) and the proofreading complex (nsp14-nsp10) that can correct mismatched base pairs during replication. Structures and functions of SARS-CoV-2 RTC have been actively studied, yet little is known about SARS-CoV-2 nsp14-nsp10. Here, we purified, reconstituted, and characterized the SARS-CoV-2 nsp14-nsp10 proofreading nuclease in vitro. We show that SARS-CoV-2 nsp14 is activated by nsp10, functioning as a potent RNase that can hydrolyze RNAs in the context of single- and double-stranded RNA and RNA/DNA hybrid duplex. SARS-CoV-2 nsp14-nsp10 shows a metal-dependent nuclease activity but has different metal selectivity from RTC. While RTC is activated by Ca2+, nsp14-nsp10 is completely inhibited. Importantly, the reconstituted SARS-CoV-2 nsp14-nsp10 efficiently removed the AA mismatch at the 3'-end of the primer, enabling the stalled RTC to restart RNA replication. Our collective results confirm that SARS-CoV-2 nsp14-nsp10 functions as the RNA proofreading complex in SARS-CoV-2 replication and provide a useful foundation to understand the structure and function of SARS-CoV-2 RNA metabolism.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA, Viral
/
Viral Nonstructural Proteins
/
Exoribonucleases
/
Viral Regulatory and Accessory Proteins
/
SARS-CoV-2
/
COVID-19
Limits:
Humans
Language:
English
Journal:
Protein Expr Purif
Journal subject:
Molecular Biology
Year:
2021
Document Type:
Article
Affiliation country:
J.pep.2021.105894
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