key: cord-0737368-6t11mudp authors: Yan, Liming; Yang, Yunxiang; Li, Mingyu; Zhang, Ying; Zheng, Litao; Ge, Ji; Huang, Yucen; Liu, Zhenyu; Wang, Tao; Gao, Shan; Zhang, Ran; Huang, Yuanyun; Guddat, Luke W.; Gao, Yan; Rao, Zihe; Lou, Zhiyong title: Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading date: 2021-05-24 journal: Cell DOI: 10.1016/j.cell.2021.05.033 sha: b497c59a35bdcedc8df4ff103e1d448eb689867e doc_id: 737368 cord_uid: 6t11mudp 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 Replication-Transcription Complex (RTC) in a form identified as Cap(0)-RTC, which couples a Co-transcriptional Capping Complex (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) (7MeGpppA) 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. In mCap(0)-RTC, one nsp10/nsp14 complex associates with Cap(-1)'-RTC 164 composed of one nsp7, two nsp8, one nsp9, one nsp12, two nsp13 and a paired 165 template-primer RNA, being mediated by the contact of nsp9 and nsp12 NiRAN with 166 nsp14 (Figures 1A and 1B) . The architecture of Cap(-1)'-RTC is generally similar to a 167 previously reported structure (Yan et al., 2020a) . Nsp9 and nsp12 NiRAN make 168 contacts with nsp14 ExoN to stabilize the association of nsp10/nsp14 in Cap(0)-refers to the helicase molecule that binds the 5' extension of template (Yan et al., 172 2020a; Yan et al., 2020b) . 173 In dCap(0)-RTC, two Cap(0)-RTC protomers are oriented by a two-fold axis to 174 form a dimer ( Figures 1A and 1C , Figure S5B In N7-CCC, the rod-shaped nsp10/nsp14 complex vertically inserts into and is 194 stabilized by a canyon region formed by nsp9 and nsp12 NiRAN (Figures 2A and 195 2B). Nsp9 and nsp10 flank nsp14 ExoN from two sides to stabilize the assembly. Table S2 ). The side chain nitrogen atoms of nsp9 R111 form hydrogen bonds with the 206 imidazole group of nsp14 H95 and the carbonyl of nsp14 T103 and a side chain nitrogen 207 of nsp14 K13 forms a hydrogen bond with nsp12 Q224. The side chain nitrogen atoms of 208 nsp14 R98 contact with nsp12 Q81 and nsp12 E84. The overall structure of nsp10/nsp14 209 complex in Cap(0)-RTC shows strong similarities with that reported in the crystal 210 structures of nsp10/nsp14 complex(Ma et al., 2015) ( Figure S6A ). The residues of 211 nsp14 interacting with nsp10 includes nsp14 T5, nsp14 D10, nsp14 T21, nsp14 T25, nsp14 H26, 212 are consistent with the reported crystal structure (Ma et al., 2015) . Sequence 214 comparison shows that these key interacting residues are highly conserved in 215 SARS-CoV-2, SARS-CoV, MERS-CoV and RaTG13 ( Figure S6B ), suggesting a 216 common mechanism for N7-CCC formation in CoVs. The N-terminus of nsp9 inserts 217 into the catalytic center of nsp12 NiRAN, which is similar to that observed in Cap(-218 1)'-RTC (Yan et al., 2020a) , but the bound GDP-Mg 2+ in the nsp12 NiRAN catalytic 219 center is missing. It is noteworthy that a recent study suggests that nsp9 is a 220 substrate for nsp12 NiRAN NMPylation, but in another study it is debated that nsp7 221 is the substrate (Conti et al., 2020; Slanina et al., 2021) . We hypothesize that the 222 post-translation modification of nsp9 by nsp12 NiRAN might play a role in complex 223 formation. 224 The inter-protomer interactions of dCap(0)-RTC are contributed by nsp7, nsp8-2, 227 nsp12 NiRAN/thumb/palm/fingers, nsp13-2 ZBD, nsp14 ExoN and N7-MTase at five 228 separate regions ( Figure 3A , Table S3 ). 229 In region 1, nsp14 V460, nsp14 Y465, nsp14 L479 and nsp14 V483 of nsp14 N7-MTase in 230 protomer A forms a hydrophobic interface with the residues nsp13-2 I79, nsp13-2 F81 and 231 nsp13-2 F90 of nsp13-2 ZBD' in protomer B ( Figure 3B ). Three hydrogen bonds are 232 observed between nsp14 D464, nsp14 S470 and nsp14 Y517 of nsp14 N7-MTase in 233 protomer A and nsp8-2 Y12 (in nsp8-2'), nsp12 S904 (in nsp12 thumb') and nsp13-2 N86 (in 234 nsp13-2 ZBD') in protomer B ( Figure 3C) . Notably, the loop region of nsp14 N7-235 MTase spanning residues nsp14 S454-nsp14 D464, which cannot be traced in the 236 reported crystal structure, is well defined with unambiguous cryo-EM density ( Figure 237 S6A, right panel). We reason that the contact of this region with nsp13-2 ZBD' in the 238 other Cap(0)-RTC protomer helps to stabilize its conformation. 239 In region 2, the residues of nsp14 N7-MTase (including nsp14 L406, nsp14 K433, 240 nsp14 Q441 and nsp14 I502) and ExoN (residue nsp14 D172) in protomer A constitute a set 241 of inter-molecular interactions with the residues of nsp12 thumb' (including 242 nsp12 Q886, nsp12 R889, nsp12 E917 and nsp12 E919) and nsp12 fingers' (including 243 nsp12 K426 and nsp12 E431) in protomer B ( Figure 3D Figures 4C and 4G) . Moreover, the loop region spanning residues nsp13-2 G203-nsp13-277 2 A208 of nsp13-2 1B in dCap(0)-RTC contacts nsp13-1 ZBD and nsp8-1 ( Figure 4J ). 278 In this region, the side chain of nsp13-2 D204 hydrogen bonds with N η1 of nsp8-2 R75 in 279 the central long helix of nsp8-2, and the carbonyl oxygen of nsp13-2 G206 contacts with 280 the side chain of nsp13-1 Q11 in the ZBD. Previous studies on SARS-CoV-2 RTCs have 281 shown that nsp8-1 participates in the assembly of C-RTC to arm the paired template- In total, 3 µL of protein sample at 3 mg/mL (added with 0.025% DDM) was 754 applied onto a H2/O2 glow-discharged, 200-mesh Quantifoil R0.6/1.0 grid 755 (Quantifoil, Micro Tools GmbH, Germany). The grid was then blotted for 3.0 s with a 756 blot force of 0 at 8°C and 100% humidity and plunge-frozen in liquid ethane using a 757 Vitrobot (Thermo Fisher Scientific, USA). Cryo-EM data were collected with a 300 758 keV Titan Krios electron microscope (Thermo Fisher Scientific, USA) and a K3 direct 759 electron detector (Gatan, USA). Images were recorded at 22500× magnification and 760 calibrated at a super-resolution pixel size of 0.82 Å/pixel. The exposure time was set 761 to 2 s with a total accumulated dose of 60 electrons per Å 2 . All images were 762 automatically recorded using SerialEM. A total of 12,704 images were collected with 763 a defocus range from -2.0 µm to -1.0 µm. Statistics for data collection and refinement 764 are in Table S1 . The methods for processing are described in Figure S2 based on complex integrity. This particle set was used to do Ab-Initio reconstruction 775 in five classes, which were then used as 3D volume templates for heterogeneous 776 refinement, with 135,801 particles converged into dCap(0)-RTC complex class and 777 80,256 particles converged into mCap(0)-RTC complex class. Next, these particles 778 were imported into RELION 3.03 (Scheres, 2012) to perform local classification to 779 obtain one class particle with final resolution 3.35 Å and 3.78 Å, respectively. The 780 methods are described in Figure S2 and S4. 781 101 150 151 259 260 442 443 596 601 nsp13 4 31 50 249 250 365 366 581 582 620 621 679 680 815 A (25 mM HEPES, pH 7.0, 4 mM MgCl 2 , 10% glycerol, 4 mM DTT) and buffer B (25 700 mM HEPES, pH7.0, 1 M NaCl, 4 mM MgCl 2 , 10% glycerol, 4 mM DTT nsp10/nsp14 complex was concentrated to 8 mg/mL and stored at 4 °C. The Nsp9 was cloned into a 707 modified pET-28b-SUMO with the N-terminus of a fusion of 6× His-tag. The protein 708 was expressed in E. coli strain BL21 (DE3) and homogenized with an ultra-high-pressure cell disrupter at 4 °C. The lysate was 711 centrifuged at 12,000 rpm for 30 min to remove cell debris. The fusion protein was 712 purified by Ni-NTA (Novagen, USA) affinity chromatography and by application to a 713 Model building and refinement To build the structure of SARS-CoV-2 Cap(0)-RTC complex, we started with the 784 model of the SARS-CoV-2 nsp12 and nsp7-8 complex (PDB: 7BTF) These were 786 individually placed and rigid-body fitted into the cryo-EM map using UCSF ) with the guidance of the cryo-EM map, and with real space refinement using The data validation statistics are shown in Table S1 In Figures S2 and S4, the resolution estimations of cryo-EM density maps are 793 based on the 0.143 Fourier Shell Correlation (FSC) criterion New tools for the analysis and validation of cryo-EM maps and atomic 798 models In 800 vitro reconstitution of SARS-coronavirus mRNA cap methylation RNA 3'-end mismatch 802 excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 803 exoribonuclease complex Methionine-Binding Residues in Coronavirus nsp14 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