Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target

Abstract

A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key 5 component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-Å. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 10 possesses a newly identified β-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp. 15One Sentence Summary: Structure of 2019-nCov RNA polymerase. infections and 4291 fatalities have been confirmed globally. 2019-nCoV is reported to be a new member of the betacoronavirus genus and is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) and to several bat coronaviruses (4). Compared to SARS-CoV and 5 MERS-CoV, 2019-nCoV appears to exhibit faster human-to-human transmission, thus leading to the WHO declaration of a Public Health Emergency of International Concern (PHEIC)(1, 3).CoVs employ a multi-subunit replication/transcription machinery, being assembled by a set of non-structural proteins (nsp) produced as cleavage products of the ORF1a and ORF1ab viral polyproteins (5) to facilitate virus replication and transcription. A key component, the RNA-10 dependent RNA polymerase (nsp12), catalyzes the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of 2019-nCoV, possibly with the assistance of nsp7 and nsp8 as co-factors (5,6). Therefore, nsp12 is a primary target for the nucleotide analog antiviral inhibitors, e.g. remdesivir which shows potential to treat 2019-nCoV infections (7,8). To inform drug design we have determined the structure of nsp12, in complex with its cofactors nsp7 15 and nsp8 by cryo-Electron Microscopy (Cryo-EM) using two different protocols, one in the absence of DTT (Dataset-1) and the other in the presence of DTT (Dataset-2).The bacterially expressed full-length 2019-nCoV nsp12 (residues S1-Q932) was incubated with nsp7 (residues S1-Q83) and nsp8 (residues A1-Q198), and the complex was then purified (fig. S1). Cryo-EM grids were prepared using this complex and preliminary screening revealed 20 excellent particle density with good dispersity. After collecting and processing 7,994 micrograph movies, we obtained a 2.9-Å resolution 3D reconstruction of one nsp12 monomer in complex with on...

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

“…The structure of the apo RdRp complex contains one nsp12, one nsp7 and two nsp8, with an overall arrangement resembling those seen in the SARS-CoV and the recently solved structure of SARS-CoV-2(13, 21) ( Figure 2). Different from the SARS-CoV RdRp structure but similar to the recent SARS-CoV-2 RdRp structure, our structure reveals that nsp12 also contains an Nterminal β-hairpin (residues 31-50) and an extended nidovirus RdRp-associated nucleotidyltransferase domain (NiRAN, residues 115-250) (22), with seven helices and a three β-strands (13,21). Following the NiRAN domain is an interface domain (residues 251-365) composed of three helices and five β-strands, which is connected to the RdRp domain (residues 366-920).…”

“…As such, RdRp has been a subject of intensive efforts of structural biology. The structures of nsp7, nsp8, and the complex of nsp12-nsp7-nsp8 have been determined (13,(18)(19)(20)(21), providing an overall architecture of the RdRp complex assembly.…”

The global population of SARS-CoV-2 is composed of six major subtypes

“…The WSP nsp 12-[967] resulted in a proline in eleven subtypes of SARS-CoV-2 and a leucine in others five subtypes at the aa residue #323 of the NSP12 (RNA-dependent RNA polymerase, RdRP) protein. It is located at the Interface domain of RdRP of SARS-CoV-2, which is responsible for the connection between the nidovirus RdRP-associated nucleotidyltransferase domain (NiRAN) and the “Right hand” polymerase domain 18 . The S protein mediates viral entry into host cells by first binding to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) in the S1 subunit and then fusing the viral and host membranes through the S2 subunit 19–22 .…”

Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

“…Treatment of infections caused by betacoronoaviruses have focused on three therapeutic strategies; vaccination with the spike glycoprotein of the SARS-Cov-2 envelope (Wrapp et al, 2020), and small-molecule targeting of conserved viral enzymes (e.g. the Mpro protease (Zhenming et al 2020) (Yang et al, 2005) and the RNApolymerase (Yan et al 2020). Nevertheless, some of the betacoronaviral non-structural proteins appear important for viral replication within SARS-CoV and influence pathogenesis (Frieman et al, 2012).…”