key: cord-0935074-pet6o0g3
authors: Park, Gina J.; Osinski, Adam; Hernandez, Genaro; Eitson, Jennifer L.; Majumdar, Abir; Tonelli, Marco; Henzler-Wildman, Katie; Pawłowski, Krzysztof; Chen, Zhe; Li, Yang; Schoggins, John W.; Tagliabracci, Vincent S.
title: The mechanism of RNA capping by SARS-CoV-2
date: 2022-02-15
journal: Res Sq
DOI: 10.21203/rs.3.rs-1336910/v1
sha: f45d0ee6b7d03fdbb4290213dac489ddcb9ff458
doc_id: 935074
cord_uid: pet6o0g3

The SARS-CoV-2 RNA genome contains a 5′-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response(1-4). How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 (7Me)GpppA(2′-O-Me)-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain(5) of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp14(6) and nsp16(7) methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system(8) that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

The NiRAN domain RNAylates nsp9 111 Given the ability of the NiRAN domain to transfer NMPs to nsp9 using NTPs as substrates, we 112 wondered whether the NiRAN domain could also utilize 5′-pppRNA in a similar fashion (Fig. 2a) . 113 We synthesized a 5′-pppRNA 10-mer corresponding to the first 10 bases in the leader sequence 114 (LS10) of the SARS-CoV-2 genome (hereafter referred to as 5′-pppRNA LS10 ) (Extended Data 115 Table 1 ). We incubated 5′-pppRNA LS10 with nsp9 and nsp12 and analysed the reaction products 116 by SDS-PAGE. Remarkably, we observed an electrophoretic mobility shift in nsp9 that was time-117 dependent, sensitive to RNAse A treatment and required an active NiRAN domain, but not an 118 active RdRp domain (Fig. 2b) . Intact mass analyses of the reaction products confirmed the 119 incorporation of monophosphorylated RNA LS10 (5′-pRNA LS10 ) into nsp9 (Fig. 2c) . The reaction 120 was dependent on Mn 2+ (Extended Data Fig. 5a ) and required a triphosphate at the 5′-end of the 121 RNA (Extended Data Fig. 5b ). Substituting Ala for Asn1 reduced the incorporation of RNA LS10 122 into nsp9 (Fig. 2d) . We also observed NiRAN-dependent RNAylation of nsp9 using LS RNAs 123 ranging from 2 to 20 nucleotides (Fig. 2e) . Mutation of the first A to any other nucleotide markedly 124 reduced RNAylation (Fig. 2f) . Thus, the NiRAN domain RNAylates the N-terminus of nsp9 in a 125 substrate-selective manner. Fig. 6a ) 24, 25 . Because the NiRAN domain transfers 5′-pRNA to nsp9, we 132 hypothesized that this protein-RNA species may be an intermediate in a similar reaction 133 mechanism to that of the VSV system. To test this hypothesis, we purified the nsp9-pRNA LS10 134 species by ion exchange and gel filtration chromatography and incubated it with GDP in the 135 presence of nsp12. Treatment with GDP deRNAylated nsp9 in a NiRAN-dependent manner, as 136 judged by the nsp9 electrophoretic mobility on SDS-PAGE (Fig. 3a) and its molecular weight 137 based on intact mass analysis (Fig. 3b) . The reaction was time-dependent, (Fig 3c) , preferred Mg 2+ 138 over Mn 2+ (Extended Data Fig. 6b ) and was specific for GDP--and to some extent GTP--but 139 not the other nucleotides tested (Fig. 3d) . Interestingly, although inorganic pyrophosphate (PPi) 140 6 was able to deAMPylate nsp9-AMP, it was unable to deRNAylate nsp9-pRNA LS10 (Fig. 3e) . (See 141 Discussion) 142 We used Urea-PAGE to analyse the fate of the RNA LS10 during the deRNAylation reaction. 143 Treatment of nsp9-pRNA LS10 with nsp12 and [a-32 P]GDP resulted in a [ 32 P]-labelled RNA species 144 that migrated similarly to GpppA-RNA LS10 produced by the Vaccinia capping enzyme (Fig. 3f) . 145 The reaction was dependent on a functional NiRAN domain but not an active RdRp domain. To 146 confirm the presence of a GpppA-RNA cap, we digested the RNA produced from the nsp12 147 reaction with P1 nuclease and detected GpppA by high performance liquid chromatography/mass 148 spectrometry (HPLC/MS) analysis (Fig. 3g) . Thus, the NiRAN domain is a GDP 149 polyribonucleotidyltransferase (GDP-PRNTase) that mediates the transfer of 5′-pRNA from nsp9 150 to GDP. 151 In our attempts to generate GpppA-RNA LS10 in a "one pot" reaction, we found that GDP inhibited 152 the RNAylation reaction (Extended Data Fig. 6c) . However, the formation of GpppA-RNA LS10 153 could be generated in one pot provided that the RNAylation occurs prior to the addition of GDP 154 (Extended Data Fig. 6c, d) . 155 Nsp14 and nsp16 catalyse the formation of the cap-0 and cap-1 structures 156 The SARS-CoV-2 genome encodes an N7-MTase domain within nsp14 6 and a 2′-O-MTase in 157 nsp16, the latter of which requires nsp10 for activity 7 . Nsp14 and the nsp10/16 complex use S-158 adenosyl methionine (SAM) as the methyl donor. To test whether NiRAN-synthesized GpppA-159 RNA LS10 can be methylated, we incubated 32 P-labelled GpppA-RNA LS10 with nsp14 and/or the 160 nsp10/16 complex in the presence of SAM and separated the reaction products by Urea-PAGE 161 (Fig. 4a) . We extracted RNA from the reaction, treated it with P1 nuclease and CIP, and then 162 analysed the products by thin layer chromatography (TLC) (Fig. 4b) . As expected, the NiRAN- GpppA-RNA LS10 to form the cap-0 and cap-1 structures, respectively (Fig 4c) . Thus, the SARS- 173 CoV-2 7Me GpppA2′-O-Me-RNA capping mechanism can be reconstituted in vitro using virally 174 encoded proteins. 175 Efficient translation of mRNAs is dependent on eIF4E binding to the 7Me GpppA-RNA cap 26 . To 176 test whether the SARS-CoV-2 RNA cap is functional, we incubated [ 32 P]-labelled 7Me GpppA-177 RNA LS10 with GST-tagged eIF4E. We observed [ 32 P]-labelled RNA in GST pulldowns of 178 [ 32 P] 7Me GpppA-RNA but not the unmethylated derivative (Fig. 4d) . Thus, the 7Me GpppA-RNA cap 179 generated by SARS-CoV-2 encoded proteins is a substrate for eIF4E in vitro, suggesting that the 180 cap is functional. 182 We determined a cryo-EM structure of the nsp7/8/9/12 complex and observed a nsp9 monomer 183 bound in the NiRAN active site (Fig. 5a, Extended Data Fig. 7-9 , Extended Data Table 2 ). The 184 native N-terminus of nsp9 occupies a similar position to previously reported structures using a 185 non-native N-terminus of nsp9 (Fig. 5b, c) 21 . Our cryo-EM analysis was hindered by the preferred 186 orientation of the complex and sample heterogeneity, yielding final maps with high levels of 187 anisotropy, with distal portions of nsp9 missing, and weak density for the N-lobe of the NiRAN 188 domain (Extended Data Fig. 7, 8) . Therefore, we used our model and the complex structure by 189 Yan et al. 21 (PDBID: 7CYQ) to study the structural basis of NiRAN-mediated RNA capping.

The first four residues of nsp9 extend into the NiRAN active site, forming electrostatic and 191 hydrophobic contacts in and around a groove near the kinase-like active site (Fig. 5d) . Asn1 of 192 nsp9 is positioned inside of the active site, primed for transfer of 5′-pppRNA onto its N-terminus.

Although the terminal NH2 group of nsp9 is the substrate for RNAylation, the local quality of the 194 structures is not high enough to distinguish its exact position. We have modelled the nsp9 acceptor

NH2 pointing towards what appears to be the phosphates of the nucleotide analogue UMP-NPP in 196 the active site (Fig 5b) . In the structure by Yan et al. 21 , Asn1 was assigned an opposite 197 conformation and there are unmodeled residues (non-native N-terminus; NH2-Gly-Ser-) visible in 198 the density maps, distorting local structural features (Fig. 5c , arrow) 27 .

Asn2 of nsp9 is in a negatively charged cleft around the NiRAN active site, and contacts Arg733, 200 which extends from the polymerase domain and is partially responsible for positioning nsp9 (Fig.   201 5e). Both Leu4 and the C-terminal helix of nsp9 form hydrophobic interactions with a β-sheet (β8-202 β9-β10) in the N-lobe of the NiRAN domain (Fig. 5e, f) . The N-terminal cap of the nsp9 C-terminal 203 helix also forms electrostatic interactions with a negatively charged pocket on the surface of the 204 NiRAN domain (Fig. 5f) . Nsp12 lacking the RdRp domain (DRdRp; 1-326) neither RNAylates 205 nsp9 nor processes nsp9-pRNA LS10 to form GpppA-RNA (Fig. 5g) . Likewise, deleting the C-206 terminal helix on nsp9 (ΔC; 1-92) and Ala substitutions of Asn1 and Asn2 abolished RNAylation 207 (Fig. 5h) . 208 The NiRAN domain resembles SelO, with an RMSD of 5.7 Å over 224 Cα atoms (PDB ID: ); however, like in SelO, Asp208 is next to the metal binding Asn209 (PKA; N171) and may 214 act as a catalytic base to activate the NH2 group on the N-terminus of nsp9 (Fig. 5i) . 215 In canonical kinases, the b1-b2 G-loop stabilizes the phosphates of ATP 28 . In contrast, the NiRAN 216 domain contains a b-hairpin insert (b2-b3) where the b1-b2 G-loop should be (Extended Data 217 Fig. 10b ). This insertion not only makes contacts with the N-terminus of nsp9, but also contains a 218 conserved Lys (K50) that extends into the active site and stabilizes the phosphates of the bound 219 nucleotide. Likewise, Arg116 also contacts the phosphates of the nucleotide. SelO contains a 220 similar set of basic residues pointing into the active site that accommodate the flipped orientation 221 of the nucleotide to facilitate AMPylation (Extended Data Fig. 10b) . Notably, Lys73, Arg116 222 and Asp218 in SARS-CoV-1 nsp12 are required for viral replication 5 .

The kinase-like residues of the NiRAN domain and the N-terminus of nsp9 are essential for 224 SARS-CoV-2 replication 225 To determine the importance of the NiRAN domain and the N-terminus of nsp9 in viral replication, 226 we used a DNA-based reverse genetics system that can rescue infectious SARS-CoV-2 (Wuhan-227 Hu-1/2019 isolate) expressing a fluorescent reporter 8 (Extended Data Fig. 11a) . We introduced 228 single point mutations in nsp9 (N1A, N1D and N2A) and nsp12 (K73A, D218A and D760A) and 229 9 quantified the virus in supernatants of producer cells by RT-qPCR to detect the viral N gene. We 230 observed a 400 to 4000-fold reduction in viral load for all the mutants compared to WT (Fig. 5j,   231 Extended Data Fig. 11a ). To account for the possibility of a proteolytic defect in the mutant viral 232 polyprotein, we tested whether the main viral protease nsp5 (M Pro ) can cleave a nsp8-nsp9 fusion 233 protein containing the Asn1/Asn2 mutations in nsp9. The N1D mutant failed to be cleaved by 234 nsp5, suggesting that the replication defect observed for this mutant is a result of inefficient 235 processing of the viral polyprotein. However, the N1A and N2A mutants were efficiently cleaved 236 by nsp5 (Extended Data Fig. 11b, c) . Collectively, these data provide genetic evidence that the 237 residues involved in capping of the SARS-CoV-2 genome are essential for viral replication.

We propose the following mechanism of RNA capping by CoV: during transcription, the nascent 240 5′-pppRNA binds to the NiRAN active site, in either a cis (Fig. 6a) or trans (Fig. 6b ) manner and 241 5′-pRNA is subsequently transferred to the N-terminus of nsp9 forming a phosphoramidate bond 242 (Fig. 6c, panels 1 and 2) . The nsp13 protein produces GDP from GTP, which binds the NiRAN 243 active site and attacks RNAylated nsp9, releasing capped RNA and regenerating unmodified nsp9 244 (Fig. 6c, panels 3 and 4) . Subsequently, nsp14 and nsp16 perform sequential N7 and 2′-O 245 methylations, forming a fully functional 7Me GpppA2′-O-Me-RNA cap.

SARS-CoV-2 nsp12 is thought to initiate transcription/replication starting with an NTP, or a short 247 5′-pppRNA primer 29 . Cryo-EM structures of the RTC suggest that the dsRNA product makes its 248 way out of the RdRp active site in a straight line, supported by the nsp8 helical stalks 21,30,31 . In a 249 cis capping model, the helical duplex with nascent 5′-pppRNA would then need to unwind, flex 250 90°, and extend into the NiRAN active site ~70 Å away (Fig. 6a) . More likely, a separate RTC 251 complex could perform capping in trans (Fig. 6b) . Notably, Perry et al. 32 propose that the nascent The SARS-CoV-2 capping mechanism is reminiscent of the capping mechanism used by VSV, 262 although there are some differences. The VSV large (L) protein is a multifunctional enzyme that In summary, we have defined the mechanism by which SARS-CoV-2 caps its genome and have 283 reconstituted this reaction in vitro using non-structural proteins encoded by SARS-CoV-2. Our Fractions containing YIPP were pooled, concentrated, and stored as above. were resolved in a 15% TBE-Urea PAGE gel. The gel was then stained with toluidine blue O and 739 the 32 P signal detected via autoradiography.

The other half of the GTase reactions were treated with 10 U/ml P1 nuclease for 1 h at 37°C.

Reactions were then split in half again with one half treated with 1 U/ml Quick CIP for 30 min at 1H-1H TOCSY pulse train using a DIPSI-2 sequence and a field strength of 10KHz was tagged at 787 the end of the HSQC sequence to observe signals from 1H nuclei that are further away from 31P.

Given the lower sensitivity of this experiment, each FID was accumulated with 4096 scans and a 789 repetition delay of 1sec was used for a total recording time of 2 days and 14 hours. Methyltransferase control reaction.

Reactions with 14 C-labelled SAM were conducted as above, with two differences: cold GDP was To form nsp12/7/8 core complex (RTC), native nsp12, nsp7 and nsp8 were incubated in 1:2:4 968 molar ratio and run over Superdex 200 increase 10/300 GL to separate unassociated monomers.

The purified complex was concentrated using spin concentrators (Amicon 10k MWCO, Sigma- 

All 1066 oligonucleotides were synthesized by LGC Biosearch Technologies. RT was performed at 50°C 1067 for 5 minutes, followed by inactivation at 95°C for 2 minutes, and 40 cycles of PCR (95°C for 3 1068 seconds, 60°C for 30 seconds

BHK-21J cells (a generous gift from C. Rice) were grown in MEM (Gibco) supplemented with 1071

VeroE6-C1008 cells (ATCC) were transduced with lentiviral vector 1072

MEM supplemented with 10% FBS, 1X NEAA, 1073 and 8 µg/ml Blasticidin

Molecular mechanisms of coronavirus RNA capping and methylation

Structures and functions of 1080 coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug 1081 design

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

Discontinuous and non-1085 discontinuous subgenomic RNA transcription in a nidovirus

Discovery of an essential nucleotidylating activity associated with a 1088 newly delineated conserved domain in the RNA polymerase-containing protein of all 1089 nidoviruses

Functional screen reveals SARS coronavirus nonstructural protein nsp14 as 1091 a novel cap N7 methyltransferase

In vitro reconstitution of SARS-coronavirus mRNA cap methylation

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and 1096 coronavirus toolkit for COVID-19 research

Coronavirus biology and 1099 replication: implications for SARS-CoV-2

A pneumonia outbreak associated with a new coronavirus of probable bat 1102 origin

Remdesivir for the Treatment of Covid-19 -Final Report

Molnupiravir, an Oral Antiviral Treatment for COVID-19. medRxiv

Protein AMPylation by an Evolutionarily Conserved Pseudokinase

Structure of the SARS-CoV nsp12 polymerase bound 1110 to nsp7 and nsp8 co-factors

A novel protein kinase-1113 like domain in a selenoprotein, widespread in the tree of life

Coronavirus replication-transcription complex: Vital and selective 1116 NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit

Innate Immune Evasion by Human Respiratory RNA Viruses

mRNA capping: biological functions and 1121 applications

2'-O methylation of the viral mRNA cap evades host restriction by IFIT 1123 family members

Multiple enzymatic activities associated with severe acute respiratory 1125 syndrome coronavirus helicase

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and 1128

Transcription Complex Reveals an Intermediate State in Cap Synthesis

Mass spectrometric based 1131 detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Protein-primed RNA synthesis in SARS-CoVs and structural basis for 1134 inhibition by AT-527. bioRxiv

Unconventional mechanism of mRNA capping by the RNA-1137 dependent RNA polymerase of vesicular stomatitis virus

RNA Synthesis and Capping by Non-segmented Negative Strand 1140 RNA Viral Polymerases: Lessons From a Prototypic Virus

The mRNA 5' cap-binding protein eIF4E and control of 1143 cell growth

NMPylation and de-NMPylation of SARS-CoV-1146 2 nsp9 by the NiRAN domain

Protein kinases: evolution of dynamic regulatory proteins

CoV-er all the bases: Structural perspectives 1151 of SARS-CoV-2 RNA synthesis

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 1154

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-1156

CoV-2 polymerase reveals mechanisms for capping and proofreading

An atomistic model of the coronavirus replication-transcription complex 1159 as a hexamer assembled around nsp15

Histidine-mediated RNA transfer to GDP for 1162 unique mRNA capping by vesicular stomatitis virus RNA polymerase

Structure of the L Protein of Vesicular Stomatitis Virus from Electron 1165

A SARS-CoV-2 protein interaction map reveals targets for drug 1167 repurposing

methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs

NMRPipe: a multidimensional spectral processing system based on 1172 UNIX pipes

NMRFAM-SPARKY: enhanced software for 1174 biomolecular NMR spectroscopy

The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for 1177

Replication of MERS-CoV and SARS-CoV-2

Automated electron microscope tomography using robust prediction 1180 of specimen movements

RELION: implementation of a Bayesian approach to cryo-EM structure 1182 determination

MotionCor2: anisotropic correction of beam-induced motion for 1184 improved cryo-electron microscopy

Real-time CTF determination and correction

SPHIRE-crYOLO is a fast and accurate fully automated particle picker 1189 for cryo-EM

cryoSPARC: algorithms for 1191 rapid unsupervised cryo-EM structure determination

PHENIX: a comprehensive Python-based system for macromolecular 1197 structure solution

MolProbity: all-atom structure validation for macromolecular 1200 crystallography

FATCAT 2.0: towards a better 1203 understanding of the structural diversity of proteins

Deciphering key features in protein structures with the new 1206

ENDscript server

MnCl2, 1 mM UMP-NPP. Copper Quantifoil 1.2/1.3 mesh 300 grids were used to freeze 3.5 μL of 974 sample at 100% relative humidity using Vitrobot mk. IV (Thermofisher). 975 Cryo-EM data collection 976 Prior to data collection, sample grids were screened on a Talos Artica microscope at the Cryo Table S3 . 1051 Virus production 1052 To generate virus from DNA-based infectious clones, 3 µg of plasmid were transfected into 2 1053 individual 6 wells of 400,000 BHK-21J cells using X-treme Gene9 Transfection Reagent (Sigma).