key: cord-0806642-35rouo1i
authors: Yan, Liming; Ge, Ji; Zheng, Litao; Zhang, Ying; Gao, Yan; Wang, Tao; Huang, Yucen; Yang, Yunxiang; Gao, Shan; Li, Mingyu; Liu, Zhenyu; Wang, Haofeng; Li, Yingjian; Chen, Yu; Guddat, Luke W.; Wang, Quan; Rao, Zihe; Lou, Zhiyong
title: Cryo-EM structure of an extended SARS-CoV-2 replication and transcription complex reveals an intermediate state in cap synthesis
date: 2020-11-14
journal: Cell
DOI: 10.1016/j.cell.2020.11.016
sha: a6a87bc18830cb7be5db7c0670d701eeb73d7514
doc_id: 806642
cord_uid: 35rouo1i

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transition towards cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA binding protein nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N-terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5’ extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC towards mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the 23 replication and transcription complex (RTC). Here, we present a structural snapshot of 24 SARS-CoV-2 RTC as it transition towards cap structure synthesis. We determine the 25 atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp8 2 -nsp12-nsp13 2 -26 RNA and a single RNA binding protein nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, 27 allowing nsp9 N-terminus inserting into the catalytic center of nsp12 NiRAN, which then 28 inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, 29 catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that 30 anchors the 5' extension of template RNA shows a remarkable conformational shift, 31 resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer 32 SARS-CoV-2 has a positive-sense single-stranded RNA (ssRNA) genome (Ziebuhr, 46 2005 ). The replicase gene open reading frames 1a (ORF1a) and ORF1b are translated 47 into large polyproteins, which are proteolytically processed by two viral proteases to yield 48 16 mature nonstructural proteins (nsps) (Ziebuhr, 2005) . For efficient proliferation, a set of 49 nsps assemble a replication and transcription complex (RTC) to synthesize the negative-50 strand template, the positive-strand genomic RNA and subgenomic mRNAs. 51

The cap structure at the 5' end of viral mRNAs plays essential roles in the lifecycle of a 52 virus by promoting initiation of translation, protecting mRNAs and to help the virus escape 53 host immune recognition (Daffis et al., 2010) . The formation of cap structure in CoVs 54 involves four sequential enzymatic reactions: (i) an 5´ RNA triphosphatase (RTPase) in 55 nsp13 removes the γ-phosphate of 5'-triphosphate end (pppA) of the nascent mRNA to 56 generate 5'-diphosphate end (ppA) ; (ii) an 57 J o u r n a l P r e -p r o o f SARS-CoV-2 cap(-1)'-RTC structure -2020.08.14 and the poor quality regions are mainly distributed beyond residue E145, possibly due to 99 lack of intermolecular interaction. Nevertheless, the good quality of the density for the N-100 terminal part of nsp13, particularly in the interaction regions with other components in the 101 extended E-RTC, allows us to rigid-body fit two nsp13 molecules into the density (Figure 102 S1, Table S1 ). 103

The extended E-RTC is composed of one nsp7, two nsp8 (identified as nsp8-1 and 104 nsp8-2(Gao et al., 2020)), one nsp12, two nsp13 (identified as nsp13-1 and nsp13-2 as 105 indicated in Figure 2 ), a paired template-primer RNA, and an nsp9 ( Figure 2 ). The cryo-EM 106 density with good quality clearly indicates the existence of nsp9 bound close to the 107 catalytic center of nsp12 NiRAN. The template-primer RNA, nsp7, nsp8 and nsp12 108 assemble C-RTC with a similar architecture as previously reported Hillen 109 et al., 2020; Wang et al., 2020b) . The paired portion of the template-primer RNA is tightly 110 clamped by nsp12 and the helical stalks of the two nsp8 protomers. Two nsp13 protomers 111 bind to the plane region formed by the two nsp8 protomers and nsp12. Nsp13-1 binds to 112 nsp12 and nsp8-1 to stabilize the architecture of RTC, while nsp13-2 channels the 113 unpaired 5' extension of the RNA template through an RNA binding channel formed by its 114 1A, 2A and 1B domains. By comparison to E-RTC, the orientation of nsp13-2 has distinct 115 shift (will be discussed later). 116 117

In the extended E-RTC, nsp9 tightly binds with nsp12 adjacent to the catalytic center 119 J o u r n a l P r e -p r o o f SARS-CoV-2 cap(-1)'-RTC structure -2020.08.14 of nsp12 NiRAN ( Figure 3A , Table S2 ). The nsp9-nsp12 buried interface is 928 Å 2 120 compared to ~3000 Å 2 for the total surface area of nsp9. Though the overall folding of 121 nsp9 in the extended E-RTC and in the crystallographic structure (PDB: 6W9Q) (Littler et 122 al., 2020) are generally similar, nsp9 N1-nsp9 L9 move by ~16 Å , allowing it to make contact 123 with nsp12 NiRAN, the Interface and the Palm domains ( Figure S2 ). 124

The nsp9-nsp12 interface has two defined regions. Region 1 is formed by nsp9 N-125 terminus and Palm domain and a previously identified β-haripin motif in 126

NiRAN of nsp12 ( Figure 3A ). In region 1, the side chains of nsp12 D36 (β-haripin), nsp12 Y728 127 (Palm) and nsp12 R733 (Palm) form hydrogen bonds with nsp9 N2 ( Figure 3B ). The backbone 128 amide and the carbonyl oxygen atoms of nsp9 E3 interact with the carbonyl oxygen atoms of 129 nsp12 Y38 (β-haripin) and nsp12 R733 (Palm), respectively. In region 2, a flat region in nsp12 130

NiRAN interacts with the C-terminal α-helix of nsp9, which is stabilized by several 131 hydrophobic interactions and three hydrogen bonds ( Figure 3C ). In this region, nsp12 V202-132 V204-I223-V231 makes hydrophobic contacts with nsp9 L4 and nsp9 L103. The side chain of 133 nsp9 N96 forms hydrogen bonds with the carbonyl oxygen atoms of nsp12 P232 and nsp12 Y289. 134

Moreover, the backbone amide of nsp9 N96 contacts with nsp12 D291 (Interface). All these 135 interacting residues are highly conserved in SARS-CoV-2, SARS-CoV, MERS-CoV and Interestingly, crystallographic structures of CoV-encoded nsp9 have shown that nsp9 138 is presumably to form a biological dimer utilizing the interaction between the C-terminal α- J o u r n a l P r e -p r o o f SARS-CoV-2 cap(-1)'-RTC structure -2020.08.14 glycine residues (G100 and G104) in a GXXXG motif (Kleiger et al., 2002; Miknis et al., 141 2009 ) of the C-terminal α-helix were shown to play an essential role in nsp9 dimerization, 142 and the substitutions of them as glutamate residues significantly attenuated viral 143 proliferation (Miknis et al., 2009 ). In the extended E-RTC, G100 and G104 directly face 144 nsp12 and participate in the formation of the nsp9-nsp12 interface ( Figure 3C , Figure S2 , 145 Figure 4A , Figure S3A ). Its di-phosphate moiety is buried in a positively 156 charged pocket formed by the side chains of nsp12 K50, nsp12 N52, nsp12 K73 and nsp12 R116 in suggested to be the conserved catalytic residue for nidoviral NiRAN (Lehmann et al., 162 2015) , is 3.9-Å to the phosphoester oxygen between the αand β-phosphate of the bound 163 GDP. The imidazole group of nsp12 H75 stacks with the base of GDP to stabilize the 164 conformation. 165

The N-terminus of nsp9 inserts deep into the catalytic center of nsp12 NiRAN and 166 makes bonds with the bound GDP ( Figure 4A , Figure S3A ). The side chain of nsp9 N1 167 makes an interaction with α-phosphate of the bound GDP and the connecting density 168 suggests the formation of a bond. Again, the residues that contact with GDP are highly 169 conserved among SARS-CoV-2, SARS-CoV, MERS-CoV and RatG13 ( Figure 3D ). In 170 contrast to GDP binding with nsp12 NiRAN, BeF 3 is not observed in the density ( Figure  171 4A, Figure S3A AMPylation activity whereby AMP is transferred from ATP to Ser/Thr/Tyr residues on 183 protein substrates (Sreelatha et al., 2018) . Because the N-terminus of nsp9 inserts into the 184 catalytic center of nsp12 NiRAN and makes a bond with GDP, we first verified whether 185 nsp9 is a possible substrate of nsp12 NiRAN. To do this, we incubated nsp9 with nsp12 in 186 the presence of NTP, and did not detect peak corresponding to the NTP-modified N-187 terminal peptide of nsp9 in mass spectrum ( Figure 4B ). Since the residue of nsp9 that 188 contacts with bound GDP is an asparagine residue, this result is not surprising. 189

Another study has shown that NiRAN domain in nidoviral RdRp has nucleotidylation 190 activity with UTP/GTP preference (Lehmann et al., 2015) . We next assessed whether 191 SARS-CoV-2 nsp12 has nucleotidylation activity. Using a pyrophosphate assay, we 192 demonstrated that SARS-CoV-2 nsp12 indeed has the nucleotidylation activity in the 193 presence of GTP or UTP ( Figure 4C ). The mutation of the catalytic residue nsp12 K73A 194 eliminated the nucleotidylation activity of nsp12 and this activity was attenuated in the 195 presence of wildtype (wt) nsp9. Moreover, we made several mutants of residues that are 196 involved in the nsp9-nsp12 interaction, including nsp9 truncated by five N-terminal 197 residues (nsp9∆5), nsp9 N96A, nsp9 G100E and nsp9 G104E, and found that all these mutations 198 restore the nucleotidylation activity of nsp12. These results further suggest that the 199 association of nsp9 inhibits the nucleotidylation activity of nsp12. 200

We then investigated whether the nucleotidylation activity of nsp12 may function as 5' 201 end cap guanylyltransferase (GTase) to transfers a GMP to ppA to produce GpppA for the 202 further formation of cap(0) structure. In an in vitro GTase assay, we found that nsp12 203 catalyzes the transfer of GMP to ppA-RNA to form GpppA-RNA in the presence of nsp13, 204

which is known to function as a CoV RTPase to yield ppA-RNA from pppA-RNA (Ivanov et 205 al., 2004; ) ( Figure 4D ). In the presence of nsp9, the signal 206 corresponding to the radioactive-labeled GpppA-RNA is attenuated than that with the 207 absence of nsp9; however, a certain extent of the signal still exists. Since the 208 nucleotidylation activity of nsp12 still remains in the presence of nsp9 ( Figure 4C RTC shows that the conformation of nsp13-1 is conserved; however, the orientation of 219 nsp13-2 has a notable difference (Figures 5A and 5B, Figure S4 ). 220

In the extended E-RTC, nsp13-2, as a rigid body, moves downwards to the template-221 primer RNA clamped by nsp8-1 and nsp8-2. As a consequence, zinc finger 3 (ZF3) motif 222 of nsp13-2 ZBD and the adjacent loop (spanning residues G66-S80) inserts into the 223 groove that is formed by nsp8-1 and nsp8-2 to clamp the paired template-primer in the 224 extended E-RTC. The insertion part of nsp13-2 ZBD is closed to the second minor groove 225 (originated from the polymerase center of nsp12) of the paired template-primer RNA 226 ( Figure 5D , Figure S5A , Table S3 ). But in E-RTC, ZF3 is distant from this groove ( Figure  227 5C, Figure S5A ). Because ZF3 has been shown to play essential roles in nidovirus mRNA 228 synthesis without impact on the helicase activity ( Another interesting observation is that the movement of nsp13-2 ZBD moves towards 232 nsp13-1 ZBD and nsp8-1 with a large distance of 35 Å results in a hydrogen-bond formed 233 by the carbonyl oxygen atom of nsp13-2 P53 and nsp8-1 Y71 ( Figure S4D , Table S4 ). In 234 contrast, nsp8-1 Y71 interacts with nsp13-1 and has no contact with nsp13-2 in E-RTC. The 235 movement of nsp13-2 also results in a loosening of the contact between 1B domains of the 236 two nsp13s ( Figures 5E and 5F ). Figure S5B ). This groove may represent a potential RNA binding groove to 261 stabilize the conformation of the released 5' end of GpppA-RNA after catalytic reaction, 262 standing by for its subsequent processing by nsp14 and nsp16 to form the cap structure 263 and the mature mRNAs. In this context, ZF3 of nsp13-2 ZBD might function as a "brake" to 264 temporarily suspend the elongation of nascent RNA, by inserting into the minor groove of the double stranded nascent RNA-template pair. 266

According to current structural information of CoV RTCs, we propose a speculative 267 model for SARS-CoV-2 RTC to catalyze the synthesis of mRNA ( Figure 6 ). In the initial 268 step, nsp7, nsp8 and nsp12 compose the central RTC (C-RTC). In the second step, C-269 RTC recruits two nsp13 molecules and template-primer RNA to form the elongation RTC 270 Figure 4D) . 442

The components used in each lane are labeled. In lane 5-7, nsp12 is incubated with nsp9 443 in molar ratio of 1:2 (denoted as +), 1:4 (denoted as 2+) and 1:6 (denoted as 3+). The 444 reaction condition is the same as that used in Figure 4D . 445

Further information and requests for resources and reagents should be directed to and 451 will be fulfilled by the Lead Contact, Zihe Rao (raozh@tsinghua.edu.cn). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 80% 565 acetonitrile and 0.1% formic acid. The Q Exactive mass spectrometer was operated in 566 data-dependent acquisition mode using Xcalibur 2.1.2 software and there was a single full-567 scan mass spectrum in the orbitrap (300-1800 m/z, 70,000 resolution) followed by 20 data-568 dependent MS/MS scans at 27% normalized collision energy (HCD). 569

The MS/MS spectra from each LC-MS/MS run were searched against the SARS-CoV-570 two missed cleavage was allowed; Carbamidomethyl (C) were set as the fixed 573 modifications; the oxidation (M) was set as the variable modification; precursor ion mass 574 tolerances were set at 20 ppm for all MS acquired in an orbitrap mass analyzer; and the 575 fragment ion mass tolerance was set at 0.02Da for all MS2 spectra acquired. The peptide 576 false discovery rate (FDR) was calculated using Percolator provided by PD. When the q 577 value was smaller than 1%, the peptide spectrum match (PSM) was considered to be 578 correct. FDR was determined based on PSMs when searched against the reverse, decoy 579 database. Peptides only assigned to a given protein group were considered as unique. 580

The false discovery rate (FDR) was also set to 0.01 for protein identifications. 581 582

In total, 3 µL of protein solution at 3 mg/mL (added with 0.025% DDM) was applied 584 onto a H2/O2 glow-discharged, 200-mesh Quantifoil R0.6/1.0 grid (Quantifoil, Micro Tools 585 GmbH, Germany). The grid was then blotted for 3.0 s with a blot force of 0 at 8°C and 586 100% humidity and plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher 587 Scientific, USA). Cryo-EM data were collected with a 300 keV Titan Krios electron 588 microscope (Thermo Fisher Scientific, USA) and a K3 direct electron detector (Gatan, 589 USA). Images were recorded at 29000× magnification and calibrated super-resolution 590 pixel size 0.82 Å/pixel. The exposure time was set to 2 s with a total accumulated dose of 591 60 electrons per Å 2 . All images were automatically recorded using SerialEM (Mastronarde, 592 2005). A total of 4107 images were collected with a defocus range from -2.0 µm to -1.0 593 J o u r n a l P r e -p r o o f SARS-CoV-2 cap(-1)'-RTC structure -2020.08.14 µm. Statistics for data collection and refinement are in Table S1 . on the complex integrality. This particle set was used to do Ab-Initio reconstruction in six 603 classes, which were then used as 3D volume templates for heterogeneous refinement, 604 with 67,540 particles converged into one nsp12-nsp7-nsp8 2 -nsp9-nsp13 2 -RNA complex 605 class. Next, these particles were imported into RELION 3.03 (Scheres, 2012) to perform 606 local classification to obtain one class particle with final resolution 2.83 Å. 607 608 Model building and refinement 609 nsp9 (PDB: 6W9Q) were individually placed and rigid-body fitted into the cryo-EM map 612 using UCSF Chimera (Pettersen et al., 2004) . The model was manually built in Coot 613 (Emsley et al., 2010) with the guidance of the cryo-EM map, and in combination with real 614 space refinement using Phenix (Afonine et al., 2018) . The data validation statistics are 615 shown in Table S1 . 616

In Figure 4C , the activity of nsp12 NiRAN domain in pyrophosphate assays were 619 calculated by using GraphPad Prism as mean ± SD from three independent experiments 620 as indicated in the legend of Figure 4C . 621

In Figure 4D , the integrate densities of the bands in lane 4-7, which are corresponding 622 to apo-nsp12, nsp12 plus nsp9, nsp12 plus 2*nsp9, and nsp12 plus 3*nsp9 nsp13-1 nsp13-2 nsp8-1 nsp8-2 nsp9 template primer

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• Structure of SARS-CoV-2 elongation complex with nsp9 has been determined.• Nsp9 binds to the catalytic center of the nsp12 (RdRp) NiRAN domain.• The nsp12 NiRAN domain catalyzes the formation of the cap core structure (GpppA).• The structure reveals an intermediate state for RTC in cap synthesis.

Yan et al. present a cryo-EM structure of the SARS-CoV-2 replication and transcription complex that includes the single RNA binding protein nsp9. The structural snapshot provides insight into how the viral machinery catalyzes a crucial step in viral mRNA cap synthesis, which is important for virus survival.