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Related Experiment Video

Updated: Nov 7, 2025

Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro

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Reconstitution and functional characterization of SARS-CoV-2 proofreading complex.

Zhijun Ma¹, Yasin Pourfarjam¹, In-Kwon Kim¹

¹Department of Chemistry, University of Cincinnati, 301 Clifton Ct, Cincinnati, OH, 45221, USA.

Protein Expression and Purification

|May 2, 2021

Summary

The SARS-CoV-2 proofreading complex (nsp14-nsp10) was reconstituted and characterized. This complex efficiently removes RNA mismatches, enabling viral RNA replication and providing insights into SARS-CoV-2 RNA metabolism.

Keywords:

COVID-19 Proofreading RNA replication SARS-CoV-2 nsp10 nsp14

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Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Published on: May 13, 2019

Area of Science:

Virology
Molecular Biology
Biochemistry

Background:

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes a global pandemic.
Viral RNA replication relies on the replication-transcription complex (RTC) and a proofreading complex.
The function of the SARS-CoV-2 nsp14-nsp10 proofreading complex remains largely uncharacterized.

Purpose of the Study:

To purify, reconstitute, and characterize the SARS-CoV-2 nsp14-nsp10 proofreading nuclease.
To elucidate the enzymatic activity and metal ion dependency of SARS-CoV-2 nsp14-nsp10.
To investigate the role of nsp14-nsp10 in correcting replication errors and facilitating RNA synthesis.

Main Methods:

In vitro purification and reconstitution of SARS-CoV-2 nsp14-nsp10 complex.
Biochemical assays to determine nuclease activity on various RNA substrates.
Characterization of metal ion selectivity and effect on enzymatic activity.
Assessment of nsp14-nsp10's ability to remove primer-template mismatches.

Main Results:

SARS-CoV-2 nsp14, activated by nsp10, exhibits potent RNase activity against single-stranded RNA, double-stranded RNA, and RNA/DNA hybrids.
The nsp14-nsp10 complex displays metal-dependent activity with distinct metal selectivity compared to the RTC.
Ca2+ inhibits nsp14-nsp10, whereas it activates the RTC.
The reconstituted nsp14-nsp10 complex effectively removed an A:A mismatch, allowing stalled RTC to resume RNA synthesis.

Conclusions:

SARS-CoV-2 nsp14-nsp10 functions as the essential RNA proofreading complex during viral replication.
The findings provide a foundation for understanding SARS-CoV-2 RNA metabolism and potential therapeutic targets.
Characterization of nsp14-nsp10's enzymatic properties and role in error correction is crucial for viral research.