key: cord-0808514-vlozgjqo
authors: Wu, Jiqin; Wang, Haofeng; Liu, Qiaojie; Li, Rui; Gao, Yan; Fang, Xiang; Zhong, Yao; Wang, Meihua; Wang, Quan; Rao, Zihe; Gong, Peng
title: Remdesivir overcomes the S861 roadblock in SARS-CoV-2 polymerase elongation complex
date: 2021-10-08
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109882
sha: 281ce9c039c637ca68307813d11305622e629d5f
doc_id: 808514
cord_uid: vlozgjqo

Remdesivir (RDV), a nucleotide analog with broad-spectrum features, has exhibited effectiveness in COVID-19 treatment. However, the precise working mechanism of RDV when targeting the viral RNA-dependent RNA polymerase (RdRP) has not been fully elucidated. Here we solve a 3.0-angström structure of SARS-CoV-2 RdRP elongation complex (EC) and assess RDV intervention in polymerase elongation phase. While RDV could induce an “i+3” delayed termination in meta-stable complexes, only pausing and subsequent elongation are observed in the EC. A comparative investigation using an enterovirus RdRP further confirm similar delayed intervention and demonstrate that steric hindrance of the RDV-characteristic 1′-cyano at the -4 position is responsible for the “i+3” intervention, while two representative Flaviviridae RdRPs do not exhibit similar behavior. A comparison of representative viral RdRP catalytic complex structures indicates that the product RNA backbone encounters highly conserved structural elements, highlighting the broad-spectrum intervention potential of 1′-modified nucleotide analogs in anti-RNA virus drug development.

RNA viruses include numerous important human pathogens, and have caused multiple 20 epidemics of global concern in the past two decades. The Coronavirus Disease-2019 21 (COVID-19) causing a global pandemic is caused by an RNA virus, sever acute respiratory Remdesivir (RDV) is an NA with a characteristic modification at the ribose 1-position. 53 It was first developed against Ebola virus (EBoV) and entered clinical trials during recent 54 Ebola epidemics in Africa (Jacobs et al., 2016) . It has been shown to be effective against 55 several coronaviruses (CoVs) and therefore was immediately tested in searching for SARS- 56 CoV-2 drug candidates (Wang et al., 2020a) . Although RDV has been approved by the 57 European Union and the United States for COVID-19 treatment, data derived from clinical 58 trials suggest that its effectiveness is not ideal (Beigel et al., 2020; Goldman et al., 2020) . 59 Hence, to understand its precise intervention and broad-spectrum mechanism and to further 60 optimize its antiviral activity through rational design will benefit the development of NA 61 drugs against clinically important RNA viruses in general. As an adenosine analog, the NTP Through comparative analyses of catalytic complex structures from representative RdRPs, we 80 proposed a general RdRP intervention mechanism for RDV that may benefit future 81 development of potent NA drugs against RNA viruses. 84 Chain terminating NAs have exhibited potency in various polymerase systems (Elion et 85 al., 1977; Hakimelahi et al., 2001) . Classical chain terminators such as the anti-herpetic drug 86 acyclovir (ACV) do not contain a 3-hydroxyl group and will not allow further catalysis after 87 the incorporation of its nucleoside monophosphate (NMP) form (Tchesnokov et al., 2009 with UTP in a primer-dependent RdRP assay using SARS-CoV-2 nsp12-nsp7-nsp8 and a 92 J o u r n a l P r e -p r o o f T33-8/P10 RNA construct comprising a 33-mer template (T33) and a 10-mer primer (P10) 93 (Fig. 1A) . In the presence of C, U, and ATP (C/U/A), the P10 can be readily converted to a 94 17-mer product (P17) within 60 min (Fig. 1A, lanes 2-4 and 12-14) . When UTP was replaced 95 by SOF-TP (C/S/A), P10 conversion was as efficient, but very low level of extension 96 (indicated by the amount of P17) occurred after the incorporation of SOF (Fig. 1A , lanes 5-7 97 and 15-17), suggesting that SOF may act as a nonobligate chain terminator in SARS-CoV-2 98 RdRP replication . We next tested T-1105, a structural analog of FVP, in a 99 similar assay using a T35/P10 construct (Fig. 1B) . Comparing to incorporation in the terminating mechanism by RBV, and FVP/T-1105. 110 We previously solved an RDV-containing SARS-CoV-2 RdRP catalytic complex using 111 a T33-7 construct (Wang et al., 2020c) . Somewhat unexpectedly, an "i+1" 18-mer product 112 (P18) was observed in this complex likely due to both interactions between the downstream 113 stem-loop RNA region and nsp12 and the incorporation of RDV. These RNA sequence/local 114 structure-dependent interactions probably induced a pausing at this particular stage and 115 trapped the RdRP in a pre-translocation state. In a gel-based assessment, "i+3" product was 116 also observed and accompanied by extension of the "i+1" product, demonstrating the pausing 117 J o u r n a l P r e -p r o o f nature of the latter, while by contrast, this "i+1" product was not prominent in regular NTP-118 driven reactions (Wang et al., 2020c) . Here we increased the reaction duration to 4 h (vs. 1 h 119 in the previous study) using the same RNA construct. While the "i+1" product diminished 120 over time, the amount of "i+3" (P20) did not apparently decrease in the tested period, 121 indicating that at least a portion of catalytic complexes terminated or became inactive at this 122 stage (Fig. 1D, lanes 83-94; Fig. S1 ). As expected, both "i+1" and "i+3" products were not 123 pronounced in the ATP comparison set (Fig. 1D, lanes 63-74) . 195 We next assessed RDV intervention in the T56 derived EC in a time course format ( Fig.   196 4). When G, A, and UTP (G/A/U) were supplied to the purified P14-containing EC, the 197 complex was expected to synthesize a 25-mer product (P25). The reaction temperature was Both structural analysis and enzymology data support the relationship between the "i+3" by RDV was evident in these two RdRPs, although low-level accumulation of 5-mer 243 (corresponding to "i+2") and 6-mer (corresponding to "i+3") was observed for HCV NS5B 244 and DENV NS5, respectively. We then tested RDV intervention in the primer-dependent 245 Picornaviridae EV71 RdRP that also contains a serine (S417) at the SARS-CoV-2 nsp12 246 S861 equivalent position using the T33-1/P10 construct (Fig. 5B ). In the presence of CTP and opportunity to capture a RDV-induced pausing EC with such encounter, either using WT 294 nsp12 or its S861 mutants. With a similar delayed intervention mechanism induced by RDV, 295 the EV71 RdRP provides another suitable system to further dissect RDV intervention.

The delayed intervention of RDV attributed to its post-incorporation encounter with 297 SARS-CoV-2 S861 side chain is a unique mechanism different from those represented by 298 immediate chain termination and error-prone synthesis due to ambiguous basepairing. This product strand backbone is highly analogous (Fig. 7) . RdRP motif C, motif E, and the 319 aforementioned clamping thumb helix relay in the moving track, interacting with the +1/-1, -320 2, -3 to -5 positions of the backbone, respectively. Each of these three structural elements 321 contain side chains within 7 Å to the 1-carbon of the corresponding product nucleotide.

Among these residues, equivalents to SARS-CoV-2 nsp12 S861 are all spatially close to the - The current study reveals that RDV can overcome the S861 roadblock in a SARS-CoV-344 2 RdRP EC, while a similar mechanism is found in the EV71 system. However, the direct 345 encounter of the incorporated RDV and the roadblock residue has not been captured by 

The authors declare no competing interests. NS5B was prepared using a pET26b-based plasmid and E. coli Rosetta (DE3) strain. Protein 528 purification and storage for HCV NS5B were performed as previously described in the JEV 529 NS5 study (Lu and Gong, 2013) , except that a 100 mM imidazole wash step was applied 530 prior to the elution step of the Ni-affinity chromatography, and MES (pH 6.5) and Tris (pH 531 J o u r n a l P r e -p r o o f 7.5) were used as the buffering agents for the cation-exchange and gel filtration columns, 532 respectively. The yield is typically 3 mg of pure NS5B protein per liter of bacterial culture.

RNA preparation and RdRP catalytic complex assembly 534 RNA templates T33-1, T33-7, T33-8, T35, and T56 were prepared through a T7 RNA 535 polymerase-glmS ribozyme-based method as described previously (Batey and Kieft, 2007) .

RNA primer P10 was purchased from Integrated DNA Technologies. P10 were annealed to 537 T33-1/T33-7/T33-8/T35 and T56 at 1.1:1 and 3.1:1 molar ratios, respectively (Gong and 538 Peersen, 2010). The T33-1 derived P14-containing catalytic complex was assmebled and 539 purified as described previously (Wang et al., 2020c) . For T56 derived P14-containing 540 catalytic complex assembly, a typical 1.5 ml reaction system containing 12 µM nsp12, 12 µM 

All dose-fractioned images were automatically recorded using SerialEM (Mastronarde, 2005) 559 with defocus ranging from 1.2 m to 1.8 m and 6,118 movies were recorded in tif format. 

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