key: cord-0932140-j6lg57zq
authors: Peng, Qi; Peng, Ruchao; Yuan, Bin; Wang, Min; Zhao, Jingru; Fu, Lifeng; Qi, Jianxun; Shi, Yi
title: Structural basis of SARS-CoV-2 polymerase inhibition by Favipiravir
date: 2021-01-18
journal: Innovation (N Y)
DOI: 10.1016/j.xinn.2021.100080
sha: f6c2985ed8ef30c998ae115ba5925ce79a5c9e8b
doc_id: 932140
cord_uid: j6lg57zq

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into an unprecedented global pandemic. Nucleoside analogues, such as Remdesivir and Favipiravir, can serve as the first-line broad-spectrum antiviral drugs by targeting the viral polymerases. However, the underlying mechanisms for the antiviral efficacies of these drugs are far from well understood. Here we reveal that Favipiravir, as a pyrazine derivative, could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides. This drug thus inhibits viral replication mainly by inducing mutations in progeny RNAs, different from Remdesivir or other RNA-terminating nucleoside analogues that impair the elongation of RNA products. We further determined the cryo-EM structure of Favipiravir bound to the replicating polymerase complex of SARS-CoV-2 in the pre-catalytic state. This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues which may explain its capacity for mimicking both adenine and guanine nucleotides. These findings shed lights on the mechanism of coronavirus polymerase catalysis and provide a rational basis for developing antiviral drugs to combat the SARS-CoV-2 pandemic.

Coronaviridae family includes many life-threatening human pathogens, such as SARS-CoV 1 , 38

Middle East Respiratory Syndrome coronavirus (MERS-CoV) 2 and the ongoing pandemic 39 SARS-CoV-2 3,4 . Coronaviruses harbor a non-segmented positive-sense RNA genome with 40 in NTP substrate and stabilize the transition intermediate 34, 35 . Based on the previous structure 152 of post-catalytic conformation in the presence of Remdesivir 25 , we modeled two magnesium 153 ions into our structure to analyze the potential catalytic state of this polymerase. The two 154 metal atoms are coordinated by D618 (motif A) and D761 (motif C) and further bridge the 155 three phosphate groups of FTP substrate. The terminal γ-phosphate is further sequestered by a 156 salt bridge contributed by K798 ( Figure 4A ). Besides, residues R553 and R555 from motif F 157 may also interact with the βand γ-phosphate groups. These interactions together stabilize the 158 incoming nucleotide and 3'-terminus of product RNA in close vicinity to facilitate the 159 nucleophilic attack to α-phosphate by the 3'-hydroxyl oxygen of -1 nucleotide. 160 161

Since Favipiravir could mimic both adenine and guanine nucleotides for RNA synthesis, we 163 modeled the GTP into the structure to compare the potential different recognition patterns for 164 different NTP substrates ( Figure 4) . Basically, the GTP reveals a highly similar interaction 165 network with the polymerase residues to that for FTP except that an additional residue K545 166 from motif F may also be involved in interactions with the carbonyl group of the guanine base 167 ( Figure 4B ). The ATP substate could also be accommodated similarly with the amino group of 168 adenine base interacting with K545. For pyrimidine nucleotides, however, the base moiety is 169 too far away from K545 to form such interactions ( Figure 4D ). This residue is highly 170 conserved in all viral RdRps and may serve as a signature residue for discriminating purine 171 and pyrimidine substrates ( Figure S6 ). Comparing the RdRps from different viruses, 172 found the residues for stabilizing the ribose, base and catalytic metals are highly conserved 174 across all viral families, whereas the residues for accommodating the phosphate groups reveal 175 obvious diversity and may also involve residues outside the seven canonical catalytic motifs 176 of RdRp, e.g. residue R48 in HCV polymerase 33 ( Figure 4D ). 177 178

With the available structures of SARS-CoV-2 polymerase before and after catalysis 22,24,25 , we 180 were able to assemble a complete scenario of the catalytic cycle to analyze the potential 181 conformational changes of the polymerase during catalysis ( Figure 5 ). It has been established 182 in flavivirus and enterovirus RdRps that the binding of incoming NTP substrates would 183 induce active site closure and relocation of metal ions to facilitate catalysis [33] [34] [35] released. This process is accompanied by the translocation of template-product RNA duplex, 197 which resumes the polymerase to get ready for the next round of catalysis ( Figure 5E and F). and Favipiravir 26-29 . The underlying mechanisms of these nucleoside analogues reveal quite 207 obvious diversity, many of which have not been well understood. Elucidating the molecular 208 basis of interactions between viral polymerase and these drugs is of particular importance for 209 developing better antiviral drugs and minimizing the potential side effects. 210 We and other groups have previously reported the structures of SARS-CoV-2 polymerase 211 complex in the apo form or in complex with template/primer RNA strands in the 212 post-catalytic states 21-25 . The pre-catalytic conformation we captured in this study fills an 213 important knowledge gap for understanding the conformational dynamics during the complete 214 catalytic cycle of this polymerase. It is interesting to note that the key residues for NTP 215 substrate recognition are conserved across many RNA viruses from different families, and 216 J o u r n a l P r e -p r o o f that Favipiravir is efficiently incorporated into RNA products similar to other typical NTP 217 substrates. These observations suggest the potential broad-spectrum antiviral efficacies of this 218 drug for a panel of different RNA viruses by similar mechanisms. The administration of 219 Favipiravir has been shown to induce mutations in progeny virion of SARS-CoV-2 as 220 revealed by cell-based assays 32 , indicating this drug can evade the proofreading mechanism of 221 coronavirus to exert the antiviral efficacy via multiple rounds of RNA synthesis. This is in 222 sharp contrast to other RNA-terminating nucleoside analogue drugs that directly impair the 223 elongation process to produce abortive RNA products 24,31 . These differences imply the 224 diversity of the underlying mechanisms of various nucleoside analogue antiviral drugs and 225 offer clues for further engineering of these drugs to inhibit viral RNA synthesis at different 226 stages of the catalytic cycle. 227

Apart from the small molecule inhibitors, the viral infection could also be effectively 228 inhibited by antibodies that interfere with viral entry pathways. The envelope spike 229 glycoprotein has been demonstrated as a dominant antigen during coronavirus infection 38,39 . 230 Numerous therapeutic antibodies have been developed targeting this molecule to impair 231 receptor recognition or membrane fusion [40] [41] [42] [43] [44] . Because of the pressure rendered by host 232 immunity, the viral envelope proteins are prone to undergo antigenic drift, resulting in 233 immune evasion 45 . In a short term of clinical usage, antibodies are highly efficient for 234 blocking viral infections which can achieve ideal specificity and minimize side effects. For 235 the long-term therapeutic usage and perspective preparation, the small molecule inhibitors 236 targeting other conserved targets, such as viral polymerase and main protease, might be better 237 options which bear less chance to develop resistance or evasion. In addition, these targets are 238 better suited for developing broad-spectrum antiviral drugs [46] [47] [48] . Simultaneously, these 239 broad-spectrum inhibitors are also likely to cause side effects due to non-specific targeting on 240 cellular components. On the other hand, due to the relatively lower cost and convenience for 241 drug delivery, the small molecule inhibitors display remarkable advantages for large-scale 242 application among human populations, especially in the less developed areas. Therefore, the 243 drug development targeting the viral polymerase would represent a highly promising and 244 universal strategy against various emerging and re-emerging viruses. 245

In summary, we present a missing structural snapshot of coronavirus polymerase 246 replication in the pre-catalytic conformation, facilitating the extrapolation of the dynamic 247 catalytic cycle for RNA nucleotide polymerization. Importantly, we reveal the structural basis 248 of Favipiravir incorporation by SARS-CoV-2, which suggests the feasibility of developing 249 other nucleotide-mimicking antiviral drugs by utilizing non-base derived molecular entities. 

The SARS-CoV-2 nsp7, nsp8 and nsp7L8 fusion proteins were expressed in E. coli, and the 498 nsp12 polymerase subunit was expressed with the Bac-to-Bac system (Invitrogen) as 499 previously described 23 . All these proteins were purified by tandem affinity chromatography 500 and size-exclusion chromatography (SEC) accordingly. To constitute the core polymerase 501 complex, the purified nsp12, nsp8 and nsp7L8 proteins were incubated on ice overnight with 502 a molar ratio of nsp12:nsp8:nsp7L8=1:3:3. The complex was then purified by SEC using a 503 Superdex 200 increase column (GE Healthcare) equilibrated with a buffer consisting of 25 504 mM HEPES-NaOH (pH 7.5), 300 mM NaCl and 2 mM Tris (2-carboxyethyl) phosphine 505 (TCEP). The fractions for nsp12-nsp8-nsp7L8 complex were pooled and concentrated to 4 506 mg/mL for subsequence experiments. 507 508

The activity of SARS-CoV-2 polymerase complex was tested as previously described 23 with 510 slight modifications. Briefly, 40-nt template RNA strands (sequences adapted for each 511 specific NTP substrate as shown in Fig. 1 ) were annealed to a complementary 20-nt primer 512 containing a 5'-fluorescein label (5'FAM-GUCAUUCUCCUAAGAAGCUA-3', Takara). To 513 perform the primer extension assay, 1 μM nsp12, 1 μM nsp7 and 2 μM nsp8 were incubated 514 for 30 min at 30 °C with 1 μM annealed RNA and 0.5 mM individual NTP or drug in a 515 reaction buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM TCEP and 2 mM 516 MgCl 2 (freshly added prior to usage). The products were denatured by heating to 100 °C for 517 was run with 0.5×TBE buffer. Images were recorded with a Vilber Fusion imaging system. 519

The RNA products were quantified by integrating the intensity of each band using ImageJ 520 software. The significance of difference was estimated by the two-tailed student's t-test to 521 calculate the P values for each experimental group. 522 523

To prepare the Favipiravir bound polymerase complex, the purified nsp12-nsp8-nsp7L8 525 complex was mixed with annealed RNA duplex (Template: 526 5'-CUAUCCCCAUGUGAUUUUACUAGCUUCUUAGGAGAAUGAC-3', Primer: 527 5'-GUCAUUCUCCUAAGAAGCUA-3') with a molar ratio of nsp12:RNA=1:1.5. The 528 mixture was incubated on ice for 2 h in a buffer containing 25 mM HEPES-NaOH (pH 7.5), 529 150 mM NaCl, 1 mM TCEP and supplemented with 0.5 mM FTP. An aliquot of 3 μL protein 530 solution (0.4 mg/mL) was applied to a glow-discharged Quantifiol 1.2/1.3 holey carbon grid 531 and blotted for 2.5 s in a humidity of 100% before plunge-freezing with an FEI Vitrobot Mark 532 IV. Cryo-samples were screened using an FEI Tecnai TF20 electron microscope and 533 transferred to an FEI Talos Arctica microscope for data collection. The microscope was 534 operated at 200 kV and equipped with a post-column Bioquantum energy filter (Gatan) which 535 was used with a slit width of 20 eV. Cryo-EM data was automatically collected using 536 SerialEM software (http://bio3d.colorado.edu/SerialEM/). Images were recorded with a Gatan 537 K2-summit camera in super-resolution counting mode with a calibrated pixel size of 1.0 Å at 538 the specimen level. Each exposure was performed with a dose rate of 10 e -/pixel/s 539 (approximately 10 e -/Å 2 /s) and lasted for 6 s, resulting in an accumulative dose of ~60 e -/Å 2 540 which was fractionated into 30 movie-frames. The final defocus range of the dataset was 541 approximately -1.7 to -3.4 μm. 542 543

The movie frames were aligned using MotionCor2 to correct beam-induced motion and 545 anisotropic magnification 50 . Initial contrast transfer function (CTF) values were estimated 546 with CTFFIND4.1 51 at the micrograph level. Particles were automatically picked with 547 RELION-3.0 52 following the standard protocol. In total, approximately 2,895,000 particles 548 were picked from ~5,600 micrographs. After four rounds of 2D classification, ~1,002,000 549 particles were selected for 3D classification with the density map of SARS-CoV-2 polymerase 550 replicating complex (EMDB-11007) low-pass filtered to 60 Å resolution as the reference. 551

After two rounds of 3D classification, three distinguished 3D classes were identified with 552 clear features of secondary structural elements. These classes showed different extents of 553 flexibility at the distal end of RNA duplex but the main body of polymerase complex was 554 highly similar. Therefore, these 3 classes were combined (including ~329,000 particles) and 555 subjected to 3D refinement supplemented with per-particle CTF refinement and 556 dose-weighting, which led to a reconstruction of 3.2 Å resolution estimated by the 557 gold-standard Fourier shell correlation (FSC) 0.143 cut-off value. In the density map, the long 558 helices within the N-terminus of nsp8 subunits were not observed. To better resolved this 559 region, a local mask for the N-terminal region of nsp8 subunits and the RNA duplex was 560 applied to perform 3D classification without particle alignment. However, no definable class 561 with a long helix track could be identified, suggesting the flexibility of this region in the 562 structure. This phenomenon might result from the shorter RNA duplex observed in other 563 structures 22,24 that would help to stabilize the long helices. In addition, this structure was 564 restricted by some extent of preferred orientation of particles, which somehow limited the 565 attainable resolution in certain views of the final reconstruction. Basically, the density map 566 was sufficient to support faithful atomic modelling in most regions. The local resolution 567 distribution of the final density map was calculated with ResMap 53 . 568 569

The structure of Remdesivir bound SARS-CoV-2 polymerase complex (PDB ID: 7BV2) was 571 rigidly docked into the density map using CHIMERA 54 . The model was manually corrected 572 for local fit in COOT 55 . The structure of FTP was built using the "Ligand builder" plug-in in 573 COOT and manually fitted into the density. The initial model was refined in real space using 574 PHENIX 56 with the secondary structural restraints and Ramachandran restrains applied. The 575 model was further adjusted and refined iteratively for several rounds aided by the 576 stereochemical quality assessment using MolProbity 57 . The representative density and atomic 577 models are shown in Supplemental Figures S2 and S3 . The statistics for image processing and 578 model refinement are summarized in the Table S1 . Structural figures were prepared by either 579 CHIMERA 54 or PyMOL (https://pymol.org/). 

This study 599 was supported by the Strategic Priority Research Program of CAS (XDB29010000), the 600 National Science and Technology Major Project (2018ZX10101004), National Key Research 601 and Development Program of China (2020YFC0845900), the National Natural Science 602

Foundation of China (NSFC) (82041016, 81871658 and 81802010), a grant from the Bill & 603

Melinda Gates Foundation and is partially supported by the Yanqi Lake Meeting

the Academic Divisions of CAS. M.W. is supported by the National Science and Technology

Major Project (2018ZX09711003) and National Natural Science Foundation of China (NSFC) 606 (81802007). R.P. is supported by the Young Elite Scientist Sponsorship Program (YESS) by

China Association for Science and Technology (CAST) (2018QNRC001). Y.S. is also 608 supported by the Youth Innovation Promotion Association of CAS

purified the protein 612 samples and conducted biochemical studies

analyzed the data and wrote 614 the manuscript. All authors participated in the discussion and manuscript editing

The authors declare no competing interests

Correspondence and requests for materials should be addressed to Yi Shi

Supplemental Figure 1. Cryo-EM analysis of SARS-CoV-2 core polymerase complex

A representative micrograph of SARS-CoV-2 core polymerase bound to RNA and Favipiravir 626 (out of ~5,600 micrographs). (B) Gallery of 2D class average images. Four rounds of 2D 627 classification were performed. (C) Flowchart of image processing. The selected classes for 628 next-step processing are indicated by red dashed boxes. (D) Euler angle distribution of the 629 final reconstruction

Local resolution distribution of the final density map

Supplemental Figure 2. Representative density and atomic models in selected regions

Typical density for different domains, as well as some key residues involved in Favipiravir 635 recognition and catalysis are shown to reveal the quality of the final reconstruction

We thank all staff members in the Center of Biological Imaging (CBI), Institute of Biophysics 598