tetano
Editor, Senior Moderator
Protein Expr Purif
. 2021 Apr 29;105894.
doi: 10.1016/j.pep.2021.105894. Online ahead of print.
Reconstitution and functional characterization of SARS-CoV-2 proofreading complex
Zhijun Ma[SUP] 1 [/SUP], Yasin Pourfarjam[SUP] 1 [/SUP], In-Kwon Kim[SUP] 2 [/SUP]
Affiliations
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 Ca[SUP]2+[/SUP], nsp14-nsp10 is completely inhibited. Importantly, the reconstituted SARS-CoV-2 nsp14-nsp10 efficiently removed the A:A 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: COVID-19; RNA replication; SARS-CoV-2; nsp10; nsp14; proofreading.
. 2021 Apr 29;105894.
doi: 10.1016/j.pep.2021.105894. Online ahead of print.
Reconstitution and functional characterization of SARS-CoV-2 proofreading complex
Zhijun Ma[SUP] 1 [/SUP], Yasin Pourfarjam[SUP] 1 [/SUP], In-Kwon Kim[SUP] 2 [/SUP]
Affiliations
- PMID: 33933612
- DOI: 10.1016/j.pep.2021.105894
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 Ca[SUP]2+[/SUP], nsp14-nsp10 is completely inhibited. Importantly, the reconstituted SARS-CoV-2 nsp14-nsp10 efficiently removed the A:A 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: COVID-19; RNA replication; SARS-CoV-2; nsp10; nsp14; proofreading.