Change in SARS-CoV-2 RDV susceptibility after serial passage

After determining optimal culture conditions (S1 Fig), SARS-CoV-2_Engl2 was passaged serially in either 1μM or 2.5μM RDV-supplemented media for 13 passages (SARS-CoV-2_Engl2 was isolated in February 2020; Fig 1A). Viruses serving as controls were passaged in parallel in either DMSO or media to monitor for mutations due to cell culture adaptation unrelated to RDV resistance. In total, we passaged SARS-CoV-2_Engl2 in parallel in 24 distinct cultures with different selective pressures (4 different conditions and 2 different virus inputs; Fig 1A). We monitored for cytopathic effect (CPE) during passaging of the cultures. CPE was observed in 7 of the 12 lineages passaged in RDV, with the loss of 5 lineages between p1 and p4 (Fig 1A and 1B). We observed general adaptation of the viruses to VeroE6 cells, with an increase in overall viral titers by 0.5 to 1 log₁₀ (Fig 1B) as well as a change in plaque phenotype (S2A Fig) after 13 passages.

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Fig 1. Continuous passage of SARS-CoV-2_Engl2 in RDV selects for partial resistant populations.

(A) Schematic of the experimental layout to select for RDV resistant viruses. The passage (p) number of the input virus SARS-CoV-2_Engl2 is given. We consider p1 as the stock supplied by Public Health England, it was passaged twice in VeroE6 prior to selection in RDV. SARS-CoV-2_Engl2 p3 is the input stock that was sequenced and used for analysis. Each condition is given a different colour and the amount of virus used at the start of the experiment is indicated. Populations that failed to amplify (no CPE or/and virus titre) are indicated by the empty circles. All lineages sequenced are indicated. (B) Virus titers (pfu/ml) at p1, p4, p7 and p10. 6 lineages per condition and two different virus inputs; 1000 pfu (solid circle) and 2000 pfu (open circle). Median for each is shown. (C) Virus growth kinetics in VeroE6 in the presence (dashed line) or absence (solid line) of 7.5μM RDV for 3 different virus populations. Data is from 2 independent experiments with 3 replicates. Error bars represent SEM. Unpaired t-tests (Holm-Šídák method; *,P< 0.05; **,P< 0.01; ***,P< 0.001. ****, P<0.0001). (D) EIDD2801 dose dependency curve. EIDD2801 treated VeroE6-ACE2-TMPRSS2 infected with 8400 pfu/ml of each virus. (E) RDV dose dependency curves determined in A549NPro-ACE2 infected with 8400 pfu/ml of each virus. (F) Bar graph of RDV EC₅₀ for different viruses in A549NPro-ACE2 with 8400 pfu/well. For all panels, error bars represent SEM.

https://doi.org/10.1371/journal.ppat.1009929.g001

Next, the replication kinetics and change in the half maximal effective concentration (EC₅₀) of RDV in a subset of passaged virus populations (Rem2.5p13.5, DMSOp13.5 and Mediap13.4) were assessed. Rem2.5p13.5 alone actively replicated in the presence of 7.5μM RDV (Fig 1C). However, titers in the presence of RDV were lower than those grown in the absence of RDV. Titers of control viruses, DMSOp13.5 and Mediap13.4 were consistently 5 log₁₀ lower when grown in the presence of RDV (Fig 1C). The Rem2.5p13.5, DMSOp13.5 and Mediap13.4 lineages displayed similar replication kinetics when cultured in the absence of RDV (Fig 1C). Furthermore, Rem2.5p13.5 lineage required a greater concentration of RDV to protect infected cells (S2B Fig) corresponding to a 2- to 2.5-fold increase in RDV EC₅₀ over a range of virus inputs in comparison with DMSOp13.5, and Mediap13.4 (S2C Fig). The partial resistance to a nucleoside analogue was specific for RDV, as we observed a minimal change in EC₅₀ of a second nucleoside analogue (EIDD2801), when comparing Rem2.5p13.5 (EC₅₀ ~9.1μM) to SARS-CoV-2_Engl2 (EC₅₀ ~8.9μM) (Figs 1D and S3C and S3D andS1 Text).

Subsequent analyses identified a second lineage, Rem1p13.5 with reduced sensitivity to RDV (Fig 1E). The EC₅₀ of Rem1p13.5 (~0.83μM) was comparable to Rem2.5p13.5 (~0.88μM), this was a 3.5- to 3.7-fold increase in EC₅₀ from the parental virus (EC₅₀~0.23μM). The RDV EC₅₀ for virus passaged in either media alone (EC₅₀~0.29–0.32μM) or DMSO (EC₅₀~0.12–0.22μM) corresponded with EC₅₀ for the parental stock virus (Fig 1F). The changes in RDV sensitivity paralleled those previously reported for MHV, SARS-CoV and EBOV resistant viruses [4,20].

Common mutations in two virus populations with partial resistance to RDV

Direct comparison of the consensus sequences from a subset of viruses passaged in different conditions with original SARS-CoV-2_Engl2 revealed two fixed non-synonymous mutations in lineages with decreased RDV susceptibility of at least 2-fold change in EC₅₀ in two independently generated populations (Rem1p13.5 & Rem2.5p13.5). These mutations were not present in viruses either passaged in absence of RDV, or the input virus (SARS-CoV-2_Engl2) or SARS-CoV-2_Wu1 (MN908947) (S1 Data). The first mutation was identified as glutamine to aspartate at amino acid 802 (E802D) in the RNA-dependent RNA polymerase (RdRp) NSP12 (Fig 2A). A glutamate at this position is highly conserved between all betacoronaviruses including SARS-CoV, MERS-CoV and unclassified sarbecoviruses (Fig 2B and S2A Data). The E802 mutation occurs within the palm sub-domains (T680 to Q815; Fig 2A), and in proximity to amino acids predicted to interact with newly synthesized RNA (C813, S814 and Q815 [22]; Fig 2A). We propose that the E802D mutation changes the steric interactions between amino acid side chains within this region resulting in minor structural changes, (Fig 2A), thereby influencing binding of nt+3 during synthesis of template RNA and allowing elongation when the active form of RDV is incorporated into the RNA. The mutation identified in NSP12 differs from amino acid residues involved with decreased RDV sensitivity in EBOV, other betacoronaviruses, (MHV, & SARS-CoV), and sites predicted in SARS-CoV-2 [19–21].

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Fig 2. Common mutations in partial RDV resistance populations.

(A) Location of E802 within structure of SARS-CoV-2 NSP12 in association with NSP7 and NSP8 (PDB ID 6YYT). Three focused panels are WT (upper) and two potential confirmations of E802D. H-bonds are indicated by light blue line. (B) Conservation of E802 amino acid across coronaviruses. Accession numbers for the coronavirus sequences are in the materials and methods. (C) NSP6 I168 amino acid is not conserved across coronaviruses.

https://doi.org/10.1371/journal.ppat.1009929.g002

The second mutation was an isoleucine to threonine substitution (I168T) in NSP6, a highly conserved protein involved in restricting autophagosome expansion [23]. This site is not conserved across coronaviruses with either an isoleucine (SARS-CoV-2), or valine (SARS-CoV & MERS-CoV) or leucine (MHV) in this position (Fig 2C and S2 Data). We predict this mutation may alter the structure of the transmembrane and extracellular domains (S4 Fig).

A single mutation in NSP12 confer partial resistance to RDV in rSARS-CoV-2_Wu1

To ascertain whether a mutation of NSP12 E802 was sufficient to mediate partial RDV resistance, we introduced either an E802D or E802A mutation at this site into SARS-CoV-2_Wu1 by reverse genetics and recovered infectious virus. Whilst we thought it was unlikely to play a role, we also recovered virus with I168T mutation in NSP6 either alone or in combination with the NSP12 mutations (E802D or E802A). All rescued viruses with specific point mutations replicated similarly to the rescued wild-type virus (rSARS-CoV-2) in human lung cells, Calu-3, with comparable replication kinetics and achieving similar peak virus titers (Fig 3A; rSARS-CoV-2 vs rNSP12-E802D, p.adj = 0.44; rSARS-CoV-2 vs rNSP12-E802A, p.adj = 0.41; rSARS-CoV-2 vs rNSP6-I168T, p.adj = 0.51; rSARS-CoV-2 vs rNSP6-I168T+NSP12-E802A, p.adj = 0.50, rSARS-CoV-2 vs rNSP6-I168T+NSP12-E802D, p.adj = 0.50; RM One-Way Anova with Dunnett’s Test). This indicates that these mutations did not provide a replicative advantage in a representative cell-line without drug exposure. In our drug screen assays, both the E802D and E802A mutations in NSP12 recapitulated partial resistance observed in the virus populations continually passaged in RDV (Fig 3B). We observed a 2.14- to 2.54-fold change in RDV EC₅₀; from 2.61μM for rSARS-CoV-2 to 5.58μM and 6.62μM for the E802A and E802D mutants, respectively (Fig 3B and S1 Table). This change in RDV sensitivity was evident over a range of virus inputs for both NSP12 mutants (S5A Fig). NSP6 I168T substitution did not confer decreased sensitivity to RDV (Fig 3B), with comparable EC₅₀ values to rSARS-CoV-2 (S1 Table).

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Fig 3. NSP12 E802 mutation recapitulates change in RDV susceptibility.

All viruses were derived by reverse genetics and have a SARS-CoV-2_Wu1 backbone with specific point mutations as indicated. (A) Virus replication kinetics of rescued viruses with single mutation in either NSP12, NSP6 or both in Calu-3. Data is from 3 independent experiments with 3 replicates, there was no significant difference between growth of the mutants versus the wild-type rSARS-CoV-2. (B) RDV dose-dependent inhibition for each mutant virus. Post-treatment with RDV, VeroE6-ACE2-TMPRSS2 were infected with 500 pfu/ml of each virus. Mutations in NSP12 decrease the sensitivity to RDV. (C) RDV dose effect on virus titers at 24 h (left) and 48 h (right). Calu-3 were treated with decreasing doses of RDV and infected with an MOI~0.01. Data from 2 independent virus stocks with 2 replicates except for rSARS-CoV-2 and rNSP12-E802A. All error bars are SEM. (D) Co-infection competition assay virus titres for the input (p0) and each subsequent passage (p1, p2 and p3) expressed as genome copies/ml. VeroE6-ACE2-TMPRSS2 were infected with different ratios of rNSP12-E802D to rSARS-CoV-2. The assay does not discriminate between rSARS-CoV-2 and rNSP12-E802D. Data from 3 independent infections. (E) Co-infection competition assay with different input ratios (1:9 and 9:1) of rSARS-CoV-2 WT and rNSP12E802D in VeroE6-ACE2-TMPRSS2. The percentage of each virus population over successive passages is shown (Data from 3 independent infections).

https://doi.org/10.1371/journal.ppat.1009929.g003

Indeed, viruses bearing both the NSP12 and NSP6 mutations were more sensitive to RDV in comparison to NSP12 single mutant viruses (rNSP6-I168T+NSP12-E802D, EC₅₀ 3.21μM; rNSP6-I168T+NSP12-E802A, EC₅₀ 3.89μM). Importantly, introduction of NSP6 and/or NSP12 mutations did not significantly affect sensitivity to an alternate nucleoside analogue EIDD2801 (S1 Table and S5B Fig). These data confirm results obtained with other viruses demonstrating EIDD2801 sensitivity was not influenced by mutations conferring decreased RDV sensitivity [20,24]. We further assessed the anti-viral activity of RDV in Calu-3. While a RDV dose-dependent reduction in titer for all viruses was observed, rNSP12-E802D and rNSP12-E802A titers were consistently higher than wild-type and rNSP6-I168T at 24h and 48h pi (Fig 3C) over a range of concentrations. Interestingly, at 24h pi, a slight shift in an increase rNSP6-I168T infectious titer was observed in comparison with wild type, though this effect disappeared by 48h.

To examine whether the viruses containing the NSP12 E802D mutation would persist and outcompete the WT in the absence of RDV, an in vitro co-infection competition assay over multiple passages was undertaken. VeroE6-ACE2-TMPRSS2 were infected with two different MOI ratios of rSARS-CoV-2 to rNSP12-E802D (1:9 and 9:1), samples were harvested every 24 h and used to infect fresh monolayers of cells. This was repeated for 3 passages. While both stocks had high infectious titres (rSARS-CoV-2, ~2.5 x 10⁷ pfu/ml and rNSP12-E802D, ~8.13 x 10⁷ pfu/ml), there was a difference in the equivalent genome copies (GE)/ml, with rNSP12-E802D a ~0.5 log₁₀ higher (rSARS-CoV-2, 7.16 x 10⁸ GE/ml and rNSP12-E802D, 2.90 x 10⁹ GE/ml). Therefore, we used different ratios of MOI rather than GE/ml equivalents for the initial infection. We monitored for virus replication by qRT-PCR and found an increase in titres at each passage (Fig 3D). We next examined the change in the ratio of wild type to E802D in the viral populations at each passage using the GridION (Oxford Nanopore Technologies). Regardless of input ratio, rSARS-CoV-2 out competed viruses containing the rNSP12-E802D mutation (Fig 3E). This would indicate the E802D mutation incurs a fitness cost in the absence of RDV and is unlikely to be sustained in the circulating population.

Prevalence of RDV mutations in circulating populations

We next examined the available SARS-CoV-2 genome sequences in the GISAID database (n = 844376 as of March 2021) to determine the frequency of replacements at NSP12 E802 and NSP6 I168. We only identified 36 (0.004%) viral sequences with a mutation at E802; E802D was the predominant replacement (n = 24), with the remaining sequence either E802A (n = 4), E802G (n = 3), E802Q (n = 1) or E802X (n = 1). The sequences containing these mutations were mostly geographically and temporally dispersed suggesting they did not share homology, though there was a small cluster of sequences (E802D, n = 9) from Germany within the same time frame. The 4 sequences with E802A substitution from May 2020 from the same geographic region (United Arab Emirates) but there is no evidence of further transmission. As one of these sequences, hCoV-19/Scotland/CVR2716/2020 was isolated from a patient who was not treated with RDV, this suggests mutation of E802 can arise in the community in the absence of drug selection.

The same pattern of very low observed global frequency of the E802 substitutions was also found at sites known to confer partial RDV resistance in other coronaviruses [20]. There were a handful of sequences with changes at either F480 (n = 13) or V557 (n = 35), and as with the E802 mutations, the sequences were mainly geographically and temporally dispersed. There was a cluster of sequences containing the V557I substitution observed in the Netherlands within a week but there was no information on whether it was the same patient or whether they were treated with RDV. When examining these mutations in respect to the global amino acid diversity along in NSP12 sequence, the region around E802 shows greater diversity than the area around F480 or V557 (S6A Fig). This suggests that strong purifying selection is acting in the regions of these sites, and they maybe more constrained by other dimensions of selection in vivo.

Replacement of NSP6 I168 occurred in 347 sequences with isoleucine replaced with threonine, valine, leucine, glycine or methionine. These data indicate that in absence of widespread selective pressure, substitutions of either NSP12 E802 or NSP6 I168 are currently rare global events. However, the identification of these sequences in the genome databases demonstrate that these viruses are viable and could potentially acquire a resistant phenotype when a stronger selective pressure is applied.

In vitro mutations provide insight into spike adaption

We next focused on those mutations arising in the in vitro passaged virus populations that were likely not directly linked to RDV resistance. The consensus sequences of a subset of the passaged stocks displayed a total of 41 distinct non-synonymous mutations and 10 synonymous mutations across the genome compared to the parental SARS-CoV-2_Engl2 sequence (Fig 4A). Importantly, we did not observe any previously identified mutations in the proof-reading ExoN (NSP14) that would change the sensitivity of the virus to RDV (Fig 4A and S1 Data). Deletions of ExoN have been demonstrated to increase RDV sensitivity for other coronaviruses [20]. While there was clear positive selection pressure across the entire genome (S2 Table), there were no major differences in the number of mutations that accumulated in any specific population, and in the ratio or type of transition vs transversion change (Figs 4B and S6B). Although, Rem2.5p13.5 displayed a slight elevation in non-synonymous changes (Fig 4C), we are unable to draw conclusions on the effect of RDV concentration on virus mutation rate due to small number recovered populations selected in RDV (S1 Text). Further comparative analysis over with the SARS-CoV-2_Wu1 sequence identified a further 2 synonymous and 8 non-synonymous mutations present in the original SARS-CoV-2_Engl2 population (Fig 4A). SARS-CoV-2_Engl2 was a 50:50 mix of two virus populations with 5 of the mutations present at a frequency ~50%, all but one of these became fixed in all passaged populations by p13 (Fig 4A).

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Fig 4. Sequence analysis of continuous in vitro passaged SARS-CoV-2_Engl2.

(A) Alignment of serially passaged viruses and SARS-CoV-2_Engl2 to SARS-CoV-2_Wu1. Non-synonymous (pink) and synonymous (green) changes from Wuhan-1 are highlighted. Light pink are sites fixed at 50% in SARS-CoV-2_Engl2 and black box is a deletion mutation in serially passaged virus. Positions of mutations are indicated, and mutations only found in RDV selected populations are in bold. (B) Synonymous vs non-synonymous changes observed in continually passaged virus populations compare to input SARS-CoV-2_Engl2. (C) Transversion vs transitional changes observed in continually passaged virus populations compare to input SARS-CoV-2_Engl2. (D) Number of in vitro passaged viruses with non-synonymous changes in Spike in comparison to SARS-CoV-2_Engl2. Mutation fixed in the consensus genomes (dark blue) are compared to the total number of viruses with evidence of the mutation at sub-consensus levels (light blue). Amino acid residues in common with the emerging variants of concern (Alpha (UK, B.1.1.7), Gamma (Brazil, P.1); and Beta (South Africa, B.1.351) are highlighted by a star. (E) Worldwide diversity of Spike protein sites of circulating SARS-CoV-2 variants. The average number of different substitutions at each codon is calculated along a 20 amino acid residues wide sliding windows. Data was calculated using only substitutions observed in a minimum of 5 sequences from the publicly available SARS-CoV-2 genomes (n = 1384). The position of the amino acid substitutions in the in vitro passaged viruses are indicated at the bottom, residues are red are shared with variants of concerns, black are specific to in vitro virus, residues in italics were synonymous. Mutations in amino acid residues that are also mutated in the variants of concern Alpha (UK, B.1.1.7), Gamma (Brazil, P.1), & Beta (South Africa, B.1.351) are shown in purple triangles.

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Most of the collective 22 mutations observed in all sequences occurred within the spike (S) open reading frame. All of the in vitro non-synonymous substitutions appeared within multiple lineages the circulating viruses (GISAID data accessed March 2021, S3 Data) with the exception of I68R, N709H and D985G. Unlike observations in other studies [25–27], the furin-like cleavage site was preserved in all but one of the passaged populations. DMSOp13.2 displayed a 24nt deletion of the entire furin-like cleavage site at a high but unfixed frequency of 77% after 13 passages (S1 Data). Importantly, in our in vitro passaged viruses we observed both synonymous and non-synonymous substitutions occurring at the same sites within spike (H69R, E484D, N501T, H655Y, P681P) as those identified in the emerging SARS-CoV-2 variants of concern (Alpha (B.1.1.7): Δ69/70, N501Y, P681H; Gamma (P.1): E484K, N501Y, H655Y; Beta (B.1.351): E484K, N501Y) (Fig 4). Except for synonymous P681P, these substitutions were not present in SARS-CoV-2_Engl2 (S1 Data and S1 Text).

It is important to stress these emerging variants of concern, collectively, share a combination of three amino acid mutations in spike receptor binding domain (RBD): N501Y common to all and K417N and E484K in the Brazil and South African variants. We observe substitutions at 2 out of 3 of these sites in our in vitro evolution lines. The probability of such a large overlap (5 codons) between the substitutions observed in vitro, and variants of concern defining mutations without a common selective pressure driving convergence, was exceptionally small (P = 3.1x10^-5; S6C Fig). This demonstrates commonality in the fitness landscape that these in vitro populations and the circulating lineages are evolving under (driving evolutionary convergence). We further examined the global distribution of all circulating amino acid replacements within spike to determine whether our in vitro substitutions occurred within hot spots for change. There were 1384 replacements observed in a minimum of 5 sequences (n = 242865 sequence up dated 14^th December 2020), many of these were clustered into certain regions within spike, creating visible hot spots of diversity (Fig 4E). For example, the window surrounding amino acid E484 appears to be a relative hot spot for replacement.

Of note are the four spike substitutions which occurred within the receptor binding domain (RBD) at either consensus (E484D, & N501T) or sub-consensus (G413R & Q498H) frequency. The E484 substitution appeared in the consensus sequence of Rem2.5p13.5 and Remp1p13.1 (Fig 4A), but it was present at a frequency of 20–40% in all the other viruses apart from DMSOp13.2 (S1 Data). The N501 substitution was present in one virus at consensus (Mediap13.1) and the sub-consensus of a second (Rem2.5p13.5) (Fig 4D). We noted that some in vitro substitutions in spike were changes to amino acids associated with SARS-CoV-1 spike (Q498H & N501T) adaption. Our data shows these mutations arise in vitro in the absence of any immune selection suggesting intrinsic fitness increases unrelated to immune evasion.

Cell culture

African Green monkey kidney cells VeroE6 expressing ACE-2 (VeroE6-ACE2) alone and with TMPRSS2 (VeroE6-ACE2-TMPRSS2), BHK-21, and A549NPro expressing ACE-2 (A549NPro-ACE2) were grown in DMEM-Glutamax supplemented with 10% fetal calf serum (FCS; Gibco) and non-essential amino acids (NEAA; Gibco). VeroE6 were a kind gift from Professor M. Bouloy (Institut Pasteur, France), BHK were sourced from the ATCC (ATCC CCL-10) and, VeroE6-ACE2, and VeroE6-ACE2-TMPRSS2 have been described elsewhere [66] A549NPro cells (a kind gift from Prof. R. Randall, University of St. Andrews, UK) were transduced to stably express ACE-2 (A549NPro-ACE2) as described [66]. Calu-3, a human lung epithelial cell line was grown in DMEM Glutamax, supplemented with 20%(v/v) FCS and NEAA was sourced from the ATCC (ATCC HTB-55). All cells were maintained at 37°C in 5%(v/v) CO₂, humidified incubator.

Viruses

All initial work with SARS-CoV-2 was undertaken with the clinical isolate SARS-CoV-2_Engl2 strain (hCoV-19/England/02/2020 (England-02), GISAID accession: EPI_ISL_407073) kindly supplied by Public Health England. Validation of the mutations was undertaken in rescued SARS-CoV-2 based on Wuhan-Hu-1 (MN908947) sequence. All virus stocks were grown in VeroE6 cells as described, supplemented with 4%(v/v) FCS, and for the drug screens regardless of cell type the final concentration of FCS was 2%(v/v). For virus growth curves, FCS concentration was decreased to 2%(v/v) in VeroE6 while in Calu-3 FCS concentration was 10%(v/v).

Plaque assays

SARS-CoV-2 titers were determined by plaque assay in either VeroE6 or VeroE6-ACE2-TMPRSS2. Cells were seeded at 2.5 x 10⁵ cells/well in 12-well plates and plaque assays performed as described [66].The plates were fixed in 8% [w/v] formaldehyde in PBS, stained with Coomassie Blue staining solution (0.1%(w/v) Coomassie Brilliant Blue R-250; 45%(v/v) methanol; 10%(v/v) glacial acetic acid) and imaged with a photo scanner (Epson Expression 1680 Pro).

Chemical inhibitors

RDV (GS-5734; HY-104077) and EIDD-2801 (HY-135853) were purchased from MedChemExpress, diluted to final concentration of 10 mM stock solutions in 100% (v/v) dimethyl sulfoxide (DMSO, Sigma) and stored at -80C.

Plasmids, virus rescue and validation of RDV resistant viruses

Reverse genetics plasmids used to rescue SARS-CoV-2_Wu1 have been described previously [66]. Briefly, specific point mutations in SARS-CoV-2 cDNAs in fragment 3 (nt11475 T to C for NSP6 I168T) and fragment 5 (nt 15845 A to C and nt 15846 G to C for NSP12 either E802A, or nt15846 G to C for NSP12 E802D) were synthesized by Biobasic and assembled into the full-length molecular clone to give pCCI-4K-SARS-CoV-2-NSP12_E802D, pCCI-4K-SARS-CoV-2-NSP12_E802A, pCCI-4K-SARS-CoV-2-NSP6_I168T, pCCI-4K-SARS-CoV-2-NSP6_I168T-NSP12_E802D-NSP6, pCCI-4K-SARS-CoV-2-NSP6_I168T-NSP12_E802D-NSP6. The pCCI-4K based plasmids containing the full-length SARS-CoV-2_Wu1 sequence with the mutations were propagated in TransforMax™ EPI300™ Electrocompetent E. coli and purified by using a Macherey Nagel Endotoxin free plasmid purification kit. For virus rescue, BHK-21 cells were transfected with 3 ug of plasmids using Lipofectamine LTX reagent as per manufacturer’s recommendation. Cell culture supernatant was harvested 48h and 72h post transfection (p0) and used to infected VeroE6 to generated p1 stock of the recovered viruses.

SARS-CoV-2 compound sensitivity studies and EC₅₀ calculations

To test for a change in sensitivity to RDV of our continually passaged SARS-CoV-2_Engl2 viruses and reverse genetic derived viruses (rNSP12-E802D, rNSP12-E802A, rNSP6-I168T, rNSP6-I168T-NSP12-E802D, rNSP6-I168T-NSP12-E802A and rSARS-CoV), two different compound screen layouts were tested (S7 Fig). Cells were seeded at 1.2 x 10⁴ cells/well for VeroE6, VeroE6-ACE2, and VeroE6-ACE2-TMPRSS2 and 1.3 x 10⁴ cells/well for A549NPro-ACE2 cell lines in a 96-well format. Two-fold serial dilutions of RDV starting at either 25 or 50 μM were added to the cells and incubated for a minimum of 2h prior to addition of virus. Depending on plate layout, either a set amount of virus or 2-fold serial dilutions of the virus were added, and plates incubated in a 37°C humidified incubator in 5% CO₂ for 72h. For the mixed array plate layout, each drug dilution and virus combination were in triplicate on each plate and repeated twice. Plates were fixed in 8%(w/v) formaldehyde for 1h and stained in Coomassie Brilliant Blue solution. Washed and airdried plates were scanned using the Celigo (Nexcelcom) and the total intensity of each well was determined. The total intensity values from the Celigo scan were used to calculate the EC₅₀ values using Prism GraphPad v8 and v9. Analysis programs used included transformation of concentration (log₁₀), normalization of transformed data and non-linear regression (log(agonist) vs. normalized response—variable slope).

Selection of RDV resistant viruses

Continual passages of SARS-CoV-2 were performed in the presence of RDV in VeroE6 cells. Assays were undertaken in a 12 well plate with each condition in triplicate; two RDV concentrations (1μM and 2.5μM) and two virus inputs (1000 & 2000 pfu) were used, and controls for cell culture adaption of the virus including media alone or 0.2%(v/v) DMSO were undertaken in parallel (Fig 1). These concentrations were kept constant throughout the experiment. Time between each amplification was dependent of visible CPE from passage 1 to passage 5 (p1 to p5), there was up to 7 days between each amplification. For these passages, 100 μl of each amplification was used to generate the subsequent passage. It became evident after passage 4 (p4) that some of the conditions used resulted in loss of infectious virus. After passage 5, 10 μl of culture media was used for passaging the virus in wells that showed evident CPE. Where CPE was not evident, 100 μl of cell culture was blindly passaged. After each passage the cell media was harvested and stored at -80°C. Samples from p4, p7, p10 and p12 were also harvested for titration and cell monolayer was harvested in TrizolLS for future analysis. Prior to sequencing, each condition was tested to determine its ability to grow in the presence of RDV.

Virus replication kinetics

To compare the replication kinetics of the parental stock, cell culture adapted and RDV resistant mutant SARS-CoV-2, monolayers of VeroE6 cells were treated with 7.5μM RDV and infected at a MOI~0.1. The inoculum was removed after 1h, cells washed and fresh media containing either DMSO or RDV added. Growth curves in VeroE6 were undertaken twice in triplicate. rSARS-CoV-2_Wu1 and mutant viruses virus growth was also assessed in Calu3 cells. For this, Calu-3 were seeded 7 days prior to infection, media was changed every 2–3 days prior to infection. The cells were infected with a MOI~0.01 (based on VeroE6-ACE2-TMPRSS2 titres) for 1h, inoculum then aspirated, cells washed, and fresh media added. All infections for virus growth curves were asynchronous. Cell culture media from each well were collected at 24, 48 and 72h post infection (p.i.) and cell-free virus titers were determined by plaque assay in VeroE6 cells. Each experiment was performed at least thrice with a triplicate of the virus each time (mutant viruses n = 3, wt n = 6). Virus titers at each time were determined by plaque assay as described above.

To assess the influence of RDV on the growth of rSARS-CoV-2 and mutant viruses, Calu-3 were seeded 7 days prior to infection in a 24 well format (2 x 10⁵ cells/well), media was changed every 2–3 days. Two hours prior to infection 2-fold serial dilutions of RDV from 2μM to 0.125μM were added to cells. Subsequently, cells were infected at a MOI of 0.01 asynchronously for 1h, inoculum aspirated, and media containing the appropriate amount if RDV added. Samples were taken 24h and 48h pi. Titers were determined by plaque assay. Data from 2 independent virus stocks with 2 replicates except we were missing one of the replicates for wild-type and a 48h pi replicated for rNSP12-E802A at 48h pi.

RNA purification from virus stock

Clarified supernatant from p13 was added to TrizolLS and total RNA was extracted as described [70] for sequence analysis.

Sequence of virus stock using Illumina MiSeq

Sequencing libraries composed of overlapping amplicons across the SARS-CoV-2 genome were prepared utilizing the ARTIC Network protocol (https://artic.network/ncov-2019). Amplicons were paired-end sequenced (2x250 nt) on an Illumina MiSeq as described previously [71]. Reads were trimmed with trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and then mapped with Burrows–Wheeler Aligner [72] to the SARS-CoV-2_Wu1 (MN908947) genome, followed by amplicon primer trimming and consensus calling with iVar [73] using a minimum read coverage of 10. Consensus sequences and raw FASTQ files have been uploaded to GenBank under BioProject number PRJNA692078.

Co-infection competition assay

VeroE6-ACE2-TMPRSS2 were co-infected with different ratios (1:9, and 9:1) of rNSP12E802D and rSARS-CoV-2 WT to assess virus fitness as described by Agostini et al., (2018) [20]. Briefly, a 1:9 ratio was equivalent of MOI ~ 0.01 to a MOI of ~0.09, cells were infected for 1 h at 37°C, media aspirated, cell washed, and fresh media added. Twenty-four h post-infection, virus supernatant was harvested, and 100 μl (10% of the volume) was used to infected new VeroE6-ACE2-TMPRSS2 and the infection procedure repeated for 3 passages. RNA was extracted from the clarified supernatant after heat inactivation at 56°C for 20 min using Beckman Coulter RNAdvance Blood kit and KingFisher automated extraction system as per manufacturer’s protocols.

Quantification of SARS-CoV-2 genome copies

To examine the virus growth kinetic during the competition assay, the viral load was determined by quantitative RT-PCR based on copies of N RNA transcript. All samples were quantified using a NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs, E3006) and a previously described CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time reverse transcription PCR Panel [74,75]. 2019-nCoV N1 forward primer 5’-GAC CCC AAA ATC AGC GAA AT-3’, 2019-nCoV N1 reverse primer 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’ and 2019-nCoV N1 probe 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’ were part of the SARS-CoV-2 (2019-nCoV) CDC RUO Primers and Probes kit (IDT Technologies, 10006713). SARS-CoV-2 N RNA was quantified using a ten-fold serial dilutions of an in-house SARS-CoV-2 RNA standard to enable genome equivalent (GE) copies/ml as the is a high correlation between copy number of N and ORF1ab as targets [76] from SARS-CoV-2 RNA extracted from supernatant. All reactions were performed on ABI 7500 Fast thermal cycler.

Sequence of virus stock using GridION (Oxford Nanopore Technology)

The GridION system was used to sequence the virus populations used for the competition assay as previously described [71]. GridION reads were processed using the ARTIC bioinformatics pipeline (https://github.com/artic-network/artic-ncov2019). Briefly, reads were size filtered then aligned to the Wuhan-Hu-1 reference (MN908947) with Minimap2 [77] followed by post-alignment amplicon primer trimming and variant calling with Nanopolish (https://github.com/jts/nanopolish).

Modelling of mutations in SARS-CoV-2 NSP12

UCSF Chimera [78] to generate the ribbon structure and rotamer function [79] used to model the mutations into SARS-CoV-2_Wu1 NSP12 (PDB 6YYT [39]). Protter [80] was used visualize the effect of the I168T mutation on organization of the transmembrane domains in NSP6.

Statistics and bioinformatic

GraphPad Prism v8 & v9 software (La Jolla, CA, USA) was used to undertake statistical analysis (transformation, normalization, non-linear regression, RM one-way ANOVA (Dunn Dunnett’s multiple comparisons test), two-way ANOVA, unpaired t-test (Holm-Šídák method)) and generate figures. EMBL-EBI Clustal omega [81] was used to align the sequences of NSP6 and NSP12 from SARS-CoV-2 Wuhan1 (MN908947), betacoronavirus England1 (bCoV England1; YP009944302 & YP009944297.1), SARS-CoV-2_Engl2, SARS-CoV Tor (NP_828849.7 & YP009944371.1), human coronavirus (hCoV, HKU1, YP459941.1 & YP009944274.1), middle eastern respiratory virus (MERS; YP009047223.1 &009047234.1), murine hepatitis virus (MHV; YP009924352.1 & 009915678.1), rat coronavirus (Parker, YP009924378.1 & YP_009924378.1), Rousettus bat coronavirus (HKU9; YP009924393.1 & YP009924388.1), rabbit CoV (HKU14; YP009924419.1 & YP009924414.1), Tylonycteris bat CoV (HKU4 YP009944320.1 & YP009944315.1), and Pipostrellus bat CoV(HKU5; YP009944349.2 & YP009944344.1). The online database CoV-GLUE (http://cov-glue.cvr.gla.ac.uk/[82]) was used to monitor for replacement in the SARS-CoV-2 genome. The database was accessed on 10th of January 2021 (n = 242865) for generating the diversity plot and further on 15^th of March 2021 n = 426623). The dN/dS ratios were calculated for each sample using all ORFs concatenated together, as well as the Spike and entire ORF1ab sequences individually (with respect to the SARS-CoV-2_Engl2 sample to monitor within passage evolution), using the program SNAP (https://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html)[83].

The code used to calculate the null distribution and the probability as well as the global distribution of the mutations within in Spike can be assessed at https://github.com/omaclean/CV19IV/.

The receptor binding domain of Spike is from amino acids 319–541: B1.351 and P1 had 3 mutations in the RBD (K417N/T, E484K and N501Y) while Alpha (B.1.1.7) only had one (N501Y). We used the following amino acids replacements and deletions in Spike for each lineage-based COG-UK defined changes for each lineage; Beta (B1.351; L18F, D80A, D215G, R246L, K417N, E484K, N501Y, A701V), Gamma (P.1; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y H655Y, T1027I), Alpha (B.1.1.7; Δ69–70, D144, N501Y, A570D, P681H, T716I, S982A, D1118H). We further included the substitution observed in P1 (V1176F) and deletion in B1.351 (Δ242–245). There is phylogenetic uncertainty if V1176F was present in the ancestor preceding the formation of the Gamma (P.1) lineage, the deletion (Δ242–245; like other defining mutations) in Beta (B.1.351) is polymorphic. We erred on the side of inclusivity in the face of this uncertainty, making our analysis more conservative. All sequences available sequences for SARS-CoV-2 were assessed from CoV-Glu (n = 242865, last update 14^th December 2020).