# The effects of Remdesivir's functional groups on its antiviral potency and resistance against the SARS-CoV-2 polymerase

Bhawna Sama, Barbara Selisko, Camille Falcou, Véronique Fattorini, Géraldine Piorkowski, Franck Touret, Kim Donckers, Johan Neyts, Dirk Jochmans, Ashleigh Shannon, et al.

► To cite this version:

Bhawna Sama, Barbara Selisko, Camille Falcou, Véronique Fattorini, Géraldine Piorkowski, et al. The effects of Remdesivir's functional groups on its antiviral potency and resistance against the SARS-CoV-2 polymerase. Antiviral Research, 2024, 232, 10.1016/j.antiviral.2024.106034. hal-04924339

HAL Id: hal-04924339

https://amu.hal.science/hal-04924339v1

Submitted on 31 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# The effects of Remdesivir's functional groups on its antiviral potency and resistance against the SARS-CoV-2 polymerase

<figure><img src="image_3.png" /></figure>

Check for
updates

Bhawna Sama<sup>a</sup>, Barbara Selisko<sup>a</sup>, Camille Falcou<sup>a</sup>, Véronique Fattorini<sup>a</sup>, Géraldine Piorkowski<sup>b</sup>, Franck Touret<sup>b</sup>, Kim Donckers<sup>c</sup>, Johan Neyts<sup>c</sup>, Dirk Jochmans<sup>c</sup>, Ashleigh Shannon<sup>a</sup>, Bruno Coutard<sup>b</sup>, Bruno Canard<sup>a,d,*</sup>

## ARTICLE INFO

### Keywords:

SARS-CoV-2

Nucleotide analogue

Remdesivir

Polymerization

Excision

MoA

Resistance

## ABSTRACT

Remdesivir (RDV, Veklury®) is the first FDA-approved antiviral treatment for COVID-19. It is a nucleotide analogue (NA) carrying a 1'-cyano (1'-CN) group on the ribose and a pseudo-adenine nucleobase whose contributions to the mode of action (MoA) are not clear. Here, we dissect these independent contributions by employing RDV-TP analogues. We show that while the 1'-CN group is directly responsible for transient stalling of the SARS-CoV-2 replication/transcription complex (RTC), the nucleobase plays a role in the strength of this stalling. Conversely, RNA extension assays show that the 1'-CN group plays a role in fidelity and that RDV-TP can be incorporated as a GTP analogue, albeit with lower efficiency. However, a mutagenic effect by the viral polymerase is not ascertained by deep sequencing of viral RNA from cells treated with RDV. We observe that once added to the 3' end of RNA, RDV-MP is sensitive to excision and its 1'-CN group does not impact its nsp14-mediated removal. A >14-fold RDV-resistant SARS-CoV-2 isolate can be selected carrying two mutations in the nsp12 sequence, S759A and A777S. They confer both RDV-TP discrimination over ATP by nsp12 and stalling during RNA synthesis, leaving more time for excision-repair and potentially dampening RDV efficiency. We conclude that RDV presents a multi-faced MoA. It slows down or stalls overall RNA synthesis but is efficiently repaired from the primer strand, whereas once in the template, read-through inhibition adds to this effect. Its efficient incorporation may corrupt proviral RNA, likely disturbing downstream functions in the virus life cycle.

## 1. Introduction

The emergence of SARS-CoV-2 responsible for COVID-19 and its subsequent global pandemic highlighted the need for broad-spectrum antiviral drugs aimed at controlling the virus especially in the early phases of an outbreak (Li et al., 2023). These antivirals have the potential to be designed in advance, stockpiled, and utilized during the initial stages of a developing epidemic, either prophylactically when given to contact cases or therapeutically alongside other control measures. Direct-acting antivirals (DAAs) are specifically directed against conserved viral proteins/enzymes that often lack cellular homologues. Consequently, any virus emerging from a known virus family will carry these conserved essential enzymes, allowing for their inhibition to be

evaluated beforehand.

The most successful DAAs so far belong to a few classes of viral enzyme inhibitors, prominently those targeting proteases and polymerases. The latter constitutes the target of existing successful therapies against human immunodeficiency virus (HIV), hepatitis C virus (HCV), and recently, SARS-CoV-2 (De Clercq and Li, 2016; Malone et al., 2022; Shannon and Canard, 2023; Li et al., 2023).

Remdesivir (RDV; GS-5734, brand name Veklury®) is the first U.S. Food and Drug Administration (FDA) approved drug used for COVID-19 treatment (Rubin et al., 2020). It is a phosphoramidate nucleotide drug (ProTide) of the adenosine analogue, GS-441524 developed by Gilead Sciences before the emergence of SARS-CoV-2 and repurposed against it (Mackman, 2022). RDV is a potent inhibitor of several RNA

<footnote><sup>a</sup> Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, UMR7257, Marseille, France</footnote>

<footnote><sup>b</sup> Unité des Virus Émergents (UVE), Aix-Marseille Université, Università di Corsica, IRD 190, Inserm 1207, IRBA, France</footnote>

<footnote><sup>c</sup> KU Leuven - Rega Institute, Leuven, Belgium</footnote>

<footnote><sup>d</sup> European Virus Bioinformatics Center, Leutragraben 1, 07743, Jena, Germany</footnote>

<footnote>* Corresponding author. Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, UMR7257, Marseille, France.</footnote>

<footnote>E-mail address: bruno.canard@univ-amu.fr (B. Canard).</footnote>
viruses in infected cell cultures (Sheahan et al., 2017; Malin et al., 2020) and early administration of the drug during the course of SARS-CoV-2 infection can curb disease progression (Beigel et al., 2020; Gottlieb et al., 2022; Russo et al., 2022).

Upon cellular uptake, RDV is converted to its 5'-triphosphate form (RDV-TP<fnref n="1" />) that mimics an adenosine triphosphate (ATP) and is thus incorporated into the SARS-CoV-2 viral RNA by the RNA-dependent RNA polymerase (RdRp) nsp12 which is demonstrated in biochemical assays (Gordon et al., 2020b; Dangerfield et al., 2020). Atomic resolution structural studies of the SARS-CoV-2 replication/transcription complex (RTC) bound to RNA have shown RDV-MP incorporated into the elongating RNA chain, Watson-Crick base-pairing with uracil in the template strand and forming base-stacking interactions with the base upstream in the primer strand (Yin et al., 2020; Bravo et al., 2021). However, the precise mechanism by which RDV-MP causes the inhibition of viral RNA synthesis is still not entirely understood.

The mode of action (MoA) of a nucleotide analogue (NA) as an antiviral can often be inferred from its chemical structure (Shannon and Canard, 2023). NAs are first converted to their active 5'-triphosphate form (NA-TP) for its incorporation by the viral polymerase, potentially leading to the blunt arrest of viral RNA synthesis. Chain terminating NAs that lack a 3'-OH group are known as obligate RNA chain terminators, while those having one are non-obligate chain terminators, causing immediate or delayed termination of RNA elongation. These non-obligate terminators mainly carry other ribose substitutions. Alternatively, NAs once incorporated into viral RNA can convey an altered genetic message. These NAs largely usually carry base modifications. This is the case for Molnupiravir where massive incorporation of its active β-D-N4-hydroxy cytidine 5'-triphosphate form into viral RNA yields overly mutated, non-functional genomes, explaining its antiviral effects (Yip et al., 2022).

The situation is not as straightforward for RDV. Indeed, RDV carries several chemical attributes confusing immediate inference of its MoA. It has a modified base, a 3'-OH, and a 1'-CN ribose substituent whose contributions to either stopping RNA synthesis or corruption of the genetic message, or both, are not entirely understood. Attempts to define the MoA of RDV against SARS-CoV-2 have led to several observations. First, RDV-TP was shown to temporarily stall RNA elongation at i+3, i.e. following the incorporation of the third nucleotide after an RDV-MP at position i. At i+3, a steric clash occurs between the 1'-CN group of RDV and the side chain of the RdRp residue Ser861, causing a translocation barrier (Gordon et al., 2020a, 2020b; Wang et al., 2020; Kokic et al., 2021; Bravo et al., 2021). In these biochemical assays, it was also observed that this stalling is easily overcome at physiological NTP concentrations. The stalling might activate backtracking as detected through single-molecule, magnetic-tweezers in vitro assays following RDV-MP incorporation (Seifert et al., 2021), potentially allowing ExoN repair activity (see below) (Malone et al., 2021). Second, it was shown that UTP incorporation opposite a templating RDV-MP was impeded, termed template-dependent inhibition (Tchesnokov et al., 2020).

The genomes of coronaviruses carry a viral 3'-to-5' exonuclease (ExoN) gene. The ExoN (nsp14) is involved in RNA synthesis fidelity through replication-associated mismatch repair and can remove NAs from the nascent viral RNA, (Minskaia et al., 2006; Smith et al., 2013; Eckerle et al., 2010; Ogando et al., 2019). Nsp14 interacts with nsp10 to form a stable complex that enhances its ExoN activity ((Bouvet et al., 2012; Ma et al., 2015; Ferron et al., 2018)). The presence of this proofreading activity in CoV RTCs is known to impact the general potency of several antiviral NAs (Smith et al., 2013). Like mispaired nucleotides, incorporated NAs are expected to be excised (proofread) from neo-synthesized RNA. However, it remains unclear whether the potency of RDV originates from resistance to excision due to altered recognition

of its scaffold by the nsp14-nsp10 ExoN complex, as studies present conflicting findings (Shannon et al., 2020a; Jockusch et al., 2020; Zhang et al., 2021; Dangerfield and Johnson, 2022; Chinthapatla et al., 2023).

The overall MoA of RDV remains difficult to ascertain. However, evaluating the respective contributions of each potential mechanism is crucial for designing DAAs with improved potency. Despite this, there is still a lack of comprehensive understanding of RDV's MoA against SARS-CoV-2, together with the specific roles of its chemical modifications relative to its natural adenosine counterpart.

Studying drug resistance may indirectly illuminate the MoA of a corresponding drug. Typically, drug resistance arises with the emergence and selection of mutations in enzyme genes targeted by the compound that reduces drug interactions and/or jeopardizes the mechanism(s) of action. This is commonly encountered during antiviral therapies. It not only reduces the drug potency but also offers important insights into drug targets and gives information on the MoA. Several RDV-resistant mutations in nsp12 have been reported after serial passaging of SARS-CoV-2 and related Murine Hepatitis Virus (MHV) (Agostini et al., 2018; Szemiel et al., 2021; Stevens et al., 2022). Moreover, modest RDV resistance has emerged in immunocompromised patients receiving RDV treatment, and as expected, several mutations indeed map in the nsp12 RdRp domain (Martinot et al., 2021; Gandhi et al., 2022; Hogan et al., 2023).

Here, we have addressed the RDV MoA from multiple perspectives. Firstly, we measured the incorporation and excision properties of RDV-TP as a substrate for the SARS-CoV-2 RTC. We utilized the active RdRp complex (also called minimal RTC (mRTC)) comprised of nsp12 along with two accessory proteins, nsp7 and nsp8 and the active ExoN complex made of nsp14 and nsp10, respectively. To precisely separate and address the role of distinct chemical modifications on the antiviral effect, we made use of RDV-TP devoid of either its 1'-CN group on the ribose [RDV(-CN)-TP] or its pseudo-adenine base, i.e., ATP carrying a 1'-CN group [ATP+CN]. Secondly, we studied the in-vitro mutagenic potential of RDV in SARS-CoV-2 infected cells by viral genome sequencing. Finally, we isolated RDV-resistant viruses and reconstituted their corresponding recombinant resistant mRTC to assess their modified interactions with RDV-TP. Our results show that RDV's antiviral mode of action in SARS-CoV-2 results from the combination of the base and ribose modifications leading to corruption of proviral RNA.

## 2. Materials and methods

### 2.1. Ribonucleotides and analogues

Ribonucleotides (rNTPs: ATP, GTP, UTP, CTP) were purchased as 100 mM solutions from Cytiva. GS-5734 (RDV) was purchased from BLD Pharm. GS-441524 (Remdesivir; RDV nucleoside) was obtained from MedChem Express (Cat. no.: HY-103586). Ribonucleotide analogues: Remdesivir triphosphate (RDV-TP), RDV-TP without 1'-CN group (RDV(-CN)-TP), ATP with 1'-CN group (ATP+CN) were purchased from TopPharm. Inc. They were pure as judged by HPLC. AT-9010 was a gift from ATEA Pharmaceuticals, synthesized and used as described in (Shannon et al., 2022).

### 2.2. Biochemical assays

#### 2.2.1. Synthetic oligonucleotides

HPLC-purified RNA substrates (Supplementary Table 1); primers, templates and self-priming hairpin (HP) RNAs were purchased from Biomers.net. The lyophilized substrates were resuspended in 10 mM HEPES (pH 7) buffer containing 10 µM EDTA. The template used in the assays was a 20-nt template (T(A)20) corresponding to the 3'-end of the SARS-CoV and SARS-CoV-2 genome before the poly A tail. This template served as the reference to design other suitable templates. The templates were annealed to a common 5' Cy5 labeled 10-nt primer (P10). Annealing of primer-template pairs was performed with a molar ratio of

<footnote n="1">¹ We will refer to RDV-TP for the nucleotide as a RdRp substrate, and RDV-MP once it has been incorporated into the RNA strand.</footnote>
1:1.5 of primer:template in presence of 110 mM of KCl at 70 °C for 10 min, then cooling back to room temperature before storing at -20 °C.

### 2.2.2. Plasmid mutagenesis

Site-directed mutagenesis for the RDV resistance mutants S759A, A777S, and double mutation of S759A & A777S were done in the nsp12 construct cloned in pJ404 plasmid. The nsp12 sequence was that of the Wuhan-Hu-1 isolate, into which a P323L mutation was introduced using site directed mutagenesis. In our RDV-resistant biochemical assays, WT, S759A, A777S, and S759A + A777S nsp12 contained the P323L substitution. It is located far from the polymerase active site, at the interface of nsp12 and nsp8 and should have no influence on nucleotide selectivity (Plante et al., 2021). Mutagenesis was performed using the overlapping, outward facing primers with Phusion-High Fidelity DNA Polymerase (NEB) as described by the manufacturer. A DpnI (NEB) digestion was done following the PCR cycle. The clones were sequenced to confirm the point mutation(s).

### 2.2.3. Protein expression and purification

SARS-CoV nsp12 and co-factors nsp7L8 and nsp8 were expressed and purified exactly as reported previously (Shannon et al., 2020b). The expression and purification protocols of SARS-CoV-2 proteins were adapted from previous reports of (Shannon et al., 2022) for nsp12 (WT or mutants), 7, 8 and of (Ahmed-Belkacem et al., 2022) for nsp14, 10 and are described below briefly. The amino acid sequences for all the proteins used in this study correspond to the SARS-CoV-2 Wuhan-Hu-1 isolate (NCBI Ref: YP_009724389.1). The codon-optimized genes (for the expression in *Escherichia coli* (*E. coli*)) were purchased from Twist Bioscience.

The gene of nsp12 was cloned in the pJ404 vector with coding sequences for N-terminal 8x-histidine (8His) tag followed by the TEV cleavage site (MRGSHHHHHHHHHGSENLYFQ). NEBExpress® competent *E. coli* cells (New England Biolabs, Cat. no. C2523) containing pGT-f2 chaperone plasmid (Takara Bio) were transformed by the expression plasmid. Bacteria were grown in LB media in the presence of ampicillin (100 µg/mL) and chloramphenicol (17 µg/mL) at 37 °C until the OD₆₀₀ reached 0.5-0.6. The protein expression was then induced with 100 µM IPTG and 10 µg/mL tetracycline. Ethanol was added to 2% and the bacterial culture was left to grow overnight at 17 °C and 200 rpm. Following the incubation, the bacteria were harvested by centrifugation (13,900×g, 10 min, 4 °C) and the pellets were stored at -80 °C until purification. The frozen cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 5 mM MgSO₄, 1% CHAPS, 10% glycerol, 5 mM TCEP, 1 mM PMSF, 1 mg/mL lysozyme, 10 µg/mL DNase I, 0.2 mM benzamidine). NaCl was added gradually up to 1 M final concentration helping in disruption of the cells as well as in the removal of bacterial nucleic acids non-specifically bound to nsp12. Following, the clarification of the lysate by centrifugation (30,000×g, 30 min, 4 °C), the supernatant was loaded on well equilibrated cobalt IMAC resin column (TALON® Superflow™, Cytiva). The column was washed with 10 column volumes (CV) of wash buffer (50 mM Tris-HCl pH 8, 1M NaCl, 5 mM TCEP, 5 mM imidazole, 10% glycerol). Recombinant protein was eluted with elution buffer (50 mM Tris-HCl pH8, 300 mM NaCl, 5 mM TCEP, 500 mM imidazole, 10% glycerol). The histidine tag was removed by TEV protease cleavage (1:10 w/w ratio to TEV:protein) performed overnight at 4 °C in dialysis buffer (50 mM Tris-HCl pH8, 200 mM NaCl, 1 mM DTT, 10% glycerol). Here, the cleaved nsp12 was used in the case of nsp12 containing P323L substitution, i.e., for the RDV-resistant biochemical analysis. The cleaved protein was allowed to pass through the cobalt resin and the flowthrough was collected. Protein was further purified on a HiLoad 26/60 Superdex 200 size exclusion chromatography column (Cytiva) equilibrated in 25 mM HEPES pH 8, 300 mM NaCl, 5 mM MgCl₂, 5 mM TCEP, 10% glycerol. The fractions were pooled after examining the purity through SDS-PAGE. Following the purity assessment, the protein was concentrated and the concentration was determined using Nanodrop Spectrophotometer. Aliquots were

made and stored at -80 °C until use.

For the expression and production of nsp7 and nsp8, the coding sequences were cloned in pQE30 plasmid fused at their 5' end to an hexahistidine and TEV cleavage site coding sequences (MRGSHHHHHHHHHGSENLYFQ). NEBExpress® Iq competent *E. coli* cells (New England Biolabs, Cat. no. C3037) were transformed by the expression plasmids. Bacteria were grown in LB media in the presence of ampicillin (100 µg/mL) and chloramphenicol (17 µg/mL) at 37 °C. At an OD₆₀₀ range of 0.5-0.6, the induction was performed with 100 µM IPTG and the bacteria were left to grow overnight at 17 °C. After the end of incubation period, the bacterial cultures were centrifuged and the pellets were stored at -80 °C. The frozen pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 5 mM MgSO₄, 10% glycerol, 0.1% Triton X-100) supplemented with 5 mM benzamidine. Following the addition of lysis buffer and its incubation for 45 min at 4 °C, the lysate was then sonicated and clarified. The supernatant was loaded on equilibrated cobalt IMAC resin column (TALON® Superflow™, Cytiva). The column was then washed with 10 CV of wash buffer (50 mM Tris-HCl pH8, 300 mM NaCl, 10 mM Imidazole) followed by a second wash with buffer supplemented with 1 mM NaCl. Protein was eluted with elution buffer (50 mM Tris-HCl pH8, 300 mM NaCl, 500 mM imidazole). The 6His tag was removed following the protocol described above in the case of nsp12. Protein was further purified by size exclusion chromatography (16/60 Superdex 200 column, Cytiva) in the final buffer consisting of 25 mM HEPES pH 8, 300 mM NaCl, 5 mM MgCl₂, 5 mM TCEP, 10% glycerol (same as that of nsp12). The fractions were pooled after determining their purity through SDS-PAGE. Following the purity assessment, the protein was concentrated and the aliquots were stored at -80 °C until use.

The coding sequences of nsp10 and nsp14 were cloned in pET-28 plasmid with an 8His tag fused to their 5' end followed by the TEV cleavage site coding sequences (MGGSHHHHHHHHHGSENLYFQ). T7 Express competent *E. coli* (New England Biolabs, Cat. no. C2566) containing the expression plasmids were grown in LB media with kanamycin (50 µg/mL) at 37 °C. Once the OD₆₀₀ reached 0.5, the induction was done with IPTG (500 µM and 50 µM for nsp10 and nsp14 respectively) and the bacteria were left to grow at 17 °C, overnight in the shaker incubator. The cells were harvested and stored at -80 °C, until use. The frozen pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 15 mM imidazole, 5 mM MgSO₄, 10% glycerol, 0.1% Triton X-100) supplemented with 5 mM benzamidine, 1 mM PMSF, 0.25 mg/mL lysozyme, 10 µg/mL DNase I. The cells were sonicated after the incubation in lysis buffer for 20 min at 4 °C. The clarification of lysate was done by centrifugation and the supernatant was loaded on an equilibrated cobalt IMAC resin column (TALON® Superflow™, Cytiva). The washing was with 10 CV of wash buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 15 mM imidazole, 5 mM MgSO₄, 10% glycerol, 1 mM beta-mercaptoethanol) followed by a second wash with buffer supplemented with 1 M NaCl. Protein was eluted with the buffer supplemented with 250 mM imidazole. The purity was determined by SDS-PAGE and the elution fractions were pooled. Nsp10 was transferred by dialysis into the final buffer of 50 mM Tris pH 8, 300 mM NaCl, 5 mM MgSO₄, 1 mM beta-mercaptoethanol, 10% glycerol. For nsp14, the protein was further purified on to a HiLoad 16/60 Superdex 200 size exclusion chromatography column (Cytiva) in the final buffer consisting of 50 mM Tris-HCl pH 6.8, 300 mM NaCl, 0.5 mM TCEP, 5% glycerol. Following the purity assessment, the protein was concentrated and the aliquots were stored at -80 °C until use. The SDS-PAGE of all purified SARS-CoV-2 proteins used in biochemical assays are shown in Fig. S1.

### 2.2.4. RNA elongation and excision assays

The SARS-CoV mRTC was prepared as described in (Shannon et al., 2020b). The mRTC was prepared by first mixing nsp7 and nsp8 (final concentration 60 µM in complex buffer (25 mM HEPES, pH 8, 150 mM NaCl, 10% glycerol, 5 mM TCEP, 5 mM MgCl₂). After incubation for 30
min at room temperature (RT), nsp8 and nsp12 were added individually to a final concentration of 10 µM nsp12, 30 µM nsp7 and 60 µM nsp8 (1:3:6) and this final complex was further incubated for 15 min at RT. Concentrations were adjusted to 2 µM nsp12 in reaction buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM MgCl₂). An elongation complex was then formed by adding the same volume of 2 µM primer/template in reaction buffer. After 10 min incubation at 30 °C, reactions were started by adding the same volume of a NTP and/or NA-TP mix in the reaction buffer. Final concentrations in the reactions were 0.5 µM nsp12 (1.5 µM nsp7 and 3 µM nsp8; (1:3:6)) and 0.5 µM P/T unless stated otherwise in the figure legends. Composition and concentration of NTP/NA-TP are given in the figure legends for each reaction.

Samples were taken at given time points (see figures) and mixed 1:3 (v/v) with FBD (formamide, 10 mM EDTA and bromophenol blue). For the 0 time point, first RNA and mRTC mix was added to the FBD stop solution and then the NTP/NA-TP mix was added to this mixture. Samples were heated for 10 min at 70 °C and cooled on ice for 2 min before analysis by denaturing polyacrylamide gel electrophoresis [(PAGE); 20% acrylamide, 7M Urea] in TBE buffer at 45 W. Separated product bands were then visualized using an Amersham™ Typhoon™ Biomolecular Imager (Cytiva).

For the excision reaction, SARS-CoV-2 mRTC reaction mixtures were heat-denatured after the polymerase reaction for 10 min at 70 °C. HP was re-annealed at 30 °C for up to 30 min. The excision enzyme complex was prepared by mixing the nsp14 and nsp10 in 1:4 M ratio (Bouvet et al., 2012) in 10 mM HEPES buffer (pH 7.5) and incubated at RT for 5 min. In the re-annealed HP, excision complex was added to 12.5 nM nsp14 and 50 nM nsp10, incubated at 30 °C and a time course was followed for 5, 10, 30, and 60 min. The reaction was stopped in FBD and analyzed, as explained above.

### 2.2.5. Quantification of product bands and data analysis after elongation

The quantification of substrate and elongated product bands were done using the ImageQuant software (version 10.2) from Cytiva. Product yield was determined by dividing the intensity of the product by the total intensity of the product(s) and remaining primer, and multiplying by the input 500 nM concentration of RNA (if not stated otherwise). For initial velocity calculation in nM/min the linear phase of product generation was monitored for which time points were chosen according to incorporation efficiency (natural NTP or misincorporation). Only the linear steady state phase of the reaction was considered. Graphs were generated using GraphPad Prism 10 software.

### 2.2.6. Quantification of product bands and data analysis after excision

The quantification of excision was done as given above for elongation, i.e. that product yield was determined by dividing the intensity of the excision product bands by the total intensity of the product(s) and the remaining polymerization product, then multiplying by the input 500 nM concentration of RNA (if not stated otherwise). When the polymerization after 30 min did not yield 100% product, the value of elongated product at the beginning of the excision was adjusted according to the actual yield. The quantity of each excision product in nM was multiplied by the number of nt excised to generate this product. Finally, the sum of excised nts at each time point was plotted against time. The slope of the initial linear phase corresponds to the initial velocity values in nM excised nt per min (R² around 0.99, end of linearity normally reached after 30 or 60 min). In the case of HP_AC (Fig. 7) where two incorporations of RDV-MP and AMP+CN were observed, the percentage of double-incorporated product was considered and the number of excised nts for each product band adjusted for this percentage of product band.

## 2.3. *In-vitro* infection assays

We have performed two major types of *in-vitro* infection assays; RDV mutagenesis analysis (2.3.1) and the selection of RDV resistant viruses

(2.3.2), where different conditions are used as indicated in the text below. An antiviral assay was also performed prior to the mutagenesis assay to determine the effective concentrations of RDV to conduct the following experiments.

### 2.3.1. RDV mutagenesis analysis in SARS-CoV-2 infected cells

#### 2.3.1.1. Cell lines and virus strain.
All cells were grown at 37 °C with 5% CO₂ with 1 % penicillin/streptomycin (5000 U/mL and 5000 µg/mL, respectively; Life Technologies) and supplemented with 1% non-essential amino acids (Life Technologies) in cell-line specific media as described below. Huh7.5 (RRID CVCL-7927) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) High glucose (4500 mg/L) (Life Technologies) with 7 0.5% heat-inactivated fetal calf serum (FCS; Life Technologies).

SARS-CoV-2 strain BavPat1 was obtained from Pr. C. Drosten through EVA GLOBAL (https://www.european-virus-archive.com/). To prepare the virus working stock, a 25 cm² culture flask of confluent VeroE6/TMRPSS2 cells growing with MEM medium with 2.5% FCS was inoculated at a multiplicity of infection (MOI) of 0.001. Cell supernatant medium was harvested at the peak of replication and supplemented with 25 mM HEPES (Sigma-Aldrich) before being stored frozen in aliquots at -80 °C. All experiments with infectious virus were conducted in a biosafety level (BSL) 3 laboratory.

#### 2.3.1.2. Antiviral assays; EC₅₀ and CC₅₀ determination.
One day before infection (Day -1), 5 × 10⁴ Huh7.5 cells per well were seeded in 100 µL assay medium (containing 2.5% FCS) in 96-well culture plates (Corning). The next day (Day 0), eight 2-fold serial dilutions of RDV (GS-5734; BLD Pharm) in triplicate were added to the cells (25 µL/well, in assay medium). Four virus control wells were supplemented with 25 µL of assay medium. After 15 min, 25 µL of a virus mix diluted in medium was added to wells. The amount of virus working stock used was calibrated prior to the assay, based on replication kinetics, so that the viral replication was still in the exponential growth phase for the readout as previously described (Delang et al., 2016; Touret et al., 2020, 2019). Plates were incubated 2 days at 37 °C prior to quantification of the viral genome by real-time RT-PCR. To do so, 100 µL of viral supernatant was collected in S-Block (Qiagen) previously loaded with VXL lysis buffer (Qiagen) containing proteinase K and carrier RNA. RNA extraction was performed using the Qiacube HT automat and the QIAamp 96 DNA kit HT following manufacturer instructions. Viral RNA was quantified by real-time RT-qPCR (GoTaq 1-step qRt-PCR, Promega) using 3.8 µL of extracted RNA and 6.2 µL of RT-qPCR mix and standard fast cycling parameters, i.e., 10 min at 50 °C, 2 min at 95 °C, and 40 amplification cycles (95 °C for 3 s followed by 30 s at 60 °C). Quantification was provided by four 2 log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10² to 10⁸ copies/reaction). RT-qPCR reactions were performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) and analyzed using QuantStudio 12K Flex Applied Biosystems software v1.2.3. Primers and probe sequences, which target SARS-CoV-2 N (nucleocapsid) gene, were: forward primer (Fw): GGCCGCAAATTGCACAAT; reverse primer (Rev): CCAATGCGCGACATTCC; Probe: FAM-CCCCCAGCGCTTCAGCGTTCT-BHQ1. Viral inhibition was calculated as follows: 100 * (quantity mean VC- sample quantity)/quantity mean VC. The 50% and 90% effective concentrations (EC₅₀, compound concentration required to inhibit viral RNA replication by 50%) were determined using logarithmic interpolation as previously described (Touret et al., 2019, 2020). For the evaluation of the 50% cytotoxic concentrations (CC₅₀), the same culture conditions as for the determination of the EC₅₀ were used, without addition of the virus, and cell viability was measured using CellTiter Blue® (Promega) following manufacturer's instructions. CC₅₀ was determined using logarithmic interpolation. All data obtained were analyzed using GraphPad Prism 9 software.
### 2.3.1.3. Genome analysis via sequencing to determine the mutagenic effect of RDV.
An amplicon of 2578 bp from position 14,880 to position 17,458 (GenBank reference NC_045,512) was produced using the SuperScript IV One-Step RT-PCR System kit (Thermo Fisher) and specific primers (SARS-14880F: TGATTGTTACGATGGTGGCTGT and SARS-17458R: GTGCAGGTAATTGAGCAGGGTC). Amplification was done using 3 µL of RNA, 500 nM of each primer and the SuperScript™ IV One-Step RT-PCR System kit (Thermo Fisher) (12.5 µL of 2X Platinum™ SuperFi™ RT-PCR Master Mix and 0.5 µL of SuperScript™ IV RT Mix) in a 25 µL final volume. The thermal profile used was 2 min at 50 °C for reverse transcription, 2 min at 98 °C, 40 cycles consisting of denaturation at 98 °C, 10 s; hybridisation at 56 °C, 10 s; and elongation 68 °C, 1 min 30s ending with final elongation 68 °C 5 min. PCR products were pooled in equimolar proportions after purification using Nucleofast PCR Plate kit (Macherey Nagel). After Qubit quantification using Qubit® dsDNA HS Assay Kit and Qubit 2.0 fluorometer (Thermo Fisher) amplicons were fragmented by sonication in fragments of ~250 bp long. Libraries were built adding barcodes, for sample identification, and primers to fragmented DNA using AB Library Builder System (Thermo Fisher). To pool the barcoded samples at an equimolar ratio, a quantification step by real time PCR using Ion Library TaqMan™ quantitation kit (Thermo Fisher) was performed. An emulsion PCR of the pools and the loading on a 520-chip were done using the automated Ion Chef instrument (Thermo Fisher). Sequencing was performed using the S5 Ion torrent technology (Thermo Fisher) following manufacturer's instructions. Consensus sequence was obtained after trimming (reads with quality score <0.99, and length of <100 bp were removed; and the 30 first and last nucleotides were removed from the reads) and mapping of the reads on a reference (BavPat1) using CLC genomics workbench software v.20 (Qiagen). Parameters for reference-based assembly consisted of match score = 1, mismatch cost = 2, length fraction = 0.5, similarity fraction = 0.8, insertion cost = 3, and deletion cost = 3. Quasi species with frequency over 0.01% were analyzed.

### 2.3.2. Selection of RDV resistant viruses

#### 2.3.2.1. Cell lines and virus strain.
The African green monkey kidney Vero E6 cell line was purchased from ATCC (Cat. no. CRL-1586™) and maintained in DMEM (Gibco, Cat. no. 41965-039) supplemented with 10% v/v heat-inactivated FCS. A549-ACE2-TMPRSS2, overexpressing human ACE2 and human TMPRSS2 receptors were purchased from Invivogen (Cat. no. a549d-cov2r) and cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS HI, Biowest) and the required selective antibiotics (blasticidin 10 µg/mL, hygromycin 100 µg/mL, puromycin 0.5 µg/mL and zeocin 100 µg/mL) at 37 °C and 5% CO₂.

The SARS-CoV-2 GHB (EPI ISL407976|2020-02-03) isolate was obtained from a Belgian patient returning from Wuhan in February 2020. The isolate was passaged 7 times on VeroE6 cells, which introduced two series of amino acid deletions in the spike protein (Boudewijns et al., 2020).

#### 2.3.2.2. Genotypic analysis via Illumina sequencing to determine RDV resistance mutations.
RNA was extracted from cell culture supernatant, using the NucleoSpin RNA virus kit (Macherey-Nagel) according to the manufacturer's instructions. Whole genome sequencing was outsourced to Eurofins Genomics (ARTIC SARS-CoV-2 WGS, Konstanz, Germany), who performed reverse transcription, enrichment of the viral genome using a primer set similar to the ARTIC primers (>200 primer pairs, covering the full 29.9 kb viral genome), generation of libraries, Illumina sequencing (2 x 150 bp read mode), and sequence cleaning to remove adapters and poor-quality bases. Sequences were further analyzed using Geneious Prime software (v2022.2.1) by mapping to the SARS-CoV-2 RefSeq NC_045,512, and variant calling as described by the software manufacturer (https://help.geneious.com/hc/en-us/articles/36004

5070991-Assembly-of-SARS-CoV-2-genomes-from-tiled-amplicon-Illumina-sequencing-using-Geneious-Prime). For each sample, >0.9*10⁶ reads that could be aligned with the SARS-CoV-2 genome were obtained with >99.5% of untrimmed bases being of high-quality, and a mean coverage of >3000 reads.

#### 2.3.2.3. Antiviral assay; EC₅₀ and CC₅₀ determination for the RDV resistant SARS-CoV-2 isolate.
The EC₅₀ was determined on A549-ACE2-TMPRSS2 cells as follows. On day -1, the test compounds were serially diluted in 100 µL assay medium (DMEM supplemented with 2% v/v FCS) in 96-well plates. In a next step, 50 µL cells (25,000 cells/well) was added and the plates were incubated (37 °C, 5% CO₂ and 95% relative humidity) overnight. On day 0, 50 µL of SARS-CoV-2 virus at an MOI of 0.001 tissue culture infectious dose (TCID₅₀) per cell was added and the plates were stored in a humidified incubator at 37 °C and 5% CO₂. In absence of antiviral activity, the cells undergo a cytopathic effect. Cell viability was determined 4 days p.i. using a viability staining with MTS (Jochmans et al., 2012). The percentage of antiviral activity was calculated by subtracting background and normalizing to the untreated-uninfected control wells, and the EC₅₀ was determined using logarithmic interpolation.

## 3. Results

### 3.1. Incorporation of RDV-TP and stalling of RNA elongation by SARS-CoV-2 mRTC

We measured RDV-TP incorporation in the presence of all native nucleotides by the active SARS-CoV-2 minimal RTC (mRTC consisting of nsp12, 7, and 8) using a SARS-CoV-specific 20-nt RNA template (T(A)20) annealed to a fluorescently labeled 10-nt RNA primer (P10). RNA elongation and RDV-MP incorporation products were resolved using denaturing PAGE and revealed using a fluorescence imager (Fig. 1). Increasing the concentration of RDV-TP relative to native nucleotides leads to a change in migration pattern of the product bands, indicating that RDV-TP is able to compete with native nucleotides as a substrate for the RdRp, resulting in RDV-MP incorporation in the extending RNA chain (Fig. 1(a)). The presence of 1'-CN and pseudoadenine base likely changes the migration behavior of the RDV-MP incorporated into RNA in comparison with that of its natural counterpart ATP, as double bands and a broadened final product mixture are observed. Quantification of primer and product bands does not show a significant inhibition of the elongation reaction upon exposure to increasing concentrations of RDV-TP. At high RDV-TP concentrations (250 µM) (Fig. 1(a)), faint bands of prematurely terminated products (at position 15, A15/R15) appear. However, RDV-TP does not act as a significant chain terminator, in contrast to our control, the GTP analogue and non-obligate chain terminator AT-9010 (Shannon et al., 2022), where the accumulation of intermediate product bands are seen (Fig. 1(b), AT14 and AT17).

We subsequently verified the incorporation of RDV-MP in the absence of ATP (Fig. 1(c)). In control reactions lacking both ATP and RDV-TP, GTP is mis-incorporated once instead of ATP, and elongation stops afterward. In the presence of RDV-TP, we observe repeated incorporations and extension beyond RDV-MP (named R in the panel legends) up to the full-length product (R20). Next, we tested the potential for RDV-TP incorporation in the place of GTP (Fig. 1(d)). In the absence of GTP and RDV-TP, a terminated RNA product is observed at position 13 (A13); the next incorporation should be that of GTP. There is clearly no mis-incorporation of ATP instead of GTP. In contrast, in the presence of RDV-TP, the A13/R13 product is extended pointing to an incorporation of RDV-TP instead of GTP. Our results thus indicate that RDV-TP is incorporated as an ATP analogue preferentially but also to a lesser extent as a GTP analogue (discussed in detail in the result section 3.3).
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Incorporation of RDV-TP in nascent RNA by SARS-CoV-2 mRTC. RNA sequences of the template (T(A)20) and the primer (P10) used in the study are shown in the top panel with the expected RNA product (when all the natural NTPs are provided). a) A PAGE gel is shown with the product analysis of polymerization reactions at indicated time-points by SARS-CoV-2 mRTC (nsp12:nsp7:nsp8 in the ratio of 1:3:6) with all NTPs at 50 µM concentration either in absence of RDV-TP or with 10 µM, 50 µM, 250 µM of RDV-TP. Product bands are labeled at the right and left side of the gel image. b) Incorporation of AT-9010, a GTP analogue and non-obligate chain terminator, in the presence of all NTPs at 50 µM each. c) Reactions run in the absence of ATP (GTP, UTP and CTP only), in the absence (right) or presence (left) of RDV-TP (50 µM each NTP/NA-TP). d) Left: elongation with ATP, UTP, CTP, Right: RDV-TP is also added (50 µM each).</figcaption></figure>

As previously described (Gordon et al., 2020b), when there is no competition between RDV-TP and ATP, a stalling in RNA elongation is observed at R15 ($i+3$) when RDV-MP is incorporated at position 12 ($i$) (Fig. 1(c)). The same phenomena of incorporation and arrested RNA extension at $i+3$ is observed when the first nucleotide to be incorporated is RDV-TP (Fig. S2). Observed stalling is moderate in our tests because of the high concentration of the following nucleotide (see below). We also tested RDV-TP incorporation and elongation with SARS-CoV mRTC and obtained similar results as with SARS-CoV-2 (Fig. S3).

In conclusion, we confirm that RDV-TP is readily incorporated and extended in the RNA product by the SARS-CoV-2 mRTC, predominantly as a substitute for ATP. A minor and transient RdRp stalling at position $i+3$ is observed when using RDV-TP, in accordance with a previous report (Gordon et al., 2020b). RDV-TP also has some propensity to act as a GTP analogue. In order to understand which specific chemical attributes of RDV-TP contribute to each of these distinct effects, we next investigated the respective contributions of the adenine pseudo-base and the 1'-CN group towards selectivity at the SARS-CoV-2 nsp12 active site.

## 3.2. The effect of 1'-CN group and pseudo-base on nucleotide analogues (NAs) in RdRp stalling

We tested two unique NA-TPs specifically designed to separate the respective contribution of chemical differences between ATP and RDV-TP. We made use of RDV-TP lacking the 1'-CN group (RDV(-CN)-TP), and ATP carrying a 1'-CN group (ATP+CN) (Fig. 2).

Fig. 3(a) shows the incorporation of ATP+CN at varied concentrations (10–250 µM) in competition with four native NTPs (50 µM) by the SARS-CoV-2 mRTC. ATP+CN shows a similar incorporation pattern as RDV-TP (Fig. 1(a)). The ATP+CN incorporation also results in broader mixture of final products with a smeary appearance on gels, inferring that 1'-CN group(s) internal to RNA change its migration behavior and eventually of final RNA products. When RDV-(CN)-TP is used (Fig. S4 (a)), this phenomenon of broader product mixture is not observed suggesting that the altered migration of elongation products indeed arises due to the 1'-CN group and not because of the pseudo-base configuration. In addition, the presence of full-length RNA products in the absence

of ATP indicates that RDV(-CN)-TP also acts as an ATP analogue. However, in contrast to RDV-TP, it does not cause any visible stalling (Fig. S4(b), also see Fig. 3(b)).

We then compared the RdRp stalling effect of the three NA-TPs at 10 µM in absence of ATP (Fig. 3(b)). Stalled $i+3$ products (position G14) are seen for both ATP+CN, and RDV-TP, while, again, no stalling is observed with RDV(-CN)-TP. Hence, the RdRp stalling is a consequence of the 1'-CN group. Interestingly, the stalling caused by the 1'-CN ribose modification is enhanced when used in combination with a natural ATP base (ATP+CN), compared to the pseudo-base of RDV-TP. Therefore, the pseudo-base seems to provide more flexibility for overcoming the $i+3$ translocation barrier caused by incorporation of an NA-TP carrying a 1'-CN group. For both RNA templates tested (Figs. 1 and 3), the next nucleotide for incorporation after the $i+3$ stall is RDV-TP or ATP+CN (templating U). Comparing the intensity of stalled products at position $i+3$ in Fig. 1(c) (50 µM RDV-TP, position 15)) with Fig. 3(b) (10 µM RDV-TP or ATP+CN, position 14) reveals a more pronounced stalling effect at lower nucleotide concentrations. Thus, as previously reported, this stalling caused by the 1'-CN group can be overcome by increasing the concentration of the next templated nucleotide (Kokic et al., 2021; Bravo et al., 2021; Gordon et al., 2020b; Wang et al., 2020).

In conclusion, NTP analogues; RDV-TP and ATP+CN containing a 1'-CN group are readily incorporated by the SARS-CoV-2 mRTC. The 1'-CN is responsible for the transient RdRp stalling at position $i+3$ especially when the following nucleotide is present at low concentrations, multiple fold lower than that of intracellular NTP (millimolar range, (Traut, 1994), although the actual concentration in viral factories is unknown). This effect is further enhanced for an A(+CN): U pair compared with a R: U pair. Therefore, the 1'-CN group causes the SARS-CoV-2 mRTC to stall, and the stalling is further influenced by the base. This finding, together with the observation that RDV-TP may serve as a GTP analogue (Fig. 1 (d)) prompted us to investigate the efficiency of incorporation of these NA-TPs instead of ATP, GTP, CTP or UTP.

## 3.3. The role of 1'-CN group and pseudo-base in NAs incorporation efficiency

We made use of one primer (P10) annealed to different templates
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Chemical structures of triphosphate forms of nucleoside analogues (NA-TPs); Remdesivir triphosphate (RDV-TP), Remdesivir triphosphate lacking 1'-CN group (RDV(-CN)-TP), Adenosine triphosphate with 1'-CN (ATP+CN) used in the study.</figcaption></figure>

(T20) to compare the efficiency of incorporation of RDV-TP, RDV(-CN)-TP, ATP+CN and ATP (control) opposite different templating bases (Template U, C, G, or A) at the first position by the SARS-CoV-2 mRTC (Figs. 4 and S5). Figs. 4 and S5 show series of four panels giving the denaturing PAGE analysis of the incorporation of RDV-TP (Fig. 4(A), first series), ATP+CN (Fig. 4(A), second series), RDV(-CN)-TP (Fig. S5 (B)), and ATP (Fig. S5(C)) instead of ATP (panel a), GTP (panel b), CTP (panel c) and UTP (panel d). In several cases, multiple incorporations are observed as the following template positions may allow further incorporations according to the promiscuity of each tested NA-TP. We quantified all the product bands in relation to the primer at each time point allowing us to determine initial velocities ($v_i$) of incorporation (nM/min) of all the three NA-TPs and ATP at the first position as represented in the form of graphs (Fig. 4(B)).

Fig. 4(B), panel a shows that RDV-TP, RDV(-CN)-TP, ATP+CN and ATP are incorporated opposite U with similar efficiency at both concentrations tested (10 and 50 µM). As described before, the RDV pseudo-base acts as a bona fide analogue of the adenine base in terms of incorporation efficiency, even resulting in a lower apparent Km value of RDV-TP in comparison to ATP with the SARS-CoV-2 RdRp (Gordon et al., 2020b). The presence of the pseudo-base and addition of the 1'-CN group has no influence on the initial velocity of incorporation opposite U at 10

µM and 50 µM of NA-TPs compared to ATP. Interestingly, the 1'-CN group increases the likelihood of incorporation in the place of GTP, i.e., the chance of incorporation of RDV-TP and ATP+CN opposite template C (Fig. 4(b)). Whilst incorporation of RDV(-CN)-TP and ATP in lieu of GTP is negligible, both RDV-TP and ATP+CN can be accepted as a GTP substitute, albeit at lower incorporation rates than when substituting for ATP. Multiple mis-incorporations/incorporation products are observed in the corresponding PAGE analysis of RDV-TP instead of GTP (Fig. 4(A), first series, (b)). Since the template is U/C rich, multiple incorporations are possible after the first incorporation event. In contrast, elongation almost completely stops after incorporation of ATP+CN as a GTP substitute (second series, (b)). The natural base seems to prevent further chain elongation despite the fact that the next NTP to be incorporated is an ATP.

As expected, considerably lower initial velocities are observed when NA-TPs are used instead of CTP (Figs. 4(c) and S5,(c)) and UTP (Figs. 4(d) and S5(d)). For RDV-TP (at 50 µM), the initial velocity observed is around 40-fold lower when substituting for a pyrimidine (CTP and UTP), compared with its incorporation as an ATP analogue.

In conclusion, RDV-TP is rapidly and competitively incorporated in the place of ATP, as previously reported (Gordon et al., 2020b; Dan-gerfield et al., 2020). It can also be incorporated as a GTP analogue, albeit with lower efficiency. Remarkably, the RDV-TP promiscuity is mainly due to its 1'-CN group, as we observe incorporation instead of GTP in case of ATP+CN as well. Overall, the combined effect of pseudo-base and the 1'-CN ribose modification on RDV-TP promotes multiple incorporations and not only as an ATP analogue. These results prompted us to investigate whether such promiscuity could occur in infected cells, i.e., if RDV could be mutagenic to the SARS-CoV-2 viral RNA (vRNA).

### 3.4. Nucleotide sequencing of SARS-CoV-2 viral RNA extracted from RDV-treated infected cells

We infected Huh7.5 cells with the ancestral SARS-CoV-2 BavPat1 strain in the presence of increasing concentrations of RDV (GS-5734) (Fig. S6). RDV inhibited viral replication with a half-maximal effective concentration ($EC_{50}$) of 0.017 µM in Huh7.5 with no cytotoxicity observed up to 20 µM. It is worth noting that the differences in $EC_{50}$ in Huh7.5 from what was previously described in VeroE6 (Do et al., 2021; Mackman, 2022) is likely due to the presence of a more efficient RDV efflux in VeroE6 than in Huh7.5 (Zhu et al., 2022), rather than different RDV metabolism.

The potential mutagenic effect of RDV was next assessed by comparing SARS-CoV-2 quasi-species at frequency over 0.01% in the absence and at different concentrations of RDV. Viral RNA extracted from the dose-response experiments with RDV concentrations in the range of the $EC_{50}$ (Fig. S6, chosen points are depicted by blue arrows) were selected for the evaluation of the quasi-species by sequencing. The data revealed that RDV induces a significant increase in the number of mutations at the frequency threshold of 0.01% compared to the control with no RDV (Fig. 5(a)). At a frequency above 0.02%, no significant difference is observed between the control and the virus exposed to RDV, suggesting that the direct or indirect mutagenic effect of RDV is modest.

Interestingly, the analysis of the mutations observed in quasi-species above 0.01% frequency revealed an increase in mutations arising from increased A-to-G and U-to-C transitions (Fig. 5(b)). Such transitions can be explained as RDV being read as a G in the template strand, and not RDV-TP being mis-incorporated in the place of GTP by the polymerase opposite a templating C.

In conclusion, the quasi-species data indicate that RDV may have a moderate mutagenic effect on the proviral genome templates, much lower than that of Molnupiravir where the MoA is known to be through excessive mutagenesis of viral genomes (Kabinger et al., 2021; Yip et al., 2022). The observed change in the nucleotide profile (A-to-G and U-to-C
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Incorporation of ATP+CN into RNA by SARS-CoV-2 mRTC and the comparison of incorporation among tested NA-TPs. The top panel displays the sequence of the RNA template (T<sub>(U)20</sub>, primer (P10) with the expected sequence of the product, if all the natural NTPs are present. a) All NTPs are provided at 50 µM concentration in the absence of ATP+CN (left corner) or at 10 µM, 50 µM, 250 µM of ATP+CN. b) GTP, UTP, CTP are used at 50 µM and either ATP+CN (left), RDV-TP (middle) and RDV(-CN)-TP (right) at 10 µM. In case of ATP+CN and RDV-TP, when these NA-TPs are incorporated at position 11 (i), stalled RNA product at position 14, G (i+3) is visible and labeled on the right.</figcaption></figure>

transitions) indicates that it is caused upon reading of the incorporated RDV-MP in the template strand (see Discussion).

**3.5. The influence of 1'-CN group and pseudo-base in nsp14-mediated excision of NAs from RNA**

To assess the potential excision of RDV-MP and chemical analogues from the RNA by the virally encoded proofreading machinery, we first incorporated each NA-TP/NTP by the SARS-CoV-2 mRTC into a self-annealed RNA hairpin (5'-6FAM-HP RNA). We then measured the excision upon challenge of the extended RNA substrate by the SARS-CoV-2 nsp14/nsp10 active ExoN complex. RNA substrates, HP_AU (see the sequence, Fig. 6(a)) and HP_AAU (Fig. S7(a)) were designed to have either one or two incorporation(s) of RDV-TP or ATP analogues, respectively. Firstly, the extension of HP by the active RdRp complex was tracked in the presence of only one NA-TP or NTP for 30 min. Following the polymerization reaction, the SARS-CoV-2 mRTC was heat-denatured and the excision of the re-annealed HP substrate was subsequently followed by addition of the active ExoN complex (12.5 nM nsp14/50 nM nsp10 to 500 nM HP) for 60 min.

The denaturing PAGE gels of extended and excised HP_AU (Fig. 6(b)) and HP_AAU (Fig. S7(b)) substrates show first the polymerization reaction profile (0, 10, 30 min) and then the excision profiles (5, 10, 30, 60 min). As controls, we used these HP RNAs in the absence of NA-TP/NTP (left) and in the presence of GTP allowing mis-incorporation of GTP opposite U (right). We observe that all the extended HPs are hydrolyzed, indicating that none of the NAs block exonuclease activity. The degradation of RNA is visible as long as the 3'-end is engaged in a stable RNA duplex; this observation is in agreement with previous studies on the specificity of SARS-CoV/CoV-2 ExoN activity for dsRNA (Bouvet et al., 2012; Chinthapatla et al., 2023). In the presence of higher concentrations of the ExoN complex we observe the complete disappearance of the extended HP band (data not shown). In order to compare the excision efficiencies, we quantified all the substrate and cleavage bands present for each condition and determined velocities during the initial phase of excision (vᵢ in nM excised nt/min). Initial velocities were linear up to 30–60 min (data not shown). Since the stem part is identical for each

extended HP, we surmise that any changes in vᵢ values is the direct consequence of the terminal incorporation. The determined initial velocities were plotted for each tested condition for HP substrates; HP_AU (single terminal incorporation) and HP_AAU (two successive incorporations) (Fig. 6(c); HP_AU (left) and HP_AAU (right)).

Both substrates exhibit comparable tendencies for excision under all tested conditions, indicating that the excision velocity is not altered significantly by one or two consecutive incorporations of the tested NA-TPs. The initial velocities are identical to the control of no NA-TP/NTP and for HP extended by RDV(-CN)-TP, ATP and GTP while lower vᵢ values are obtained for HP incorporated with RDV-MP and AMP+CN. This effect is more pronounced when using an 1'-CN NA-TP with a natural base compared to the pseudo-base, as the reduction in initial velocity is more noticeable for AMP+CN excision.

We also made use of HP_AC with an incorporation of C following a single incorporation of the molecules as ATP (Fig. 7). Using this substrate, we observe that RDV-TP and to a lesser extent ATP+CN is incorporated twice. In contrast, this is not seen for RDV(-CN)-TP, ATP or GTP controls, thus indicating that the 1'-CN group promotes misincorporation even opposite G in this sequence context (Fig. 7(b)). Quantification of excision efficiency including the second incorporation (or misincorporation) reveals no difference between HP_AC substrates with one or two terminal extensions (Fig. 7(c)). All substrates were found to be comparably well excised.

In conclusion, we observe that none of the tested NA-TPs drastically impact the ExoN activity. The excision pattern remains similar for both single and double incorporation(s). We evidence statistically significant ExoN inhibition for AMP+CN in the context of HP_AU and HP_AAU (please refer to the statistically significant p-values in Fig. 6) but not in the case of HP_AC where AMP+CN is incorporated as a perfect match and followed by a mismatch for the second incorporation. The mismatched RDV-MP and AMP+CN as a CMP substitute are better excised than the perfect match. Overall, our experiments indicate the potency of RDV in SARS-CoV-2 infected cells is unlikely to be a result of poor excision by the viral proofreading complex.
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Incorporation efficiencies of RDV-TP, RDV(-CN)-TP, ATP+CN as ATP, GTP, CTP and UTP analogues by SARS-CoV-2 mRTC. In display at the top panel, all four sets of primer/templates (P10/T20) combinations used in the assay below. These have variations only in the template sequence at the position 11 and 12. The RNA product sequence is shown as a reference when all native nucleotides are provided. In each experimental condition, only one NA-TP/NTP; RDV-TP, RDV(-CN)-TP, ATP+CN, ATP is added to visualize single incorporation. A) In the first series, only RDV-TP is provided at 10 µM and 50 µM and the elongation reaction is monitored at indicated time-points for Template U (a), Template C (b), Template G (c), Template A (d). In the second series, the same experiment is done as in the first series with ATP+CN. Multiple incorporations are visible depending on the promiscuity of provided NTP/NA-TP. B) Initial velocities (nM/min) of incorporation of each NA/NTP used in the study are calculated for the indicated template (at the top of the gel of the first series) and at the indicated concentrations (bottom of the graph). In (B), note that the scale of the y-axis (a) is different from (b), (c) and (d). The data presented represents the average of two replicates, with error bars representing standard deviations (SD).</figcaption></figure>

### 3.6. *In-vitro selection and characterization of a SARS-CoV-2 isolate resistant to RDV*

Drug resistance mutations often point not only to the viral target of a given compound, but also may help to understand its MoA. Recent studies report the emergence of SARS-CoV-2 RDV resistance in immunocompromised patients ((Martinot et al., 2021; Gandhi et al., 2022; Hogan et al., 2023) which prompted us to investigate the resistance development to RDV and whether this could be aligned to the biochemical properties and mechanisms studied above.

We passaged the SARS-CoV-2-GHB isolate in VeroE6 cells in the presence of increasing concentrations of GS-441524, the RDV nucleoside. The selection started at a concentration of 0.5 µM as we had previously determined the EC₅₀ of GS-441524 in this cell system to be around 0.78–0.89 µM (Do et al., 2021). For each next passage, the virus

was cultured at the same concentration, a 3x higher concentration and a 3x lower concentration. The culture demonstrating virus breakthrough at the highest concentration, as observed by a significant cytopathic effect (CPE), was then used for the next passage. We started this experiment in triplicate but for two trials we could not significantly increase the GS-441524 concentration above 10 µM by passage 15 (day 52). These cultures were therefore not further analyzed. In one culture however, the concentration of GS-441524 could be increased gradually to 20 µM at passage 14 (day 49) (Fig. S8) and maintained for 6 additional passages (until day 71).

For further analysis, a new virus stock (p21) was grown on VeroE6 cells in the presence of 5 µM GS-441524, and whole genome sequencing (Illumina) was performed on RNA purified from this stock. This showed two dominant nucleotide changes (>80% of the reads), U15715G and G15769U, that result in amino acid changes S759A and A777S on the
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Nucleotide changes mediated by Remdesivir in SARS-CoV-2 infected cell (Huh7.5 cell line). (a) Changes and their position on the genome in the quasi species for virus control (Vc) or SARS-CoV-2 treated by Remdesivir at 0.015 µM. (b) Quantification of the nucleotide change effect of Remdesivir for quasi species above 0.01%, for any kind of mutation (all), or A-to-G and U-to-C transitions, on the tested segment. Means of two independent experiments. (***): P < 0.001, Pearson's χ2 test with Yates' continuity correction.</figcaption></figure>

nsp12 RdRp domain. Of note, the nsp12 RDV resistance mutation S759A was also reported in an independent study by (Stevens et al., 2022). Selection over the full genome of all nonsynonymous nucleotide changes showed that two other changes also became fixed in the 3C-like protease (3CL^pro^), G109R and in spike (S), K182N. As a control, SARS-CoV-2 was also passaged at the same frequency in the absence of compound, with no mutations identified in the nsp12 gene at passage 20 (data not shown).

We performed phenotyping on SARS-CoV-2 wild-type (WT) virus and the RDV resistant double mutant isolate virus in A549-ACE2-TMPRSS2 cells (Table 1). Interestingly the EC₅₀ on SARS-CoV-2 of GS-441524 in these cells is 3.6 µM, which is significantly higher than in VeroE6 cells (see above). More importantly, a >10-fold change increase in half maximal effective concentration (EC₅₀) values of GS-441524 is observed for the mutant virus. As a control, treatment with the 3CL^pro^ inhibitor Nirmatrelvir expectedly showed no differences in EC₅₀ values.

Fig. 8 shows the location of S759A and A777S on the SARS-CoV-2 nsp12 structure (PDB:7UO4 (Malone et al., 2023)). S759 is located within the catalytic motif C S<sup>759</sup>DD in the palm domain; and A777S is located in the vicinity of catalytic Motif D.

In conclusion, we identified a profile of two amino-acid substitutions

in SARS-CoV-2 nsp12, S759A and A777S located in motif C and near motif D of the RdRp, respectively, that are associated with resistance against GS-441524. This resistant isolate virus led to >14 fold loss in the sensitivity of RDV. We next aimed to determine effect of these mutations on the activity of the SARS-CoV-2 RdRp and its incorporation of RDV-TP.

### 3.7. Biochemical characterization of RDV resistance nsp12 mutations, S759A and A777S

We purified S759A, A777S, and S759A + A777S nsp12 mutants to investigate their effect individually and in combination. We first measured incorporation efficiency of RDV-TP compared with the natural substrate ATP (Fig. 9). For this, we provided either ATP or RDV-TP at a single concentration of 50 µM using the same primer-template combination as before in result section 3.3 (template-U). Single nucleotide incorporation was followed over time to measure vᵢ for both ATP and RDV-TP incorporation. This was used to calculate the ‘discrimination value’, i.e., to determine the preference for either ATP or RDV-TP incorporation, by dividing ATP vᵢ by RDV-TP vᵢ. A discrimination value of less than 1 means that incorporation of RDV-TP is faster than
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Excision of incorporated NA-TPs; RDV-TP, RDV(-CN)-TP, ATP+CN after single and double incorporations into HP_AU and HP_AAU, respectively. a) Sequence of self-priming hairpin AU (HP_AU) with 6-FAM label at the 5'-end, with the loop in grey and the stem in blue. In pink, the first incorporation (for the natural NTP) at the 3' end is shown with the templated sequence in green. b) The PAGE gel demonstrates the polymerization (0, 10, 30 min) and the excision reaction product profiles (5, 10, 30, 60 min). The polymerization reactions are carried out in the presence of only one NA-TP/NTP (specified above the gel) at 50 µM concentration or none at all (control) and is subjected to excision by nsp14: nsp10 complex. In case of RDV-TP, +1 elongation product is labeled at position 28 (R28) and the excision products are labeled below the substrate band (-R and so on). c) Quantification of excision products in terms of initial velocities are plotted for extended HP_AU (left) and HP_AAU (right). HP indicates control without NA-TP/NTP, HP-R represents RDV-TP incorporated once into HP_AU and HP-RR (on right-side graph) represents two consecutive incorporations of RDV-TP into substrate HP_AAU, etc. For HP_AU, data represents average of 2 experiments and for HP_AAU, average of 3 experiments. Error bars represent standard deviation (SD). * represents the statistical analysis (unpaired t-test) comparing the control data set (HP) with HP-A(+CN) and HP-AA(+CN) in HP_AU and HP_AAU with p values of 0.0279 and 0.0323 respectively.</figcaption></figure>

ATP while a value of more than 1 shows that ATP is incorporated faster (Fig. 9; lower panel). As in section 3.3, we found that WT RdRp incorporates ATP and RDV-TP with similar efficiencies.

The mutant RdRps incorporate both ATP and RDV-TP with an overall lower initial velocity in comparison to the WT RdRp. More importantly, both the individual S759A and A777S mutations result in around 1.4-fold discrimination against RDV-TP. The appearance of significant RDV-TP discrimination for S759A RdRp mutant has also been reported previously (Stevens et al., 2022; Malone et al., 2023). The two mutations have an additive effect as the RdRp double mutant shows a clear preference for ATP over RDV-TP, with a discrimination factor of 2.3.

Next we followed the polymerization efficiency on primer-template (*template-U*), by first providing all NTPs and then by replacing ATP with RDV-TP (Fig. 10). In contrast to the WT RdRp, for mutant S759A we observed a very strong polymerase stalling at G14 (*i*+3 position), when RDV-MP is incorporated at position 11 (*i*) (Fig. 10(b)). In this RNA scaffold, the next nucleotide templated for incorporation is RDV-MP (at *i*+4 position). To verify the influence of this templating *i*+4 nucleotide on the pronounced stalling, we tested different templates (Fig. S9). We observed that indeed the *i*+4 position plays a role in the stalling as seen for templates C and U_4R in comparison to templates U_R and CA where there is relatively little stalling. Interestingly, the number of RDV-MPs to be incorporated in the growing RNA chain also play a role in the stalling of the S759A RdRp as multiple terminated products are seen in the case

of templates C and U_4R where four RDV-MP incorporations are possible over the length of RNA. Despite the influence of the template sequence on polymerase stalling, the S759A mutant is found to be consistently more susceptible to stalling compared to the WT RdRp.

This phenomena of increased RdRp stalling is also observed in the double mutant RdRp (Fig. 10(d)) while A777S Fig. 10(c)) showed product elongation pattern similar to the WT complex. Therefore, the stalling effect is directly attributed to S759A.

In conclusion, the observed RDV resistance provided by the S759A RdRp mutation can be explained by two distinct MoAs at the enzyme level. First, as seen before (Stevens et al., 2022) S759A shows a preference for the natural ATP substrate over RDV-TP, and second, more importantly, it causes increased stalling after RDV-MP incorporation. The second MoA seems to be counter-intuitive, as increased stalling should increase RDV sensitivity. We propose that the slowing down of the RdRp allows more time to ExoN-mediated repair (see Discussion).

## 4. Discussion

Remdesivir (RDV) is one NA known to be effective against SARS-CoV-2. The potency of RDV is mainly due to the ability of its triphosphate form (RDV-TP) to compete efficiently with its natural counterpart ATP in spite of high cellular ATP concentrations (Traut, 1994). There are studies concurring that RDV-TP is a better substrate than ATP for CoV
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Excision of incorporated NA-TPs; RDV-TP, RDV(-CN)-TP, ATP+CN after single or double (mis)-incorporation into HP_AC. a) Sequence of the 27 nucleotides long RNA hairpin substrate, HP_AC used in this assay where in pink the first incorporation (in case of the natural NTP) is shown. b) PAGE shows the product bands for polymerization and excision reactions at the indicated reaction conditions. Here +1 elongation product (at position 28) is evident for all the conditions except for control but +2 product (position 29) is only visible for RDV-TP and ATP+CN indicating G:R and G:A(+CN) terminal mismatch respectively. c) Initial velocities (nM excised nt/min) are given in the graph for each condition after quantifying the excision products. HP-R[R] represents one incorporation (R) and the terminal mis-incorporation of RDV-TP [R] and is same for ATP+CN. The data represents the average of two experiments.</figcaption></figure>

<table><caption>**Table 1**
SARS-CoV-2 phenotypic resistance associated with the S759A + A777S mutation profile as determined in A549-ACE2-TMPRSS2 cells.</caption><thead><tr><th></th><th>Toxicity<br>A549-ACE2-<br>TMPRSS2 CC<sub>50</sub><br>(µM)</th><th>SARS-CoV-2-GHB<br>(WT) EC<sub>50</sub> (µM)</th><th>SARS-CoV-2-GHB<br>S759A + A777S<br>EC<sub>50</sub> (µM) [FC]<sup>c</sup></th></tr></thead><tbody><tr><td>GS-441524</td><td>&gt;50 (n = 8)</td><td>3.6<sup>a</sup> (3.3–4.7)<sup>b</sup> (n = 8)</td><td>&gt;50 [&gt;14]<sup>c</sup> (&gt;50 – &gt;50) (n = 7)</td></tr><tr><td>Nirmatrelvir</td><td>&gt;10 (n = 8)</td><td>0.079<sup>a</sup><br>(0.056–0.093)<sup>b</sup> (n = 8)</td><td>0.076 [0.96]<sup>c</sup><br>(0.072–0.11) (n = 7)</td></tr></tbody></table>

RdRps (Gordon et al., 2020a; Gordon et al., 2020b; Dangerfield et al., 2020). RDV-TP is incorporated into viral RNA, and this translates into an antiviral effect through a MoA which is the topic of the present study.

RDV-TP has been proposed to act as a delayed chain terminator for CoVs. Following its incorporation at position *i*, RNA synthesis is halted at position *i+3* (Gordon et al., 2020a, 2020b). This rather transient stalling is overcome by even micromolar range concentrations of the *i+4* nucleotide ((Gordon et al., 2020b; Bravo et al., 2021; Kokic et al., 2021)). A structural cause behind this *i+3* RdRp stalling in SARS-CoV-2 is described by several studies, in which a steric clash between the RDV-MP 1'-CN group and the side chain of S861 residue in nsp12 is evidenced. It is this clash that leads to slowing down the translocation of the ds RNA (Bravo et al., 2021; Kokic et al., 2021; Tchesnokov et al.,

2020; Wang et al., 2020).

In accordance with previous reports, we showed biochemically by comparing the stalling effect of RDV-MP, RDV(-CN)-MP and AMP+CN that stalling is the consequence of the 1'-CN group (Fig. 3(b)). However, we also found that the nucleobase contributes to this effect by influencing the strength of stalling. Stronger RdRp stalling for AMP+CN than RDV-MP incorporation can be due to the natural base being more efficiently involved in Watson-Crick bonding than the pseudo-base, whose planarity and vibrational status is different from that of a bona fide purine. However, the relevance of the stalling is an open question since it is easily overcome at NTP concentrations around 10–20 µM, about 2 logs lower than the millimolar intracellular NTP concentration range (Traut, 1994). This suggests that the stalling by RDV-MP, seen in biochemical assays as apparent delayed chain termination of RNA elongation, might not be relevant at high biological NTP concentrations nor can be identified as a sole MoA of RDV against SARS-CoV-2.

The SARS-CoV-2 polymerase is error-prone and exhibits fast replication compared to other viral replicases (Shannon et al., 2020b; Seifert et al., 2021; Campagnola et al., 2022). Interestingly, our study suggests that a 1'-CN group in an NA-TP can further increase the infidelity of this polymerase. Our biochemical assays showed that RDV-TP can also be incorporated as a GTP analogue by SARS-CoV-2 mRTC, although this was around 18-fold less at 10 µM and 2-fold less efficient at 50 µM NTP concentration (Fig. 4(B)) than as an ATP analogue, thus hinting towards it's broader purine analogue substrate properties. This promiscuity of RDV-TP is again influenced by the 1'-CN group as we observed the same phenotype in the case of ATP+CN. In comparison, ATP and RDV (-CN)-TP fail to mis-incorporate as GTP in our experimental conditions.

One unique feature of SARS-CoV-2 and other large *Nidovirales*

<footnote>a Median value.</footnote>

<footnote>b 25th–75th percentile, EC₅₀ = 50% effective concentration.</footnote>

<footnote>c FC = Fold Change of EC₅₀.</footnote>
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** SARS-CoV-2 nsp12 structure showing location of residues associated with RDV resistance. a) Stalled SARS-CoV-2 mRTC (PDB: 7UO4) with pre-incorporated RDV-TP (cyan); nsp12 (blue), nsp8 (olive green), nsp7 (orange), template strand (pink), primer strand (magenta). b) Focus of enzyme active-site with S759DD motif shown. S759 and A777 (associated with RDV resistance mutations S759A and A777S) are highlighted in yellow.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Comparison of single-nucleotide ATP or RDV-TP incorporation by SARS-CoV-2 wild type nsp12 and RDV-resistant nsp12 mutants. Initial velocities (nM/min) were measured using 0.2 µM SARS-CoV-2 mRTC and 0.5 µM P10/T(U)20 for the incorporation of ATP or RDV-TP by WT RdRp and mutant RdRps; S759A, A777S and S759A + A777S. Corresponding discrimination values (ratio vᵢ(ATP) to vᵢ(RDV-TP)) are given at the bottom of the graph. The data shows the average of two replicates. Error bars correspond to the standard deviation (SD).</figcaption></figure>

relatives is the possession of a 3'-5' ExoN activity involved in correcting replication errors (Eckerle et al., 2007, 2010). *In vitro* activity has been well demonstrated for several CoVs including SARS-CoV and SARS-CoV-2 (Minskaia et al., 2006; Bouvet et al., 2012; Ferron et al., 2018; Ogando et al., 2019; Dangerfield and Johnson, 2022). The active ExoN is composed of a complex formed by nsp14 (ExoN) and nsp10 (cofactor) (Bouvet et al., 2012; Ma et al., 2015; Ferron et al., 2018). Besides mismatches, it has been shown to excise incorporated NAs, including Ribavirin (RBV), and 5-fluorouracil (5-FU), dampening or neutralizing their antiviral potential (Smith et al., 2013).

We earlier predicted that RDV-MP excision from RNA could be impaired due to its chemical modifications (Shannon et al., 2020a). Molecular dynamics (MD) simulations also proposed that 1'-CN could alter accommodation of the terminal analogue within the ExoN active

site (Zhang et al., 2021). In the present study, we challenged this prediction by actually measuring ExoN activity on RNAs extended by NA-TPs. We observe that the ExoN has no blatant difficulty in hydrolyzing the RDV-MP, AMP+CN or RDV(-CN)-MP (Fig. 6). We did observe, however, that the 1'-CN may play a role in slowing down the ExoN but this is context dependent. It was more obvious for AMP+CN than for RDV-MP, indicating that again, pairing of the nucleobase is also relevant. We tested the mismatch or a non-Watson-Crick base paired end, C: RDV-MP or C: AMP+CN (Fig. 7) and noticed that these could be excised as easily as the perfectly paired end. Likewise, other studies have also reported that RDV-MP does not show significant resistance to excision (Jockusch et al., 2020; Dangerfield and Johnson, 2022). In contrast, another report claimed that the excision of RMP is significantly slower than a natural base-paired RNA (Chinthapatla et al., 2023). Thus, in our experimental set-up the ExoN activity towards RDV-MP is not inhibited by the presence of the 1'-CN group and it is modestly modulated by the sequence context. We surmise that the interference of the ExoN is influenced by the velocity of incorporation, putative back-tracking and possibly other factors.

In the case of Murine Hepatitis Virus (MHV), an ExoN(−) mutant was approximately 4.5-fold more sensitive to RDV compared to the WT suggesting that, incorporated RDV-TP is able to be excised by ExoN (Agostini et al., 2018). The modest ExoN effect suggests that not all incorporated RDV-MPs are excised, potentially acting on subsequent ExoN-dependent events. Subsequently, when RDV-MP escapes excision and is incorporated into RNA, it is expected to act as a template for UTP. It has been shown that this UTP incorporation opposite RDV-MP is kinetically disfavored, resulting in template-dependent inhibition by stalling the mRTC. Again, this was shown to be dependent on NTP concentrations, requiring concentrations up to 1 mM of both UTP and the following NTP (Tchesnokov et al., 2020). Strikingly, our sequencing analysis of RDV-treated genomes evidencing U to C changes, indicates that RDV-MP templated the misincorporation of cytosine (CMP). This would extend the effects of the kinetic block of a templating RDV-MP. The specific contribution of this MoA to the general mechanism of inhibition is unknown. We remain cautious about the observed mutagenesis: the early steps of deep sequencing include a reverse transcription step, whose fidelity may be affected by the presence of templating RDV-MP. It is not possible to know with certainty which polymerase (mRTC or RT) is responsible for the observed transitions. Deciphering the respective contributions of these two enzymes remains beyond the scope of the present research article. However, in any case it is certain that incorporation of RDV-MP in full-length genomes
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** RNA synthesis by RDV-resistant RdRp mutants, S759A, A777S and S759A + A777S in comparison to wild-type RdRp. The primer (P10) and template (T(U)20) sequences used in the study are indicated in the top panel. PAGE gel of polymerization reaction are shown for WT RdRp (a), S759A RdRp (b), A777S RdRp (c) and double mutant, S759A + A777S RdRp (d). In each of the panels on the left, the reaction with all the natural NTPs is shown and on the right, reactions without ATP but RDV-TP. The concentration of all NTPs and NAs are 50 µM each. For S759A (b) and S759A + A777S (d), the stalled RNA product is visible at position 14 (G14).</figcaption></figure>

occurred.

We next studied the selection and mechanistic properties conveyed by the S759A and A777S RDV resistance point mutations in the SARS-CoV-2 RdRp which led to the >14-fold resistance observed in infected cells. A serine is present in the catalytic motif C (S759DD) of almost all nidoviral RdRps instead of the canonical GDD motif found in all other (+) ssRNA viruses. This residue along with motif F, is shown to play a role in the regulation of speed and fidelity of the CoV genome replication (Ferron et al., 2021; Campagnola et al., 2022). A777 maps in the vicinity of motif D. This motif is known to play a crucial role in the closure of the

polymerase active site after the positioning of NTP for incorporation (Yang et al., 2012; McDonald et al., 2016; Selisko et al., 2018). Mutations in this region can impact the dynamics of the Motif D loop, potentially influencing the rate of incorporation. It is interesting to note that other RDV resistance mutations are reported in the surrounding of Motif D like A777S (e.g., such as at positions V792, C799, E802 (for a database follow-up, see https://covdb.stanford.edu/drms/rdrp/)), suggesting that they provide a fidelity check against RDV-TP. We demonstrate that each of these mutations independently increases the preference of ATP over RDV-TP by the SARS-CoV-2 mRTC. This effect is
additive in the double mutant.

Here we also report that in addition to the discrimination of RDV-TP relative to ATP (also shown by (Stevens et al., 2022; Malone et al., 2023)), the S759A mutation increases the propensity of the RdRp to stall after RDV-MP incorporation(s). Visualization of S759 in SARS-CoV-2 RdRp with RDV-TP in its pre-incorporation state showed that the side chain of Ser (-OH group) makes a proximal hydrogen bond (2.3 Å) with the 2'-OH of the primer, while the 1'-CN of RDV-TP is accommodated nearby at 3.6 Å (Malone et al., 2023) (see Fig. 8(b)). Consequently, replacing Ser with Ala will eliminate this amino acid:primer interaction, possibly misaligning RDV-TP and reducing its incorporation efficiency. Following RDV-MP incorporation, the misalignment effect may additionally be potentiated further down the line. When there is incorporation of RDV-MP at both the *i* and *i*+4 positions, the 1'-CN group of RDV-MP in *i* may actually have a long-range effect on the primer 3'-end, and selectively disfavor RdRp activity, explaining the strong-stop band observed using templates T(U)20 (Fig. 10) and C (Fig. S9). Overall, the increased stalling in S759A in comparison to WT RdRp might give more time for ExoN to remove the inserted RDV-MP, adding an indirect effect to the drug resistance mechanism. We propose that other RDV-TP mutations discriminating RDV-TP towards ATP could simply add up to S759A, since both effects, discrimination and stalling may work synergistically. Importantly, the increased RdRp stalling in RDV resistant mutants also implies that this cannot be considered as the RDV mode of action. If this was the case, the stalling would have been expected to decrease, rather than increase, in the resistant virus RdRp.

## 5. Conclusion

In conclusion, our study points to several simultaneous modes of action of Remdesivir and clarifies the implication of its 1'-CN group and pseudo-base. RDV-TP is readily incorporated in the growing chain as an ATP analogue, and its 1'-CN group also facilitates in its incorporation as a GTP analogue in *in vitro* primer extension assays. The 1'-CN causes stalling, an effect exacerbated by the S759A resistance mutation, whereas the pseudo-base weakens the Watson-Crick bonding compared to adenine. In agreement to infected cell data (Agostini et al., 2018), we observe that RDV-MP can be excised from elongating RNA, and the pseudo-base of the analogue contributes to this excision sensitivity. Rapid RDV-MP incorporation may go unnoticed, but when it slows down (due to transient stalling caused from low local NTP concentrations, incorporation as a GTP analogue, or multiple incorporations), RDV-MP becomes detectable and is likely excised, possibly causing further delays in RNA synthesis. However, if RDV-MP escapes excision and persists in the RNA, its reading as a templating GMP is theoretically possible, potentially accounting for a weak A-to-G and C-to-U transition pattern, as observed here in Huh7.5 cells. The incorporated 1'-CN of the templating RDV-MP presumably clashes with main chain atoms of A558 (as visualized on the PDB: 7UO4 (Malone et al., 2023);) explaining both the difficult incorporation of the cognate UTP (Tchesnokov et al., 2020) and the specific nucleotide change pattern detected here. These results are in line with resistance selection; RDV-MP incorporation and fixation into RNA is minimized for the S759A resistance mutant through discrimination and increased stalling, leading to excision. The high substrate efficiency of RDV-TP as an ATP analogue argues that high levels of resistance should be difficult to achieve. Finally, once incorporated into RNA the 1'-CN modification on RDV-MP is also most probably impacting the downstream functions of viral RNAs containing RDV-MP(s), e.g., transcription, recombination, RNA folding, translation, etc. (Shannon and Canard, 2023). As such, RDV-MP likely acts as a chemical corruptor of viral RNA to also account for its antiviral mode of action.

### CRediT authorship contribution statement

**Bhawna Sama:** Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal

analysis, Data curation, Conceptualization. **Barbara Selisko:** Writing – review & editing, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Camille Falcou:** Software, Methodology, Formal analysis. **Véronique Fattorini:** Software, Methodology, Formal analysis. **Géraldine Piorkowski:** Software, Methodology, Formal analysis. **Franck Touret:** Validation, Resources, Methodology, Formal analysis, Data curation. **Kim Donckers:** Software, Methodology, Formal analysis, Data curation. **Johan Neyts:** Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. **Dirk Jochmans:** Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Ashleigh Shannon:** Writing – review & editing, Visualization, Validation, Software, Investigation, Formal analysis. **Bruno Coutard:** Writing – review & editing, Visualization, Validation, Investigation, Funding acquisition, Formal analysis. **Bruno Canard:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

### Funding

This work was supported by grants from the Fondation pour la Recherche Médicale (Aide aux Équipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant no.101003627), the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement no. 101005077 (IMI-CARE), and the PRODEVAR project (ANRS-MIE). A part of the work was made was supported by the European Virus Archive GLOBAL (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 Research and Innovation Program under grant agreement no. 871029.

Bursary to B.Sa. was provided by Fondation de Coopération Scientifique, Méditerranée Infection.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

We thank Léa Lo Bello, Adrien Delpal, Jean Massines and Dr. Sergio Hernandez for their excellent technical assistance in the purification of proteins. We thank Prof. Olve Peersen for his careful reading of the manuscript and insightful feedback.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.antiviral.2024.106034.

### Data availability

Data will be made available on request.

### References

Agostini, M.L., Andres, E.L., Sims, A.C., Graham, R.L., Sheahan, T.P., Lu, X., Smith, E.C., Case, J.B., Feng, J.Y., Jordan, R., Ray, A.S., Cihlar, T., Siegel, D., Mackman, R.L., Clarke, M.O., Baric, R.S., Denison, M.R., 2018. Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mBio 9, e00221. https://doi.org/10.1128/mBio.00221-18.

Ahmed-Belkacem, R., Hausdorff, M., Delpal, A., Sutto-Ortiz, P., Colmant, A.M.G., Touret, F., Ogando, N.S., Snijder, E.J., Canard, B., Coutard, B., Vasseur, J.-J., Decroly, E., Debart, F., 2022. Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues. J. Med. Chem. 65, 6231–6249. https://doi.org/10.1021/acs.jmedchem.2c00120.
Beigel, J.H., Tomashek, K.M., Dodd, L.E., Mehta, A.K., Zingman, B.S., Kalil, A.C., Hohmann, E., Chu, H.Y., Luetkemeyer, A., Kline, S., Lopez de Castilla, D., Finberg, R. W., Dierberg, K., Tapson, V., Hsieh, L., Patterson, T.F., Paredes, R., Sweeney, D.A., Short, W.R., Touloumi, G., Lye, D.C., Ohmagari, N., Oh, M.R., Ruiz-Palacios, G.M., Benfield, T., Fåtkenheuer, G., Kortepeter, M.G., Atmar, R.L., Creech, C.B., Lundgren, J., Babiker, A.G., Pett, S., Neaton, J.D., Burgess, T.H., Bonnett, T., Green, M., Makowski, M., Osinusi, A., Nayak, S., Lane, H.C., 2020. Remdesivir for the treatment of covid-19 — final report. N. Engl. J. Med. 383, 1813–1826. https://doi.org/10.1056/NEJMoa2007764.

Boudewijns, R., Thibaut, H.J., Kaptein, S.J.F., Li, R., Vergote, V., Seldeslachts, L., Van Weyenbergh, J., De Keyzer, C., Bervoets, L., Sharma, S., Liesenborghs, L., Ma, J., Jansen, S., Van Looveren, D., Vercuytsse, T., Wang, X., Jochmans, R., Martens, E., Roosevelt, K., De Vlieger, D., Schepens, B., Van Buyten, T., Jacobs, S., Liu, Y., Martí-Carreras, J., Vanmechelen, B., Wawina-Bokalanga, T., Delang, L., Rocha-Pereira, J., Coelmont, L., Chiu, W., Leyssen, P., Heylen, E., Schols, D., Wang, L., Close, L., Matthijnsens, J., Van Ranst, M., Compernolle, V., Schramm, G., Van Laere, K., Saelens, X., Callewaert, N., Opdenakker, G., Maes, P., Weynand, B., Cawthorne, C., Vande Velde, G., Wang, Z., Neyts, J., Dallmeier, K., 2020. STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters. Nat. Commun. 11, 5838. https://doi.org/10.1038/s41467-020-19684-y.

Bouvet, M., Imbert, I., Subissi, L., Gluais, L., Canard, B., Decroly, E., 2012. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/np14 exoribonuclease complex. Proc. Natl. Acad. Sci. USA 109, 9372-9377. https://doi.org/10.1073/pnas.1201130109.

Bravo, J.P.K., Dangerfield, T.L., Taylor, D.W., Johnson, K.A., 2021. Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication. Mol. Cell 81, 1548–1552. e4. https://doi.org/10.1016/j.molcell.2021.01.035.

Campagnola, G., Govindarajan, V., Pelletier, A., Canard, B., Peersen, O.B., 2022. The SARS-CoV nsp12 polymerase active site is related to large-genome replication. J. Virol. 96, e00671. https://doi.org/10.1128/jvi.00671-22.

Chinthapatla, R., Sotoudegan, M., Srivastava, P., Anderson, T.K., Moustafa, I.M., Passow, K.T., Kennelly, S.A., Moorthy, R., Dulin, D., Feng, Y.J., Harki, D.A., Kirchdoerfer, R.N., Cameron, C.E., Arnold, J.J., 2023. Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease. Nucleic Acids Res. 51, 315–336. https://doi.org/10.1093/nar/gkac1177.

Dangerfield, T.L., Huang, N.Z., Johnson, K.A., 2020. Remdesivir is effective in combating COVID-19 because it is a better substrate than ATP for the viral RNA-dependent RNA polymerase. *iScience* 23. https://doi.org/10.1016/j.isci.2020.101849.

Dangerfield, T.L., Johnson, K.A., 2022. Substrate specificity and kinetics of RNA hydrolysis by SARS-CoV-2 NSP10/14 exonuclease. ACS Bio Med Chem Au 2, 600–606. https://doi.org/10.1021/acsbiomedchemau.2c00046.

De Clercq, E., Li, G., 2016. Approved antiviral drugs over the past 50 years. Clin. Microbiol. Rev. 29, 695-747. https://doi.org/10.1128/CMR.00102-15.

Delang, L., Li, C., Tas, A., Quérat, G., Albulescu, I.C., De Burghraeve, T., Guerrero, N.A. S., Gigante, A., Piorkowski, G., Decroly, E., Jochmans, D., Canard, B., Snijder, E.J., Pérez-Pérez, M.J., van Hemert, M.J., Coutard, B., Leyssen, P., Meyk, J., 2016. The viral capping enzyme nsp1: a novel target for the inhibition of chikungunya virus infection. Sci. Rep. 6, 31819. https://doi.org/10.1038/srep31819.

Do, T.N.D., Donckers, K., Vangeel, T., Chatterjee, A.K., Galay, P.A., Bobardt, M.D., Bilello, J.P., Cihlar, T., De Jonghe, S., Neyts, J., Jochmans, D., 2021. A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents. Antivir. Res. 192, 105122. https://doi.org/10.1016/j. antiviral.2021.105122.

Eckler, L.D., Becker, M.M., Halpin, R.A., Li, K., Venter, E., Lu, X., Scherbakova, S., Graham, R.L., Baric, R.S., Stockwell, T.B., Spiro, D.J., Denison, M.R., 2010. Infidelity of SARS-CoV nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS Pathog. 6, e1000896. https://doi.org/10.1371/journal.ppat.1000896.

Eckler, L.D., Lu, X., Sperry, S.M., Choi, L., Denison, M.R., 2007. High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J. Virol. 81, 12135–12144. https://doi.org/10.1128/JVI.01296-07.

Ferron, F., Sama, B., Decroly, E., Canard, B., 2021. The enzymes for genome size increase and maintenance of large (+)RNA viruses. Trends Biochem. Sci. 46, 866–877. https://doi.org/10.1016/j.tibs.2021.05.006.

Ferron, F., Subissi, L., Silveira De Morais, A.T., Le, N.T.T., Sevajol, M., Gluais, L., Decroly, E., Vonrhein, C., Bricogone, G., Canard, B., Imbert, I., 2018. Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA. Proc. Natl. Acad. Sci. U. S. A. 115, E162–E171. https://doi.org/10.1073/pnas.1718806115.

Gandhi, S., Klein, J., Robertson, A.J., Peña-Hernández, M.A., Lin, M.J., Roychoudhury, P., Lu, P., Fournier, J., Ferguson, D., Mohamed Bakhash, S.A.K., Catherine Muenker, M., Srivathsan, A., Wunder, E.A., Kerantzas, N., Wang, W., Lindenbach, B., Pyle, A., Wilen, C.B., Ogbuagu, O., Greninger, A.L., Iwasaki, A., Schulz, W.L., Ko, A.I., 2022. De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case report. Nat. Commun. 13, 1547. https://doi.org/10.1038/s41467-022-29104-y.

Gordon, C.J., Tchesnovok, E.P., Feng, J.Y., Porter, D.P., Götte, M., 2020a. The antiviral compound remdesivir potentially inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J. Biol. Chem. 295, 4773–4779. https://doi.org/10.1074/jbc.JAC12013056.

Gordon, C.J., Tchesnovok, E.P., Woolner, E., Perry, J.K., Feng, J.Y., Porter, D.P., Götte, M., 2020b. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J. Biol. Chem. 295, 6785–6797. https://doi.org/10.1074/jbc. RA12013679.

Gottlieb, R.L., Vaca, C.E., Paredes, R., Mera, J., Webb, B.J., Perez, G., Oguchi, G., Ryan, P., Nielsen, B.U., Brown, M., Hidalgo, A., Sachdeva, Y., Mittal, S., Osiyemi O., Skarinski, J., Juneja, K., Hyland, R.H., Osinusi, A., Chen, S., Camus, G., Abdelghany, M., Davies, S., Behenna-Renton, N., Duff, F., Marty, F.M., Katz, M.J., Ginde, A.A., Brown, S.M., Schiffer, J.T., Atmar, R.L., Creek, C.B., 2022. Early remdesivir to prevent progression to severe covid-19 in outpatients. N. Engl. J. Med. 386, 305–315. https://doi.org/10.1056/NEJMoa2116846.

Hogan, J.I., Duerr, R., Dimartino, D., Marier, C., Hochman, S.E., Mehta, S., Wang, G., Heguy, A., 2023. Remdesivir resistance in transplant recipients with persistent coronavirus disease 2019. Clin. Infect. Dis. 76, 342–345. https://doi.org/10.1093/cid/cia769.

Jochmans, D., Leyssen, P., Nechts, J., 2012. A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE. J. Virol Methods 183, 176–179. https://doi.org/10.1016/j.jviromet.2012.04.011.

Jockusch, S., Tao, C., Li, X., Chien, M., Kumar, S., Morozova, I., Kalachikov, S., Russo, J. J., Ju, J., 2020. Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir. Sci. Rep. 10. https://doi.org/10.1038/s41598-020-73641-9.

Kabinger, F., Stiller, C., Schmitzová, J., Dienemann, C., Kokic, G., Hillen, H.S., Höbartner, C., Cramer, P., 2021. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat. Struct. Mol. Biol. 28, 740–746. https://doi.org/10.1038/s41594-020-0651-0.

Kokic, G., Hillen, H.S., Tegunov, D., Dienemann, C., Seitz, F., Schmitzova, J., Farnung, L., Siewert, A., Höbartner, C., Cramer, P., 2021. Mechanism of SARS-CoV-2 polymerase stalling by remdesivir. Nat. Commun. 12, 79. https://doi.org/10.1038/s41467-020-20542-0.

Li, G., Hilgenfeld, R., Whitley, R., De Clercq, E., 2023. Therapeutic strategies for COVID-19: progress and lessons learned. Nat. Rev. Drug Discov. 22, 449–475. https://doi.org/10.1038/s41573-023-00672-y.

Ma, Y., Wu, L., Shaw, N., Gao, Y., Wang, J., Sun, Y., Lou, Z., Yan, L., Zhang, R., Rao, Z., 2015. Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex. Proc. Natl. Acad. Sci. USA 112, 9436–9441. https://doi.org/10.1073/pnas.1508686112.

Mackman, R.L., 2022. Phosphoramidate prodrugs continue to deliver, the journey of remdesivir (GS-5734) from RSV to SARS-CoV-2. ACS Med. Chem. Lett. 13, 338–347. https://doi.org/10.1021/acsmedchemlett.1c00624.

Malin, J.J., Suárez, I., Priesner, V., Fåtkenheuer, G., Rybniker, J., 2020. Remdesivir against COVID-19 and other viral diseases. Clin. Microbiol. Rev. 34, e00162. https://doi.org/10.1016/j.cmr.2020.01620-20.

Malone, B., Chen, J., Wang, Q., Llewellyn, E., Choi, Y.J., Olinares, P.D.B., Cao, X., Hernandez, C., Eng, E.T., Chait, B.T., Shaw, D.E., Landick, R., Darst, S.A., Campbell, E.A., 2021. Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex. Proc. Natl. Acad. Sci. USA 118, e2102516118. https://doi.org/10.1073/pnas.2102516118.

Malone, B., Urakova, N., Snijder, E.J., Campbell, E.A., 2022. Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat. Rev. Mol. Cell Biol. 23, 21–39. https://doi.org/10.1038/s41580-021-00432-z.

Malone, B.F., Perry, J.K., Olinares, P.D.B., Lee, H.W., Chen, J., Appleby, T.C., Feng, J.Y., Bilello, J.P., Ng, H., Sotiris, J., Ebrahim, M., Chua, E.Y.D., Mendez, J.H., Eng, E.T., Landick, R., Götte, M., Chait, B.T., Campbell, E.A., Darst, S.A., 2023. Structural basis for substrate selection by the SARS-CoV-2 replicase. Nature 614, 781–787. https://doi.org/10.1038/s41586-022-05664-3.

Martinot, M., Jary, A., Fafi-Kremer, S., Leducq, V., Delagreverie, H., Garnier, C., Pacanowski, J., Mékinian, A., Pirenne, F., Tiberghien, P., Calvez, V., Humbrecht, C., Marcelin, A.-G., Lacombe, K., 2021. Emerging RNA-dependent RNA polymerase mutation in a remdesivir-treated B-cell immunodeficient patient with protracted coronavirus disease 2019. Clin. Infect. Dis. 73, e1762–e1765. https://doi.org/10.1093/cid/cia1474.

McDonald, S., Block, A., Beaucourt, S., Moratorio, G., Vignuzzi, M., Peersen, O.B., 2016. Design of a genetically stable high fidelity coxsackievirus B3 polymerase that attenuates virus growth in vivo. J. Biol. Chem. 291, 13999–14011. https://doi.org/10.1074/jbc.M116.726596.

Minskaia, E., Hertzig, T., Gorbalenya, A.E., Campanacci, V., Cambillau, C., Canard, B., Ziebuhr, J., 2006. Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc. Natl. Acad. Sci. U. S. A. 103, 5108–5113. https://doi.org/10.1073/pnas.0508200103.

Ogando, N.S., Ferron, F., Decroly, E., Canard, B., Posthuma, C.C., Snijder, E.J., 2019. The curious case of the nidovirus exoribonuclease: its role in RNA synthesis and replication fidelity. Front. Microbiol. 10, 1813. https://doi.org/10.3389/fmicb.2019.01813.

Plante, J.A., Mitchell, B.M., Plante, K.S., Debбbink, K., Weaver, S.C., Menachery, V.D., 2021. The variant gambit: COVID-19's next move. Cell Host Microbe 29, 508–515. https://doi.org/10.1016/j.chom.2021.02.020.

Rubin, D., Chan-Tack, K., Farley, J., Sherwat, A., 2020. FDA approval of remdesivir — a step in the right direction. N. Engl. J. Med. 383, 2598–2600. https://doi.org/10.1056/NEJMp2032369.

Russo, P., Tacconelli, E., Olimpieri, P.P., Celant, S., Colatrella, A., Tomassini, L., Palù, G., 2022. Mortality in SARS-CoV-2 hospitalized patients treated with remdesivir: a nationwide, registry-based study in Italy. Viruses 14, 1197. https://doi.org/10.3390/v14061197.

Seifert, M., Bera, S.C., van Nies, P., Kirchdoerfer, R.N., Shannon, A., Le, T.-T.-N., Meng, X., Xia, H., Wood, J.M., Harris, L.D., Papini, F.S., Arnold, J.J., Almo, S., Grove, T.L., Shi, P.-Y., Xiang, Y., Canard, B., Depken, M., Cameron, C.E., Dulin, D., 2021. Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective. Elife 10, e70968. https://doi.org/10.7554/eLife.70968.
Selisko, B., Papageorgiou, N., Ferron, F., Canard, B., 2018. Structural and functional basis of the fidelity of nucleotide selection by flavivirus RNA-dependent RNA polymerases. Viruses 10, 59. https://doi.org/10.3390/v10020059.

Shannon, A., Canard, B., 2023. Kill or corrupt: mechanisms of action and drug-resistance of nucleotide analogues against SARS-CoV-2. Antivir. Res. 210, 105501. https://doi.org/10.1016/j.antiviral.2022.105501.

Shannon, A., Fattorini, V., Sama, B., Selisko, B., Feracci, M., Falcou, C., Gauffre, P., El Kazzi, P., Delpal, A., Decroly, E., Alvarez, K., Eydoux, C., Guillemot, J.-C., Moussa, A., Good, S.S., La Colla, P., Lin, K., Sommadossi, J.-P., Zhu, Y., Yan, X., Shi, H., Ferron, F., Canard, B., 2022. A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase. Nat. Commun. 13, 621. https://doi.org/10.1038/s41467-022-28113-1.

Shannon, A., Le, N.T.-T., Selisko, B., Eydoux, C., Alvarez, K., Guillemot, J.-C., Decroly, E., Peersen, O., Ferron, F., Canard, B., 2020a. Remdesivir and SARS-CoV-2: structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites. Antivir. Res. 178, 104793. https://doi.org/10.1016/j.antiviral.2020.104793.

Shannon, A., Selisko, B., Le, N.-T.-T., Huchting, J., Touret, F., Piorkowski, G., Fattorini, V., Ferron, F., Decroly, E., Meier, C., Coutard, B., Peersen, O., Canard, B., 2020b. Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis. Nat. Commun. 11, 4682. https://doi.org/10.1038/s41467-020-18463-z.

Sheahan, T.P., Sims, A.C., Graham, R.L., Menachery, V.D., Gralinski, L.E., Case, J.B., Leist, S.R., Pyrc, K., Feng, J.Y., Trantcheva, I., Bannister, R., Park, Y., Babusis, D., Clarke, M.O., Mackman, R.L., Spahn, J.E., Palmiotti, C.A., Siegel, D., Ray, A.S., Cihlar, T., Jordan, R., Denison, M.R., Baric, R.S., 2017. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci. Transl. Med. 9, eaal3653. https://doi.org/10.1126/scitranslmed.aal3653.

Smith, E.C., Blanc, H., Vignuzzi, M., Denison, M.R., 2013. Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS Pathog. 9, e1003565. https://doi.org/10.1371/journal.ppat.1003565.

Stevens, L.J., Pruijssers, A.J., Lee, H.W., Gordon, C.J., Tchesnovok, E.P., Gribble, J., George, A.S., Hughes, T.M., Lu, X., Li, J., Perry, J.K., Porter, D.P., Cihlar, T., Sheahan, T.P., Baric, R.S., Götte, M., Denison, M.R., 2022. Mutations in the SARS-CoV-2 RNA-dependent RNA polymerase confer resistance to remdesivir by distinct mechanisms. Sci. Transl. Med. 14, eabo0718. https://doi.org/10.1126/scitranslmedabo0718.

Szemiel, A.M., Merits, A., Orton, R.J., MacLean, O.A., Pinto, R.M., Wickenhagen, A., Lieber, G., Turnbull, M.L., Wang, S., Furnon, W., Suarez, N.M., Mair, D., Filipe, A. da S., Willett, B.J., Wilson, S.J., Patel, A.H., Thomson, E.C., Palmarini, M., Kohl, A., Stewart, M.E., 2021. In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. PLoS Pathog. 17, e1009929. https://doi.org/10.1371/journal.ppat.1009929.

Tchesnovok, E.P., Gordon, C.J., Woolner, E., Kocinkova, D., Perry, J.K., Feng, J.Y., Porter, D.P., Götte, M., 2020. Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action. J. Biol. Chem. 295, 16156-16165. https://doi.org/10.1074/jbc.AC120.015720.

Touret, F., Baronti, C., Goethals, O., Van Loock, M., de Lamballerie, X., Querat, G., 2019. Phylogenetically based establishment of a dengue virus panel, representing all available genotypes, as a tool in dengue drug discovery. Antivir. Res. 168, 109-113. https://doi.org/10.1016/j.antiviral.2019.05.005.

Touret, F., Gilles, M., Barral, K., Nougairède, A., van Helden, J., Decroly, E., de Lamballerie, X., Coutard, B., 2020. In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication. Sci. Rep. 10, 13093. https://doi.org/10.1038/s41598-020-70143-6.

Traut, T.W., 1994. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1-22. https://doi.org/10.1007/BF00928361.

Wang, Q., Wu, J., Wang, H., Gao, Y., Liu, Q., Mu, A., Ji, W., Yan, L., Zhu, Y., Zhu, C., Fang, X., Yang, Xiaobao, Huang, Y., Gao, H., Liu, F., Ge, J., Sun, Q., Yang, Xiuna, Xu, W., Liu, Z., Yang, H., Lou, Z., Jiang, B., Guddat, L.W., Gong, P., Rao, Z., 2020. Structural basis for RNA replication by the SARS-CoV-2 polymerase. Cell 182, 417-428.e13. https://doi.org/10.1016/j.cell.2020.05.034.

Yang, X., Smidansky, E.D., Maksimchuk, K.R., Lum, D., Welch, J.L., Arnold, J.J., Cameron, C.E., Boehr, D.D., 2012. Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure 20, 1519-1527. https://doi.org/10.1016/j.str.2012.06.012.

Yin, W., Mao, C., Luan, X., Shen, D.-D., Shen, Q., Su, H., Wang, X., Zhou, F., Zhao, W., Gao, M., Chang, S., Xie, Y.-C., Tian, G., Jiang, H.-W., Tao, S.-C., Shen, J., Jiang, Y., Jiang, H., Xu, Y., Zhang, S., Zhang, Y., Xu, H.E., 2020. Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir. Science 368, 1499-1504. https://doi.org/10.1126/science.abc1560.

Yip, A.J.W., Low, Z.Y., Chow, V.T.K., Lal, S.K., 2022. Repurposing molnupiravir for COVID-19: the mechanisms of antiviral activity. Viruses 14, 1345. https://doi.org/10.3390/v14061345.

Zhang, L., Zhang, D., Wang, X., Yuan, C., Li, Y., Jia, X., Gao, X., Yen, H.-L., Cheung, P.P.-H., Huang, X., 2021. 1'-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication. Phys. Chem. Chem. Phys. 23, 5852-5863. https://doi.org/10.1039/d0cp05948j.

Zhu, Y., Binder, J., Yurgelonis, I., Rai, D.K., Lazarro, S., Costales, C., Kobylarz, K., McMonagle, P., Steppan, C.M., Aschenbrenner, L., Anderson, A.S., Cardin, R.D., 2022. Generation of a VeroE6 Pgp gene knock out cell line and its use in SARS-CoV-2 antiviral study. Antivir. Res. 208, 105429. https://doi.org/10.1016/j.antiviral.2022.105429.
Supplementary Material

# The effects of Remdesivir's functional groups on its antiviral potency and resistance against the SARS-CoV-2 polymerase

Bhawna Sama<fnref n="1" />, Barbara Selisko<fnref n="1" />, Camille Falcou<fnref n="1" />, Véronique Fattorini<fnref n="1" />, Géraldine Piorkowski<fnref n="2" />,
Franck Touret<fnref n="2" />, Kim Donckers<fnref n="3" />, Johan Neyts<fnref n="3" />, Dirk Jochmans<fnref n="3" />, Ashleigh Shannon<fnref n="1" />, Bruno
Coutard<fnref n="2" />, Bruno Canard<fnref n="1" />,<fnref n="4" />*

<footnote n="1">¹Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix-Marseille Université, UMR7257, Marseille, France.</footnote>

<footnote n="2">²Unité des Virus Émergents (UVE), Aix-Marseille Université, Università di Corsica, IRD 190, Inserm 1207, IRBA, France.</footnote>

<footnote n="3">³KU Leuven - Rega Institute, Leuven, Belgium</footnote>

<footnote n="4">⁴European Virus Bioinformatics Center, Leutragraben 1, 07743 Jena, Germany</footnote>

<footnote>*Corresponding author: bruno.canard@univ-amu.fr</footnote>
<table><caption>**Supplementary Table. 1. RNA substrates**</caption><thead><tr><th>Name</th><th>Sequence (5'-3')</th><th>Length (nt)</th></tr></thead><tbody><tr><td>Primer (P10)</td><td>5'/Cy5/GUCAUUCUCC 3'</td><td>10</td></tr><tr><td>Template A (T<sub>(A)</sub>20)</td><td>5' UAGCUUCUUAGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template U (T<sub>(U)</sub>20)</td><td>5' UAGCUUCUUGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template C (T<sub>(C)</sub>20)</td><td>5' UAGCUUCUUUCGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template G (T<sub>(G)</sub>20)</td><td>5' UAGCUUCUUUGGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template U_4R</td><td>5' UAGCUUCAAUGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template U_R</td><td>5' AAGCAACAAUGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>Template CA</td><td>5' AAGCAACUACGGAGAAUGAC 3'</td><td>20</td></tr><tr><td>HP_AU</td><td>5'/6-Fam/CGAGAGCAUCGCGUAGUUUUCUACGCG 3'</td><td>27</td></tr><tr><td>HP_AA</td><td>5'/6-Fam/CGAGAGAUUCGCGUAGUUUUCUACGCG 3'</td><td>27</td></tr><tr><td>HP_AC</td><td>5'/6-Fam/CGAGAGAGUCGCGUAGUUUUCUACGCG 3'</td><td>27</td></tr></tbody></table>
a

<figure><img src="image_14.png" /></figure>

b

<figure><img src="image_15.png" /></figure>

**Fig.S1. SDS-PAGE of purified SARS-CoV-2 proteins.** a) Purified nsp12, nsp7, nsp8, nsp14 and nsp10 proteins. b) P323L nsp12 and RDV-resistant nsp12 mutants; S759A, A777S and S759A+A777S purified in P323L mutation containing nsp12.
<figure><img src="image_16.png" /></figure>

**Fig.S2. Incorporation of RDV-MP by SARS-CoV-2 mRTC.** Sequence of primer (P10) and template (T(U)20), where first incoming nucleotide will be ATP or nucleotide analogue RDV-TP is shown in the top panel above the gels. PAGE gel images show a) the elongation of RNA when all the NTPs are provided at 50 µM concentration with no RDV-TP (corner left) or 10 µM, 50µM and 250µM (corner right) of RDV-TP. At the right-hand side, the identities of product bands are indicated when either ATP or RDV-TP can be incorporated. b) All NTPs and AT-9010 are provided at 50 µM. c) Reactions contain GTP, CTP, UTP in absence (left), and in presence (right) of RDV-TP. d) Reactions contain ATP, CTP, UTP in absence (left) and in presence (right) of RDV-TP.
<figure><img src="image_17.png" /><figcaption>**Fig.S3. Incorporation of RDV-MP by SARS-CoV mRTC with RDV-MP incorporated as first nucleotide.** The sequence of RNA template (T<sub>(u)20</sub>), primer (P10) and the expected RNA product when all natural NTPs are provided is displayed in the top panel. PAGE patterns of the polymerization reactions with SARS-CoV mRTC composed of nsp12, nsp7L8 and nsp8 in a molar ratio of 1:3:3 molar ratio are shown. In a) reactions with all NTPs at 50 µM concentration in the absence of RDV-TP (corner left), or at 10 µM, 50 µM and 250 µM of RDV-TP. Panel b) shows the same reaction set-up but in the absence of ATP. Stalled RNA product G14 (*i* +3) after RDV-TP incorporation at position 11 (*i*), is clearly visible at 10 µM RDV-TP.</figcaption></figure>
<figure><img src="image_18.png" /></figure>

**Fig.S4. Incorporation of RDV(-CN)-TP in RNA by SARS-CoV mRTC incorporated as first nucleotide.** The upper panel shows the RNA template (T<sub>(u)20</sub>), primer (P10), and the expected RNA product when all natural NTPs are present. SARS-CoV mRTC corresponds to the one in Fig.S2. PAGE product patterns are shown a) obtained with all NTPs at 50 µM in the absence of RDV-TP without 1-CN group (RDV(-CN)-TP) or in the presence of RDV(-CN)-TP at 10 µM, 50 µM, or 250 µM. In panel b) the same reactions in absence of ATP are shown.
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>**Fig.S5. Incorporation of RDV(-CN)-TP and ATP on Template U, C, G and A by SARS-CoV-2 mRTC.** A) The sequences of the four primer/template (P10/T20) sets used in the subsequent biochemical assays are shown, along with the expected product sequences when all native NTPs are present. PAGE results display the polymerization reaction at the specified time points when (B) only RDV(-CN)-TP is supplied, and (C) when only ATP is supplied at varying concentrations (10, 50, 250 µM) for Template U (a), Template C (b), Template G (c), and Template A (d). Primer and product band intensities were quantified and initial velocities of incorporation were determined (refer to the table in Figure 4B).</figcaption></figure>
HUH 7.5

<figure><img src="image_22.png" /><figcaption>**Fig.S6.** Quantification of the replication inhibition induced by Remdesivir on SARS-CoV-2 measured by genome copy number in Huh7.5 with EC₅₀ value indicated. Mean ± standard deviation (SD) shown (n = 3).</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>**Fig.S7.** Excision of incorporated NA-TPs; RDV-TP, ATP+CN, RDV(-CN)-TP for two consecutive incorporations into HP_AAU. a) Sequence of 27 nucleotides long hairpin RNA, HP_AAU is shown in grey (loop), stem (blue) and green (template). In pink, the expected product is shown when the native NTP, ATP is added. b) PAGE shows the product bands for polymerization and excision reactions at the indicated reaction conditions. The +2 incorporation products are seen in all conditions except for Control (no incorporation) and GTP (+1 misincorporation, U:G). The excision products are labeled for RDV-TP reaction condition.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Fig. S8.** Passaging SARS-CoV-2-GHB (Wuhan) in VeroE6 cells in the presence of increasing concentrations of GS-441524. Selection was initiated at 0.5 µM. At every passage, several new cultures were started with the same concentration as well as a lower and a higher concentration. The passage with the highest compound concentration that could be maintained was selected for the following passage.</figcaption></figure>
Template C

Template U_4R

Template U_R

Template CA

C U U C U U C G A U

U A A C U U C G A U

U A A C A A C G A A

C A U C A A C G A A

a

<figure><img src="image_25.png" /></figure>

Time min
NA 0 1 5 10 30 60 10 5 15 30 60
NTP 50 RDV-TP no A 50 RDV-TP no A 50 RDV-TP no A 50 RDV-TP no A

Template C

Template U_4R

Template U_R

Template CA

b

<figure><img src="image_26.png" /></figure>

Time min
NA 0 1 5 10 30 60 10 5 15 30 60
NTP 50 RDV-TP no A 50 RDV-TP no A 50 RDV-TP no A 50 RDV-TP no A
**Fig. S9. Increased RNA stalling after RDV-TP incorporation by S759A RdRp in comparison to wild-type RdRp in different RNA substrates.** Four different templates of 20 nucleotides length (T20) are employed using a common primer of 10 nucleotides length (P10). The template sequences (Template C, U_4R, U_R, CA) allowing single or multiple RDV-MP incorporations are indicated above the gel. Product sequences are indicated at the left side of each reaction. RNA extension reactions by SARS-CoV-2 mRTC for wild-type RdRp (a) and S759A mutant RdRp (b) are shown at indicated time-points employing RDV-TP, GTP, CTP, UTP at 50 µM concentration each.