A novel coronavirus (COVID-19 virus) 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) is the central component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. We report the cryo-EM structure of COVID-19 virus fulllength nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-Å resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified βhairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics targeting viral RdRp.

Highlights d Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed d Conformational changes in nsp8 and its interaction with the exiting RNA are observed d Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated d Transition model from primase complex to polymerase complex is proposed

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ringshaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.assembly | packaging B unyaviruses constitute the largest segmented negative-sense single-stranded RNA (-ssRNA) virus family, which is subdivided into Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus genera (1). The genomes of Bunyaviridae members are featured by three segments, the large (L), middle (M), and small (S) segments (2, 3). The L segment encodes an RNA-dependent RNA polymerase (RdRp), the M segment encodes a precursor of glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid protein (NP). Moreover, a few Bunyaviridae members possess a nonstructural protein (NSs and/or NSm) using an ambisense coding strategy by the S and M segments (4).Similar to other -ssRNA viruses, all members of the Bunyaviridae family contain a ribonucleoprotein (RNP) complex composed of genomic RNA enwrapped by the NP (4). After entry into the cytoplasm through membrane fusion mediated by glycoproteins, the RNP is released from the virion and serves as the template with which the copackaged RdRp transcribes mRNAs from the viral genome in the RNP. In the later stage of virus replication, complementary positive-strand RNA (cRNA) is produced in the form of an RNP. The RNP serves as the template for replication that generates the viral genomic RNA in the form of an RNP ready to be packaged in the virion. Throughout...

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...

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