key: cord-259246-azt5sr9w
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: 2020-10-19
journal: bioRxiv
DOI: 10.1101/2020.10.19.345470
sha: 
doc_id: 259246
cord_uid: azt5sr9w

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 against the newly emerging viral diseases. Recent clinical trials of these two drugs for SARS-CoV-2 treatment revealed antiviral efficacies as well as side effects with different extents1–4. As a pyrazine derivative, Favipiravir could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides, which may further lead to mutations in progeny RNA copies due to the non-conserved base-pairing capacity5. Here, we 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.

proteins 10 . A total of 4 structural proteins and at least 16 non-structural proteins (nsps) as well as 8 accessory proteins are encoded by the coronavirus genome. Among them, nsp12 is the core catalytic subunit of viral RNA-dependent RNA polymerase (RdRp) complex, which executes transcription and replication of the viral genomic RNA 11 . Two cofactor subunits, nsp7 and nsp8, associate with nsp12 to constitute an obligatory core polymerase complex to confer processivity for RNA elongation 12 . To achieve complete transcription and replication, a panel of other nsps are involved to accomplish other enzymatic functions, including the nsp14-nsp10 exonuclease for proofreading [13] [14] [15] , the nsp13 helicase for RNA unwinding [16] [17] [18] [19] , the nsp14 N7methyltransferase and the nsp16-nsp10 2'-O-methyltransferase for capping [20] [21] [22] [23] [24] . Due to the key roles of polymerase complex for viral replication, it has long been thought of as a promising antiviral drug target 25 . Recently, we and other groups have determined the structures of SARS-CoV-2 core polymerase complex in both apo and RNA-bound states 26-30 , providing important information for structure-based antiviral drug design.

In the efforts to combat SARS-CoV-2 pandemic, Remdesivir was initially expected as a highly competent drug candidate for disease treatment. However, the recently disclosed outcome of clinical trials revealed controversial efficacies as well as some cases of side effects 1,2 . The clinical improvement rate of patients receiving Remdesivir within 7 days was only 3% and ~66% subjects developed adverse effects 1,2 . In contrast, another broad-spectrum antiviral Favipiravir showed good promise that 61.21% patients were clinically recovered at day 7 of treatment and 31.90% subjects were reported with side effect manifestations 3,4 . Both of them are administered as pro-drugs and should be processed into triphosphate forms as nucleotide mimicries to interfere with RNA synthesis. The active form of Remdesivir has a similar base moiety to adenine nucleotide which can be incorporated into the growing strand of RNA product using an uracil nucleotide as the template 30 . Besides, the cyanogroup in the ribose ring may cause steric clash with polymerase residues during elongation, resulting in aberrant termination of RNA synthesis 29,31 . It is yet unclear how Favipiravir could be recognized by the polymerase and disturb the faithful process of RNA production.

To investigate the mechanism for the antiviral efficacy of Favipiravir, we performed in vitro primer-extension assays using a template derived from the 3'-untranslated region (3'-UTR) of the authentic viral genome. The +1 catalytic position was inserted with different template residues accordingly to allow only one nucleotide to be incorporated in the presence of each individual nucleotide triphosphate (NTP) substrate (Fig. 1) . Even though the product strand was supposed to grow by only one nucleotide, some larger RNA products with two or three nucleotides extension were generated for each specific NTP substrate ( Fig. 1c and d) . This phenomenon suggests the SARS-CoV-2 polymerase is prone to mis-incorporate unwanted nucleotides into the product RNA, resulting in low fidelity for transferring the genomic information during transcription and replication. Similar observations were also reported recently that SARS-CoV-2 polymerase is more tolerant for mismatches between template and product residues than other viral RdRps 5 , which further highlights the requirement for the proofreading nuclease nsp14 to maintain the integrity of viral genome. Interestingly, Favipiravir could be incorporated into the RNA product with similar efficiencies to those of ATP or GTP substrates guided by U or C template residues, respectively (Fig. 1c) . In contrast, Remdesivir could only be incorporated with a U in the template strand (Fig. 1d) . These evidences demonstrate that Favipiravir is a universal mimicry for purine nucleotides, different from other specific nucleotide analogues, such as Remdesivir 29,30 and Sofosbuvir 32 .

Even though Favipiravir could be efficiently incorporated into RNA products to impair the fidelity of RNA synthesis, it did not significantly inhibit RNA production in vitro (Extended Data Fig. 1) . The presence of Favipiravir in RNA product did not immediately terminate the extension of growing strand except when repeat pyrimidine residues were encountered in the template. The RNA synthesis seemed prone to stall if multiple consecutive incorporations of Favipiravir were supposed to take place (Extended Data Fig. 1b) , consistent with the observations reported by other groups recently 5 . This evidence suggests that the presence of repetitive Favipiravir residues might distort the configuration of template-product RNA duplex to prevent its further extension. However, the consecutive incorporation of Favipiravir might take place with extremely low probabilities in the cellular environment due to the competition of ATP/GTP substrates. Thus, Favipiravir is more likely to be discretely incorporated during virus replication and induce mutations in progeny RNA copies. This hypothesis was supported by a recently reported virus-based inhibition assay which revealed that Favipiravir was able to escape the proofreading mechanism of SARS-CoV-2 replication complex and led to mutations in progeny viral genome 5 .

To uncover the structural basis of Favipiravir recognition by the polymerase, we determined the structure of SARS-CoV-2 nsp12-nsp7-nsp8 core polymerase complex in the presence of a template-product partial duplex RNA and Favipiravir at 3. Fig. 3 ). In the structure, the nsp8 subunit (nsp8.1) in the nsp7-nsp8 heterodimer is mostly unresolved, similar to the recently reported structure of Remdesivir-bound complex in the pre-translocation conformation 30 (Fig. 2a) . Besides, the N-terminal long helices of nsp8 subunits could not be visualized as well, which are supposed to form a sliding platform for template-product duplex elongation 27,29 . This might result from the insufficient length of the RNA duplex to establish extensive interactions with the helix track. This structure resolved 18 nucleotide residues in the template strand and 16 residues in the primer strand. Importantly, Favipiravir was captured in the triphosphate form (FTP) before catalysis which bound at the +1 position and paired with a C template residue ( Fig. 2b and c) . The α-phosphate of FTP is located in vicinity of the 3'-hydroxyl group of A -1 residue with a distance of ~3.3 Å and no density for a phosphate-di-ester bond could be observed (Fig. 2c) . Moreover, the density of FTP could only be visualized at lower contour levels than the other residues, suggesting the lower occupancy of Favipiravir in the complex, which might be related to the pre-catalytic state without stable covalent interactions with the product RNA. Thus, this structure provides a missing snapshot of the pre-catalytic conformation of SARS-CoV-2 polymerase, an earlier stage before the previously reported post-catalysis/pre-translocation and post-translocation conformations 27,29,30 .

As a mimicry of nucleotide substrate, FTP is recognized as an incoming NTP by conserved catalytic residues (Fig. 3a) . Residues D623 (motif A) and N691 (motif B) stabilize the ribose ring of FTP, and residue S682 (motif B) may potentially interact with both the ribose and pyrazine branch moieties. The A -1 residue of product RNA potentially further contact with the α-phosphate and ribose of FTP +1 to drag the two residues in close proximity for catalysis. The amide group of Favipiravir pairs with the template C residue and potentially forms three hydrogen bonds for base-pairing (Fig. 3a) . When paired with a U template residue, there might be two hydrogen bonds in between (Extended Data Fig. 4) . In order to capture the pre-catalytic conformation, we did not include catalytic metals in the buffers for protein purification and assembled the complex by incubation on ice to prevent catalysis (Extended Data Fig. 1c ). Yet, we observed the density for a single metal ion anchored by D761 (motif C) of nsp12 which is offset the catalytic position and may arise from the cellular environment during expression (Fig.   3a) . A similar conformation was also observed in the structure of enterovirus 71 (EV71) polymerase (PDB: 5F8I) 33 . To enable catalysis, two metal ions are required to neutralize the negative charges of phosphate groups in NTP substrate and stabilize the transition intermediate 33, 34 . Based on the previous structure of post-catalytic conformation in the presence of Remdesivir 30 , we modeled two magnesium ions into our structure to analyze the potential catalytic state of this polymerase. The two metal atoms are coordinated by D618 (motif A) and D761 (motif C) and further bridge the three phosphate groups of FTP substrate. The terminal γ-phosphate is further sequestered by a salt bridge contributed by K798 (Fig. 3a) . Besides, residues R553 and R555 from motif F may also interact with the βand γ-phosphate groups.

These interactions together stabilize the incoming nucleotide and 3'-terminus of product RNA in close vicinity to facilitate the nucleophilic attack to α-phosphate by the 3'-hydroxyl oxygen of -1 nucleotide.

Since Favipiravir could mimic both adenine and guanine nucleotides for RNA synthesis, we modeled the GTP into the structure to compare the potential different recognition patterns for different NTP substrates (Fig. 3) . Basically, the GTP reveals a highly similar interaction network with the polymerase residues to that for FTP except that an additional residue K545 from motif F may also be involved in interactions with the carbonyl group of the guanine base ( Fig. 3b) . The ATP substate could also be accommodated similarly with the amino group of adenine base interacting with K545. For pyrimidine nucleotides, however, the base moiety is too far away from K545 to form such interactions (Fig. 3d ). This residue is highly conserved in all viral RdRps and may serve as a signature residue for discriminating purine and pyrimidine substrates (Extended Data Fig. 5 ). Comparing the RdRps from different viruses, including positively-/negatively-sensed and segmented/non-segmented RNA viruses, we found the residues for stabilizing the ribose, base and catalytic metals are highly conserved across all viral families, whereas the residues for accommodating the phosphate groups reveal obvious diversity and may also involve residues outside the seven canonical catalytic motifs of RdRp, e.g. residue R48 in Hepatitis C virus (HCV) polymerase 32 (Fig. 3c ).

With the available structures of SARS-CoV-2 polymerase before and after catalysis 27,29,30 , we were able to assemble a complete scenario of the catalytic cycle to analyze the potential conformational changes of the polymerase during catalysis (Fig. 4) . It has been established in flavivirus and enterovirus RdRps that the binding of incoming NTP substrates would induce active site closure and relocation of metal ions to facilitate catalysis 32-34 . A similar scenario may also exist for coronavirus polymerase, and potentially other viral RdRps. The motif A loop in apo polymerase adopts an open conformation with the active site fully accessible for incoming NTP substrates ( Fig. 4a and b) . One of the catalytic metal ions (metal A) is anchored by D761 offset the catalytic site, whereas the other one (metal B) might be less stable and frequently exchange between the polymerase and the solvent. Therefore, the metal A atom could be visualized in several viral polymerases without the template RNA and NTP substrate in the active site, including bunyavirus (PDB: 6Z6G) 35 , and arenavirus (PDB: 6KLC and 6KLD) 36 polymerases. With the NTP substrate coming in, the motif A loop flip inward to close the active site and the side chain of D761 (motif C) rotates to relocate metal A into the catalytic site.

Besides, residue K798 (motif D) would also be re-oriented to stabilize the γ-phosphate of NTP ( Fig. 4c and d) . After catalysis, the motif A loop and residue K798 would move back to the open conformation, allowing the pyrophosphoric acid fragment to be released. This process is accompanied by the translocation of template-product RNA duplex, which resumes the polymerase to get ready for the next round of catalysis ( Fig. 4e and f) .

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

Given the better record of Favipiravir than Remdesivir in side effect manifestations 3,4 , it may represent a better option for clinical treatment of SARS-CoV-2 infections, as well as a panel of other human-infecting RNA viruses 37 . In addition, these findings provide an important basis for developing better broad-spectrum inhibitors with higher potency to combat the infection of various emerging RNA viruses. Wang 

The SARS-CoV-2 nsp7, nsp8 and nsp7L8 fusion proteins were expressed in E. coli, and the nsp12 polymerase subunit was expressed with the Bac-to-Bac system (Invitrogen) as previously described 28 . All these proteins were purified by tandem affinity chromatography and sizeexclusion chromatography (SEC) accordingly. To constitute the core polymerase complex, the purified nsp12, nsp8 and nsp7L8 proteins were incubated on ice overnight with a molar ratio of nsp12:nsp8:nsp7L8=1:3:3. The complex was then purified by SEC using a Superdex 200

increase column (GE Healthcare) equilibrated with a buffer consisting of 25 mM HEPES-NaOH (pH 7.5), 300 mM NaCl and 2 mM Tris (2-carboxyethyl) phosphine (TCEP). The fractions for nsp12-nsp8-nsp7L8 complex were pooled and concentrated to 4 mg/mL for subsequence experiments.

The activity of SARS-CoV-2 polymerase complex was tested as previously described 28 with slight modifications. Briefly, 40-nt template RNA strands (sequences adapted for each specific NTP substrate as shown in Fig. 1 ) were annealed to a complementary 20-nt primer containing a 5'-fluorescein label (5'FAM-GUCAUUCUCCUAAGAAGCUA-3', Takara). To perform the primer extension assay, 1 μM nsp12, 1 μM nsp7 and 2 μM nsp8 were incubated for 30 min at 30 °C with 1 μM annealed RNA and 0.5 mM individual NTP or drug in a reaction buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM TCEP and 2 mM MgCl2 (freshly added prior to usage). The products were denatured by heating to 100 °C for 10 min in the presence of formamide and resolved by 20% PAGE containing 9 M urea which was run with 0.5×TBE buffer. Images were recorded with a Vilber Fusion imaging system. The RNA products were quantified by integrating the intensity of each band using ImageJ software. The significance of difference was estimated by the two-tailed student's t-test to calculate the P values for each experimental group.

To prepare the Favipiravir bound polymerase complex, the purified nsp12-nsp8-nsp7L8 Each exposure was performed with a dose rate of 10 e -/pixel/s (approximately 10 e -/Å 2 /s) and lasted for 6 s, resulting in an accumulative dose of ~60 e -/Å 2 which was fractionated into 30 movie-frames. The final defocus range of the dataset was approximately -1.7 to -3.4 μm.

The movie frames were aligned using MotionCor2 to correct beam-induced motion and anisotropic magnification 38 . Initial contrast transfer function (CTF) values were estimated with CTFFIND4.1 39 at the micrograph level. Particles were automatically picked with RELION-3.0 40 following the standard protocol. In total, approximately 2,895,000 particles were picked from ~5,600 micrographs. After four rounds of 2D classification, ~1,002,000 particles were selected for 3D classification with the density map of SARS-CoV-2 polymerase replicating complex (EMDB-11007) low-pass filtered to 60 Å resolution as the reference. After two rounds of 3D classification, three distinguished 3D classes were identified with clear features of secondary structural elements. These classes showed different extents of flexibility at the distal end of RNA duplex but the main body of polymerase complex was highly similar. Therefore, these 3 classes were combined (including ~329,000 particles) and subjected to 3D refinement supplemented with per-particle CTF refinement and dose-weighting, which led to a reconstruction of 3.2 Å resolution estimated by the gold-standard Fourier shell correlation (FSC) 0.143 cut-off value. In the density map, the long helices within the N-terminus of nsp8 subunits were not observed. To better resolved this region, a local mask for the N-terminal region of nsp8 subunits and the RNA duplex was applied to perform 3D classification without particle alignment. However, no definable class with a long helix track could be identified, suggesting the flexibility of this region in the structure. This phenomenon might result from the shorter RNA duplex observed in other structures 27,29 that would help to stabilize the long helices. In addition, this structure was restricted by some extent of preferred orientation of particles, which somehow limited the attainable resolution in certain views of the final reconstruction. Basically, the density map was sufficient to support faithful atomic modelling in most regions. The local resolution distribution of the final density map was calculated with ResMap 41 .

The structure of Remdesivir bound SARS-CoV-2 polymerase complex (PDB ID: 7BV2) was rigidly docked into the density map using CHIMERA 42 . The model was manually corrected for local fit in COOT 43 . The structure of FTP was built using the "Ligand builder" plug-in in COOT and manually fitted into the density. The initial model was refined in real space using PHENIX 44 with the secondary structural restraints and Ramachandran restrains applied. The model was further adjusted and refined iteratively for several rounds aided by the stereochemical quality assessment using MolProbity 45 . The representative density and atomic models are shown in Extended Data Fig. 3 . The statistics for image processing and model refinement are summarized in the Extended Data Table 1 . Structural figures were prepared by either CHIMERA 42 or PyMOL (https://pymol.org/).

The The aligned sequences were then used for conservation analysis via the ConSurf 52 server. The figure was generated with PyMOL.

The cryo-EM density map and atomic coordinates have been deposited to the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) with the accession codes EMD-30469 and 7CTT, respectively. All other data are available from the authors on reasonable request.

Remdesivir for the Treatment of Covid-19 -Preliminary Report

We thank all staff members in the Center of Biological Imaging (CBI), Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS), for assistance with data collection. This study was