Coupling of N7-methyltransferase and 3′-5′ exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading

Abstract

The capping of mRNA and the proofreading plays essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 R eplication- T ranscription C omplex (RTC) in a form identified as Cap(0)-RTC, which couples a C o-transcriptional C apping C omplex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing a N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) ( 7Me GpppA) at 5’ end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA, but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Possible Targets of Pan-Coronavirus Antiviral Strategies for Emerging or Re-Emerging Coronaviruses

“…In addition to the inhibition of RdRp, drug hits should be evaluated for resistance to exoribonuclease (ExoN) and methyltransferase activities. NSP14 acts as (guanine-N7)-methyltransferase (N7-MTase) that catalyzes viral mRNA capping, and 3’-to-5’ proofreading ExoN that removes mis-incorporated nucleotides from the 3′ end of the nascent RNA [ 49 , 72 ], which are critical for virus replication and transcription [ 49 , 72 ]. During the capping, Cap(0)-RTC is composed of NSP12 nidovirus RdRp-associated nucleotidyltransferase (NiRAN), NSP9, NSP14, and NSP10 [ 72 ].…”

SARS-CoV-2 Portrayed against HIV: Contrary Viral Strategies in Similar Disguise

“…Conversely, the HIV-1 genome is reverse-transcribed into dsDNA after entry, a step that is catalyzed by the viral RT prebound to the viral genome ( Figure 2 a). Interestingly, this reverse-transcription process, which lacks a proof-reading step, is much more error-prone than the transcription of the SARS-CoV-2 genome which is supported by a sophisticated proof-reading mechanism [ 39 , 146 ], contributing to the comparatively broad genomic diversity of HIV-1 ( Table 1 , Figure 4 ). HIV must transport its nucleic acid to the nucleus via the nuclear pore.…”

“…However, the strength and timing of the driving immune forces, and the capacities to evade these forces are very different in both viruses. Important discriminative factors are the acute nature of SARS-CoV-2 infection, resulting in a small temporal window of active replication and adaptation, combined with a low mutation rate due to the proof-reading mechanism of the SARS-CoV-2 polymerase complex [ 146 ]. This contrasts with the chronic nature of HIV-1 infection that allows for a lifelong ongoing viral replication and adaptation with a high mutation rate in the absence of proof-reading by the HIV-1 polymerase complex.…”

SARS-CoV-2 Portrayed Against HIV: Contrary Viral Strategies in Similar Disguise

“…Conversely, the HIV-1 genome is reverse-transcribed into dsDNA after entry, a step that is catalyzed by the viral RT prebound to the viral genome (Figure 2a). Interestingly, this reverse-transcription process, which lacks a proof-reading step, is much more error-prone than the transcription of the SARS-CoV-2 genome which is supported by a sophisticated proof-reading mechanism [39,146], contributing to the comparatively broad genomic diversity of HIV-1 (Table 1, Figure 4). HIV must transport its nucleic acid to the nucleus via the nuclear pore.…”