key: cord-0900453-cgtwkgqf authors: Shi, Wei; Chen, Ming; Yang, Yang; Zhou, Wei; Chen, Shiyun; Yang, Yi; Hu, Yangbo; Liu, Bin title: A dynamic regulatory interface on SARS-CoV-2 RNA polymerase date: 2020-07-30 journal: bioRxiv DOI: 10.1101/2020.07.30.229187 sha: 63dad50349405524e1b0a71d62114e99aeaeb2ff doc_id: 900453 cord_uid: cgtwkgqf The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design. Author summary Since it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health. RdRp. These observations let us to suggest this interface as a potential new drug 48 intervention site, outside of the canonical polymerase active center, in RdRp for antiviral 49 design. Our findings would provide new insights into regulatory mechanism of this novel 50 SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and 51 benefit the global public health. Overall architecture of RdRp/RNA complex 74 To obtain the target structure, we incubated the individually purified nsp12, nsp7 and 75 nsp8 proteins in the presence of a template-primer RNA duplex and 76 adenosine-5′-triphosphate (ATP) to allow one nucleotide to be incorporated into the 3′ 77 end of the primer chain. The resulting RdRp/RNA complex was used in cryo-EM 78 imaging and structural determination. More than 4 million initial particles were 79 automatically picked from 4,750 micrograph movies and 123,246 particle projections 80 were used to reconstruct the final overall 3.4 Å resolution density map of the complex 81 (S1 Fig and S1 Table) . 82 The structure of RdRp/RNA complex contains one nsp12, one nsp7 and two nsp8 to accommodate the RNA duplex ( Fig 3A) . Notably, a ~3.1 Å movement of the RdRp wild-type or mutated complex was required for shifting RNA (Fig 4E and 4F ). Based on these structural and biochemical data, it is likely that a moderate interface 182 between nsp8(I) and nsp7 may facilitate the formation of a relatively stable polymerase 183 core and concurrently allow for certain flexibility of the nsp8(I)/nsp7 interface, which are 184 critical for highly efficient and processive RNA synthesis. This nsp8(I)/nsp7 interface 185 rearrangement appears to be translocation-coupled during nucleotide addition cycle and 186 leads to a different interface between nsp8(I) and nsp7 subunits. Thus, we suggest that the 187 nsp8(I)/nsp7 interface is an important regulatory site for RNA polymerase activity of 188 RdRp, and this regulation mechanism might also be applicable to other coronaviruses due 189 to the conservation in sequence and structure. In addition, small-molecule antiviral agents Protein expression and purification 199 The full-length genes of SARS-CoV-2 nsp7 (residues 1-83), nsp8 (residues 1-198) and Table) into AGCGCGGATGCCCAATCATTCTTGAATCGAGTCTGTGGAGT ATCGGCCGCTCGGTTGACGCCTTGTGGTACTGGGACCTCT ACCGACGTAGTTTACCGGGCCTTCGACATATACAATGACAA GGTTGCGGGGTTCGCTAAGTTTTTAAAGACTAACTGTTGTC GTTTCCAAGAAAAAGATGAAGACGACAATCTGATTGACTC GTACTTTGTAGTCAAAAGACATACATTTAGTAACTACCAAC ACGAGGAGACTATCTATAACTTGCTGAAAGACTGTCCCGC CGTCGCGAAGCACGACTTTTTTAAATTTAGAATCGATGGCG ATATGGTGCCGCATATTAGCCGCCAGAGGTTAACAAAGTAC ACAATGGCAGACCTGGTATATGCGCTACGCCACTTTGATGA AGGGAATTGTGACACCCTCAAGGAGATTTTGGTAACTTAC AATTGTTGTGACGACGACTACTTTAACAAAAAGGATTGGTA TGATTTCGTTGAAAATCCCGACATACTGCGCGTGTATGCGA ACCTGGGCGAACGCGTGCGCCAGGCGCTCCTCAAGACGG TTCAATTCTGCGATGCGATGCGCAACGCGGGCATTGTGGG CGTGCTGACCCTGGATAACCAGGATCTGAACGGCAATTGG TATGATTTCGGAGACTTCATACAAACCACTCCCGGAAGCG GCGTGCCGGTGGTGGATAGCTACTATAGCCTGCTGATGCCG ATTCTGACCCTGACCCGCGCGCTGACCGCTGAGAGTCATG TAGATACCGACTTAACCAAACCTTATATTAAATGGGATCTAT TGAAGTACGACTTTACGGAAGAGCGGTTAAAGCTTTTCGA CCGATACTTTAAATATTGGGATCAGACCTATCATCCGAACT GCGTGAACTGCCTGGATGATCGCTGCATTCTGCATTGCGCG AACTTTAACGTGCTGTTTAGCACCGTGTTTCCGCCGACCA GCTTTGGCCCGCTGGTGCGCAAGATATTCGTTGATGGCGTG CCGTTTGTGGTGAGCACCGGCTATCATTTCAGGGAGCTGG GCGTGGTGCATAACCAGGATGTGAACCTGCATAGCAGCCG CCTGAGCTTTAAAGAACTGCTAGTATACGCCGCTGACCCG GCGATGCATGCGGCGAGCGGCAACCTGCTGCTGGATAAAA GAACGACTTGTTTCTCTGTCGCGGCCCTCACAAACAATGT CGCATTCCAAACCGTGAAGCCTGGGAATTTCAATAAGGAC TTTTATGATTTTGCCGTCTCTAAGGGGTTTTTTAAAGAAGG CTCAAGCGTAGAGCTAAAGCACTTCTTTTTTGCCCAAGAT GGTAATGCTGCCATATCGGACTACGACTACTATAGGTACAA TTTACCAACTATGTGCGACATCAGGCAACTGCTGTTCGTTG TAGAGGTTGTGGATAAATATTTCGACTGCTACGACGGCGGC TGCATTAACGCGAACCAGGTGATTGTGAACAACCTGGATA AGTCCGCCGGGTTCCCGTTCAATAAGTGGGGCAAAGCCCG TTTGTACTACGATTCAATGAGCTATGAAGACCAAGACGCCC TCTTCGCTTACACTAAGAGAAATGTCATCCCTACGATCACA CAAATGAATCTGAAATATGCGATTAGCGCGAAGAATCGGG CGCGCACCGTGGCGGGCGTGAGCATTTGCAGCACCATGAC CAACCGCCAGTTTCATCAGAAACTGCTGAAGTCCATAGCG GCGACCCGCGGCGCAACAGTAGTAATTGGAACCTCAAAAT TTTATGGGGGCTGGCATAACATGCTTAAAACCGTCTACTCA GATGTCGAGAATCCGCATCTTATGGGCTGGGATTATCCGAA ATGCGATCGCGCGATGCCGAACATGCTGCGCATTATGGCGA GCCTGGTGCTGGCGCGCAAACATACCACCTGCTGCAGCCT GAGCCATCGCTTCTACCGTCTGGCGAACGAATGCGCGCAG GTGCTGAGCGAAATGGTGATGTGCGGCGGCAGCCTGTATG TTAAGCCGGGCGGCACGAGCAGCGGCGATGCGACCACCG CGTATGCGAACAGCGTGTTTAACATTTGCCAGGCGGTTACT GCAAATGTAAATGCCCTATTAAGCACGGATGGTAACAAAAT TGCTGATAAATATGTGCGCAACCTCCAGCATCGACTTTACG AGTGTTTATACCGAAATCGCGATGTAGATACTGATTTCGTA AACGAGTTTTATGCCTACCTTCGAAAACATTTCTCAATGAT GATCCTGAGCGATGATGCGGTGGTGTGCTTCAATTCAACCT ACGCCTCCCAAGGCCTCGTTGCTTCGATAAAAAACTTTAA ATCTGTATTGTACTACCAAAATAATGTTTTCATGAGTGAAG CGAAATGCTGGACCGAAACTGACCTAACGAAGGGACCTCA TGAATTCTGTTCCCAGCATACCATGCTGGTGAAACAGGGC GATGATTATGTGTATCTGCCGTATCCAGATCCGAGCCGCATT CTGGGCGCGGGGTGTTTCGTTGACGACATCGTAAAAACAG ATGGTACGTTGATGATAGAGCGCTTTGTGTCATTAGCCATA GATGCCTATCCGCTAACAAAACATCCCAATCAAGAGTACGC TGACGTTTTTCATCTGTATCTACAGTATATACGGAAGCTGCA CGACGAGCTAACGGGACACATGTTGGATATGTACTCAGTG ATGCTGACCAACGATAACACCAGCCGCTATTGGGAACCGG AATTCTACGAGGCGATGTATACTCCTCACACCGTGCTGCAG Codon-optimized SARS-CoV-2 nsp7 AGCAAGATGTCGGATGTGAAATGCACCAGCGTGGTACTTC TGTCAGTTCTACAACAACTGCGCGTTGAGTCCTCGTCGAA ACTCTGGGCGCAATGTGTACAATTACACAATGACATTTTGC TAGCCAAAGACACAACTGAAGCGTTTGAGAAGATGGTCA GTCTGCTGTCAGTCCTGCTGTCGATGCAAGGCGCGGTGGA TATTAACAAACTGTGCGAAGAAATGCTGGATAACCGCGCG ACCCTGCAG Codon-optimized SARS-CoV-2 nsp8 GCGATTGCGAGCGAATTTAGCAGCCTGCCGAGCTATGCGG CGTTTGCGACCGCGCAGGAAGCGTACGAGCAAGCGGTCG CTAATGGGGACTCGGAGGTCGTGCTCAAAAAACTAAAGA AATCCTTGAACGTGGCGAAGTCGGAGTTTGATCGCGATGC GGCGATGCAGAGAAAGCTGGAAAAGATGGCGGATCAGGC GATGACCCAGATGTATAAACAGGCGCGCAGCGAAGATAAA CGCGCGAAAGTGACCAGCGCGATGCAGACCATGCTGTTTA CCATGCTGAGGAAGCTCGACAATGATGCGCTGAACAACAT TATTAACAACGCGCGCGATGGCTGCGTGCCGCTGAACATTA TTCCGCTCACAACCGCCGCAAAGCTGATGGTAGTTATCCCT GACTACAATACCTACAAAAACACTTGCGATGGCACCACCT TTACCTATGCGAGCGCGCTGTGGGAAATTCAGCAGGTGGT GGATGCGGATAGCAAGATCGTACAGCTGAGCGAAATTAGC ATGGATAACAGCCCGAACCTGGCGTGGCCGCTGATTGTGA CCGCGCTGCGCGCGAACAGCGCGGTGAAACTGCAG Novel Coronavirus-Infected Pneumonia Clinical features of patients infected with 2019 novel 328 coronavirus in Wuhan An interactive web-based dashboard to track COVID-19 in real time Epub 2020/02/23 visualization system for exploratory research and analysis Coot: model-building tools for molecular graphics Python-based system for macromolecular structure solution MolProbity: 444 all-atom structure validation for macromolecular crystallography Addressing preferred 448 specimen orientation in single-particle cryo-EM through tilting New tools for the 451 analysis and validation of cryo-EM maps and atomic models Meeting 455 modern challenges in visualization and analysis 305 We thank the staff at the cryo-EM facility and instrument core facility in the HormelThe authors declare no competing interests.