key: cord-0890501-vn1hdjw8
authors: Wang, Quan; Wu, Jiqin; Wang, Haofeng; Gao, Yan; Liu, Qiaojie; Mu, An; Ji, Wenxin; Yan, Liming; Zhu, Yan; Zhu, Chen; Fang, Xiang; Yang, Xiaobao; Huang, Yucen; Gao, Hailong; Liu, Fengjiang; Ge, Ji; Sun, Qianqian; Yang, Xiuna; Xu, Wenqing; Liu, Zhijie; Yang, Haitao; Lou, Zhiyong; Jiang, Biao; Guddat, Luke W.; Gong, Peng; Rao, Zihe
title: Structural basis for RNA replication by the SARS-CoV-2 polymerase
date: 2020-05-22
journal: Cell
DOI: 10.1016/j.cell.2020.05.034
sha: 46f508bb717aba96ccfd8fa942fc26559916d917
doc_id: 890501
cord_uid: vn1hdjw8

Summary Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre-/post- translocated polymerase complexes. The structures show notable structural rearrangements occurring to nsp12 and its cofactors nsp7/nsp8 to accommodate the nucleic acid compared to the apo complex, while there are highly conserved residues in nsp12 positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanisms of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription/replication machinery.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, with the RNA backbone ( Figure 4C ). Although the map signal for this region is 165 relatively weak and undefined in the post-translocation structure, as mentioned 166 earlier in the structure of the pre-translocation state, the conformation of motif G and 167 its interaction with the +1 template nucleotide through S501 backbone nitrogen are 168 well resolved ( Figure 3B ). In a very recent study characterizing the role of motif G in 169 EV71 RdRP translocation, the motif G residues equivalent to K500/S501 was shown 170

to play a critical role in restricting the +1 template nucleotide (Wang, 2020). This 171 restriction, on one hand, ensures precise positioning of the templating base for 172 efficient NTP binding and catalysis, and on the other hand to control translocation by 173 setting up a rate-limiting step (Wang, 2020). 174

The interactions with RNA are manifested in broader conformational changes of the 176 palm, fingers and thumb domain of nsp12 which seem to be further propagated to 177 the two copies of nsp8. Specifically, besides the C-terminal tail (residues 926-932, 178 C-tail) next to the RNA exit channel that points toward the active site in the apo state 179 but flips out in the catalytic complexes, two α-helices of the thumb domain are 180 deflected 5 to 10 degrees to the outward of the complex relative to the RNA ( Figure  181 4D). What's interesting is that, in the pre-translocation structure, the N-terminal 182 helical domain of nsp8-1 lies on a notch formed at the top of these two helices and 183 makes a conformational change in the 845-855 loop and stabilizes the 900-910 loop which is missing in the apo and post-translocation structure ( Figures 4D and 4E) . 185

Besides, the nsp8-2 N-terminal helix which lays on the fingers has deflected by 186 about 5 degrees (Figures 4F). In the pre-translocation structure, which has a longer 187 RNA product, the most distal section of this helix appears to be able to switch 188 between two particular conformations and interact with exiting RNA (Figures 2D and  189   2E) . To further dissect the RDV-TP inhibition mechanism and to test this steric clash 232 hypothesis, we compared the behaviors of RDV-TP with ATP in our T33-1/P10-233 based assays and the WT RdRP and an S861A mutant were also compared 234 regarding the i+3 related product profiles ( Figure 6 ). We first compared the primer 235 extension profiles between the CTP/ATP and CTP/RDV-TP combinations. It turned 236 out that the latter combination led to both the regular 14-mer product (corresponding 237 to the incorporation of a C-R-C-R tetra-nucleotide) and a misincorporation-derived 238 15-mer product. This misincorporation event likely arose from a C:R misincorporation 239 as this activity is greatly reduced when the RDV-TP concentration is lowered ( consider that, with respect to nsp8-upstream RNA interactions, conformation II might 286 be the dominant state during RNA processive elongation, rather than conformation I. 287

Conformation I in our structure is therefore likely to be a transition state before the 288 RNA products getting long enough to establish the elongation-mode interactions with 289

Nsp7 and nsp8 can form a 'non-canonical primase' to generate RNA primers (Imbert 

We would like to pay an exceptional tribute to ShanghaiTech University and their 318 administrative team, as well as the Bio-Electron Microscopy Facility for their great 319 care and support to our research team to enable us to carry out this research in a 320 safe and healthy environment. It would have been impossible for us to attain this 321 achievement without their tremendous efforts in the last two months during the 322 COVID-19 pandemic. We also must express our gratitude to the campus service 323 Figures S1, S2, S3, S4 and S7 . 401 product was made (corresponding to an RNA three-nucleotide longer than that has 449 the first incorporated RDV-MP or the "i+3" product) by the WT RdRP, indicating a premature termination event. Bottom: The S861A mutant also produced the 16-mer 451 and the "i+3" products, albeit with a relatively lower amount of "i+3" termination. The 452 addition of the complementary DNA helped visualize the 16-and 17-mer bands by 453 trapping the RNA template. The same marker (M) was used as in Figure 1  454 See also Figures S1, S4 and S5 . 455 

Further information and requests for resources and reagents should be directed to 496 and will be fulfilled by the Lead Contact, Zihe Rao (raozh@tsinghua.edu.cn). 497

All unique/stable reagents generated in this study are available from the Lead 499

Contact without restriction. 500

The cryo-EM maps and atomic models have been deposited at the Electron 

Proteins were obtained through recombinant expression in E. coli BL21(DE3). 507

The SARS-CoV-2 nsp12 (GenBank: MN908947) gene was inserted into a pET22b 510

vector possessing a C-terminal 10 × His tag. Full-length SARS-CoV-2 nsp7 and 511 nsp8 genes were respectively inserted into a pET28a vector with an N-terminal 6 × 512

His tag. The plasmids were transformed into E. coli BL21(DE3). When bacterial 513 cultures were grown in LB medium to an OD 600 of 0.6-0.8 at 37℃, growth 514 temperature was reduced to 16℃ followed by the addition of a final concentration of 515 0.5 mM IPTG to induce the expression. After overnight induction, the harvested cell 516 pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 517 mM MgCl 2 , 10% glycerol) with benzonase (Yeasen) and lysed by passage through a 518 high-pressure homogenizer at 4℃. The lysate was centrifugated at 18000 rpm for 30 519 min to obtain supernatant containing recombinant protein, which was purified by Ni-520 NTA affinity chromatography. The protein bound to the Ni-NTA column was eluted 521 with lysis buffer supplemented with 300 mM imidazole and concentrated to load onto 522 a Superdex 200 10/300 Increase column (GE Healthcare, USA) in gel filtration buffer 523 (50 mM HEPES, pH 7.0, 100 mM NaCl, 4 mM MgCl 2 and 4mM DTT). The collected 524 protein fractions were concentrated to 5 mg/mL and stored at 4℃ for further use. 525

Eleven template-primer RNA constructs (Table S1) were initially screened to identify 526 sequences that could generate stalled nsp12/nsp7/nsp8-RNA catalytic complexes 527 stable enough to survive an anion exchange chromatography purification. A 528 construct comprising a 33-mer template (T33-1) and a 10-mer primer (P10) was chosen in complex assembly to generate a 14-mer product (P14)-containing catalytic 530 complex for structural studies. The RNA templates were prepared by in vitro T7 RNA 531 polymerase-glmS ribozyme-based approaches described previously (Batey and Kieft, 532

2007; Gong and Peersen, 2010) (see below for more detail), and the P10 and an 8-533 mer primer (P8) was chemically synthesized (Integrated DNA Technologies). The 534 T33-1/P10 construct was made with a 10% molar excess of P10 annealed to T33-1 535 via a 3-min incubation at 45 °C followed slow cooling to room temperature (r.t.). For 536 the P14-containing stalled catalytic complex assembly (post-translocation complex), 537 a typical 1.5-mL reaction mixture containing 12 µM nsp12, 12 µM nsp7, 24 µM nsp8, The target RNAs were purified by 12% polyacrylamide/7 M urea gel electrophoresis, 586 excised from the gel, eluted using an Elu-Trap device (GE Healthcare), ethanol 587 precipitated, dissolved in RNA annealing buffer (RAB) (50 mM NaCl, 5 mM Tris (pH 588 7.5), 5 mM MgCl 2 ), and stored at −80°C after a self-annealing process (a 5-min 589 incubation at 95 °C followed by snap cooling to minimize inter-molecular annealing). 590

The synthesis of remdesivir triphosphate (RDV-TP) was accomplished according to 

Cryo-EM structures of the SARS-CoV-2 RNA polymerase in complexes with RNA, before and after RNA translocation, reveals structural rearrangements that the RNA dependent RNA polymerase (RdRp) nsp12 and its cofactors (nsp7 and nsp8) undergo to accommodate nucleic acid binding. Further insights into how the complex is inhibited by remdesivir, and into the primase to polymerase transition, are also presented.

Oligonucleotide sequences 

Towards automated 764 crystallographic structure refinement with phenix.refine

Coronavirus Susceptibility to the Antiviral Remdesivir

Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease

SARS and MERS: recent 780 insights into emerging coronaviruses

The future of antivirals: broad-spectrum inhibitors

The PyMOL molecular graphics system

Features and development of Coot

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

UCSF ChimeraX: Meeting modern challenges in visualization and analysis

Structural basis for active site closure by the poliovirus RNA-792 dependent RNA polymerase

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from 795 severe acute respiratory syndrome coronavirus 2 with high potency

A decade after SARS: strategies for 797 controlling emerging coronaviruses

Structure of 799 replicating SARS-CoV-2 polymerase. bioRxiv

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

A second, non-canonical RNA-dependent RNA polymerase 805 in SARS coronavirus

Therapeutic efficacy of the small molecule GS-5734 against 854 Ebola virus in rhesus monkeys

Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits 857 primer-independent RNA polymerase activity

Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by 860 remdesivir

Insights into SARS-CoV 862 transcription and replication from the structure of the nsp7-nsp8 hexadecamer

A Novel Coronavirus from Patients with Pneumonia in China

The authors declare no competing interests. 352

595 became slowly clear. The reaction mixture was stirred at 0 °C for 2 hours, and a 596 suspension of pyrophosphate tributylamine salts (844 mg, 1.54 mmol) in CH 3 CN (3 597 mL) was added followed by the addition of Bu 3 N (762 mg, 4.12 mmol). The reaction 598 mixture was stirred at 0°C for 2h, the reaction was monitored by liquid 599 chromatography/mass spectrometry (LC/MS), quenched by the addition of 600 triethylammonium bicarbonate buffer (1 M, 12 mL). The resulting mixture was stirred 601 at room temperature for 30 min, and then triethylamine (1 mL) was added. The 602 mixture was stirred an additional 30 min and washed three times with EtOAc (20 mL 603 × 3). The aqueous phase was purified by Pre-HPLC to afford the product RDV-TP 604 salt (205 mg, 24 %, 3.0 eq triethylamonium) as an off-white solid. 605

In total, 3 µL of protein-RNA complex solution at 5 mg/mL (added with 0.025% DDM) 607 was applied onto an H 2 /O 2 glow-discharged, 200-mesh Quantifoil R0.6/1.0 grid 608 (Quantifoil, Micro Tools GmbH, Germany). The grid was then blotted for 3.0 s with a 609 blot force of 0 at 8°C and 100% humidity and plunge-frozen in liquid ethane using a 610Vitrobot (Thermo Fisher Scientific, USA). Cryo-EM data were collected with a 300 611 keV Titan Krios electron microscope (Thermo Fisher Scientific, USA) and a K2 612Summit direct electron detector (Gatan, USA). Images were recorded at EFTEM with 613 a 165000× magnification and calibrated super-resolution pixel size 0.82 Å/pixel. The 614 exposure time was set to 5 s with a total accumulated dose of 60 electrons per Å 2 . 615All images were automatically recorded using SerialEM (Mastronarde, 2005) . For the 616 sample of nsp12-nsp7-nsp8-RNA post-translocated catalytic complex, a total of 617 6,826 images were collected with a defocus range from 1.0 µm to 1.8 µm. For the 618 sample of nsp12-nsp7-nsp8-RNA pre-translocated catalytic complex, a total of 6,524 619 images were collected with a defocus range from 1.0 µm to 2.0 µm. Statistics for 620 data collection and refinement are in Table S2 . 621

All dose-fractioned images were motion-corrected and dose-weighted by 623 were selected after two rounds of 2D classification. 100,000 particles were used to 630 do Ab-Initio reconstruction in four classes, and then these four classes were used as 631 3D volume templates for heterogeneous refinement with all selected particles, with 632 119,662 particles converged into nsp12-nsp7-nsp8-RNA class and 183,165 particles 633 converged into nsp12-nsp8-RNA class. Next, both particle sets were used to perform 634 non-uniform refinement, yielding a resolution of 3.26 Å and 3.60 Å, respectively. For 635 the dataset of nsp12-nsp7-nsp8-RNA pre-translocated catalytic complex, a total of 636 1,330,896 particles were auto-picked using the templates selected from the previous 637 Table S2 . Characterization, Related to Figures 1, 2, 3, 5 and 6 . in Figure 1A and the downstream hairpin sequences are identical for all constructs. 677Besides the construct name (e.g. T33-1/P10), a number is assigned to each 678 construct. RNA profile on the primer-extension reactions is tested on a gel (lower 679 panels). The EV71 RdRP (3Dpol) capable of producing a homogenous P14 product 680 from the T33-6/P10 construct (number 6) was used as a reference (lanes 1 and 14) . 681Different extent of primer utilization (compare lanes 2-8 and 10-13), misincorporation 682 (lanes 2/6/7), product migration shift (lanes 4/11/12/13, brown vertical bars), and 683 formation of product template-product-containing higher-order RNA structures (lanes 684 4/11/12/13, red vertical bars) were observed. One construct did not yield the product 685 of the expected length (lane 5). The T33-1/P10 construct (number 1) was chosen for 686 the catalytic complex assembly and structural study. 687

The accumulation of the T33-7/P10-derived P18 product is related to RDV-MP 688 incorporation. Purified T33-7/P14 complex was incubated with GTP/RDV-TP (+GR) 689 or GTP/ATP (+GA) at 4 °C for various periods ("0 min" corresponds to immediate 690 reaction quench after manual mixing to initiate the reaction). For the +GR reaction, 691 the P18 product is most prominent at "0 min" and diminishes over time, suggesting 692 an RDV-induced pausing mechanism. The level of the P20 product is consistent at 5, 693 30, and 60 min time points, consistent with the RDV-induced "i+3" premature 694 termination mechanism. In the +GA reaction, both the P18 and P20 products are not 695 as prominent. Due to the formation of higher-order product-containing RNA