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

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 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.

SUBMITTER: Ma Z

PROVIDER: S-EPMC8082334 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In the early 1960's the first human coronaviruses (designated 229E and OC43) were identified as etiologic agents of the common cold, to be followed by the subsequent isolation of three more human coronaviruses similarly associated with cold-like diseases. In contrast to these "mild" coronaviruses, over the last 20 years there have been three independent events of emergence of pandemic severe and acute life-threatening respiratory diseases caused by three novel beta-coronaviruses, SARS CoV, MERS CoV and most recently SARS CoV2. Whereas the first SARS CoV appeared in November 2002 and spontaneously disappeared by the summer of 2003, MERS CoV has continued persistently to spill over to humans via an intermediary camel vector, causing tens of cases annually. Although human-to-human transmission is rare, the fatality rate of MERS CoV disease is remarkably higher than 30%. COVID-19 however, is fortunately much less fatal, despite that its etiologic agent, SARS CoV2, is tremendously infectious, particularly with the recent evolution of the Omicron variants of concern (BA.1 and BA.2). Of note, MERS CoV prevalence in camel populations in Africa and the Middle East is extremely high. Moreover, MERS CoV and SARS CoV2 co-exist in the Middle East and especially in Saudi Arabia and the UAE, where sporadic incidences of co-infection have already been reported. Co-infection, either due to reverse spill-over of SARS CoV2 to camels or in double infected humans could lead to recombination between the two viruses, rendering either SARS CoV2 more lethal or MERS CoV more transmittable. In an attempt to prepare for what could develop into a catastrophic event, we have focused on developing a novel epitope-based immunogen for MERS CoV. Implementing combinatorial phage-display conformer libraries, the Receptor Binding Motif (RBM) of the MERS CoV Spike protein has been successfully reconstituted and shown to be recognized by a panel of seven neutralizing monoclonal antibodies.

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

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