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Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

protein was generated by inserting a GSGSGS linker between nsp7-and nsp8-coding sequences. Cofactors were expressed in NEB Express C2523 (New England Biolabs) cells carrying the pRare2LacI (Novagen) plasmid in the presence of Ampicillin (100 µM/mL) and Chloramphenicol (17 µg/mL). Protein expression was induced with 100 µM IPTG once the OD600 = 0.5-0.6, and expressed overnight at 17°C. Protein was purified first through affinity chromatography with HisPur Cobalt resin (Thermo Scientific), with a lysis buffer containing 50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM Imidazole, supplemented with 20 mM MgSO4, 0.25 mg/mL Lysozyme, 10 µg/mL DNase, 1 mM PMSF, with lysis buffer supplemented with 250 mM imidazole. Eluted protein was concentrated and dialyzed overnight in the presence of histidine labeled TEV protease (1:10 w/w ratio to TEV:protein) for removal of the protein tag. Cleaved protein was purified through a second cobalt column and protein was purified through size exclusion chromatography (GE, Superdex S200) in gel filtration buffer (25 mM HEPES pH 8, 150 mM NaCl, 5mM MgCl2, 5 mM TCEP). Concentrated aliquots of protein were flash-frozen in liquid nitrogen and stored at -80°C. A synthetic, codon-optimized SARS-CoV nsp12 gene (DNA 2.0) bearing C-terminal 8His-tag preceded by a TEV protease cleavage site was expressed from a pJ404 vector (DNA 2.0) in E. coli strain BL21/pG-Tf2 (Takara). Cells were grown at 37°C in the presence of Ampicillin and Chloramphenicol until OD600 reached 2. Cultures were induced with 250 µM IPTG and protein expressed at 17°C overnight. Purification was performed as above in lysis buffer supplemented with 1% CHAPS. Two additional wash steps were performed prior to elution, with buffer supplemented with 20 mM imidazole and 50 mM arginine for the first and second washes respectively. Polymerase was eluted using lysis buffer with 500 mM imidazole and concentrated protein was purified through gel filtration chromatography (GE, Superdex S200) in the same buffer as for nsp7L8. Collected fractions were concentrated and supplemented with 50% glycerol final concentration and stored at -20 °C.

Immunofluorescence assay
Huh7-hACE2 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented with 1% FBS. 1.5 h before the viral inoculation, the tested compounds were added to the wells in triplicate. The infection proceeded for 24 h without the removal of the viruses or the compounds. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific). Hoechst 33342 was added in the final step to counterstain the nuclei. Fluorescence images of approximately ten thousand cells were acquired per well with a 10x objective in a Cytation 5 (BioTek). The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).

SARS-CoV-2 virus production and characterization
SARS-CoV-2 WT and nsp14 exoribonuclease knockout viruses were prepared using a SARS-CoV-2 infectious clone (4). Briefly, viral RNA was obtained by in vitro RNA transcription, and 40 μg RNA transcripts and 20 μg N gene RNA were co-electroporated into 8×10 6 Vero E6 cells using Gene Pulser XCell electroporation system (Bio-Rad, Hercules, CA) at a setting of 270 V and 950 μF with a single pulse. The electroporated cells were seeded to a T75 flask and immediately transfer to BSL-3 facility. Viral production was confirmed by RT-PCR. The supernatants of electroporated cells were harvested and centrifuged at 1,000 g for 10 min to remove cell debris. 250 μl supernatant was added and mixed thoroughly with 1 ml of TRIzol LS reagent (Thermo Fisher Scientific). RNA was extracted according to the manufacture's instruction and resuspended in 20 μl of nuclease-free water. RT-PCR was performed using the SuperScript® IV One-Step RT-PCR kit (Thermo Fisher Scientific).
Virus was determined by plaque assay. Approximately 1.2×10 6 Vero E6 cells were seeded to each well of a 6-well plate. The viruses were 10-fold serially diluted with 2% FBS DMEM medium and 200 μl of virus dilution was transferred to each well of the 6-well plate. After the incubation for 1 h at 37°C, 2 ml of overlay medium containing 2% FBS DMEM medium and 1% sea-plaque agarose (Lonza, Walkersville, MD), was added to the infected cells per well. After a 2-day incubation, another 2 ml of overlay medium with neutral red (final concentration 0.01%) was added onto the first overlay. After 12 h incubation, the plates were sealed with Breath-Easy sealing membrane (Sigma-Aldrich, St. Louis, MO) and plaques were counted.

SARS-CoV-2 luciferase replicon assay
SARS-CoV-2 transient luciferase replicon assay was performed as previously described (5). WT and mutant replicon RNA, and N gene mRNA were obtained through T7 in vitro transcription, and 40 μg RNA transcripts and 20 μg N gene RNA were co-electroporated into 8×10 6 Huh-7 cells using Gene Pulser XCell electroporation system (Bio-Rad) at a setting of 270V and 950 μF with a single pulse. After 10 min recovery, electroporated cells were seeded to 24-well plates, and harvested at indicated timepoints. Luciferase signal was measured using Renilla luciferase assay system (Promega) and read by Cytation 5 (Bio Tek) according to the manufacturer's protocols.

Construct fabrication.
The fabrication of the RNA hairpin has been described in detail in Ref. (6). The RNA hairpin is made of a 499 bp double-stranded RNA stem terminated by a 20 nt loop that is assembled from three ssRNA annealed together, and two handles, one of 856 bp at the 5' end and one 822 bp at the 3' end. The handles include either a 343 nt digoxygenin-labeled ssRNA or a 443 nt biotin-labeled ssRNA. Upon applied force above ~21 pN, the hairpin opens and frees a 1043 nt ssRNA template for SARS-CoV-2 replication. To obtain the different parts of the RNA construct, template DNA fragments were amplified via PCR, purified (Monarch PCR and DNA cleanup kit) and in vitro transcribed (NEB HiScribe™ T7 High Yield RNA Synthesis Kit). Transcripts were then treated with Antarctic Phosphatase and T4 Polynucleotide Kinase. RNAs were purified using the RNA Clean & Concentrator-25 kit (Zymo Research). Individual RNA fragments were annealed and ligated with T4 RNA ligase 2 (NEB) to assemble the RNA hairpin.

Stochastic-pausing model
The model is described in detail in (7)(8)(9). There are many kinetic models that are consistent with the empirical dwell-time distributions we observe, and we here work under the assumption that the probability of pausing is low enough that there is only one rate-limiting pause in each dwell-time window. This assumption washes out most details of the kinetic scheme that connects pauses and nucleotide addition, but allows us to determine the general form of the dwell-time distribution without specifying how the pauses are connected to the nucleotide addition pathway ( "# #)* + a +' 2(1 + t/1s) ,/. 7.
(Eq. 1) In the above expression, the gamma function in the first term contributes the portion /0 of dwell times that originate in the RdRp crossing the dwell time window of size 12 base pairs without pausing; the second term is a sum of contributions originating in pause-dominated transitions, each contributing a fraction / of dwell times; the third term captures the asymptotic power-law decay (amplitude +' ) of the probability of dwell-times dominated by a backtrack. The backtracked asymptotic term needs to be regularized for times shorter than the diffusive backtrack step. We have introduced a regularization at 1 s, but the precise timescale does not matter, as long as it is set within the region where the exponential pauses dominate over the backtrack. From left to right, each term of Equation 1 is dominating the distribution for successively longer dwell-times. A cut-off factor ( ) for short times is introduced to account for the fact that the dwell time window includes 12 nucleotide-addition steps, The fit results dependence on these cut-offs is negligible as long as they are introduced in regions where the corresponding term is sub-dominant. Here the cut is placed under the center of the elongation peak, guaranteeing that it is placed where pausing is sub-dominant.

Maximum likelihood estimation
The normalized version of Equation 1 is the dwell time distribution fit to the experimentally collected dwell-times { 4 } 4 by minimizing the likelihood function (10) = − ∑ ln 12 ( 4 ) 4 (Eq. 2) with respect to rates and probabilistic weights.

Dominating in a dwell-time window vs. dominating in one step
The fractions / represent the probability that a particular rate / dominates the dwell-time. We want to relate this to the probability / that a specific exit rate dominates within a one-nt transcription window. Assuming we have labelled the pauses so that /%* > / , we can relate the probability of having rate dominating in 12 steps to the probability of having it dominate in one step through 3) The first term in Equation 3 represents the probability of having no pauses longer than the 78 pause in the dwell-time window, and the second term represents the probability of having no pauses longer than the ( − 1) 78 pause. The difference between the two terms is the probability that the 78 pause will dominate. This can be inverted to yield a relation between the single-step probabilities ( / ) and the dwell-time window probabilities ( / ) This relationship has been used throughout the manuscript to relate our fits over a dwell time window to the single-step probabilities.

Maximum likelihood estimation (MLE) fitting routine
The above stochastic-pausing model was fit to the dwell time distributions using a custom Python 3.7 routine. Shortly, we implemented a combination of simulated annealing and bound constrained minimization to find the parameters that minimize Equation 2. We calculated the statistical error on the parameters by applying the MLE fitting procedure on 100 bootstraps of the original data set (11), and reported the standard deviation for each fitting parameters.

Competition between obligatory terminator nucleotide analogues and their natural nucleotide homologues
Starting with an empty active site (E), we assume that there is direct binding competition between the natural nucleotide (N) and the nucleotide analogue terminator (T, simply coined terminator) that result in either the former bound (Nb) or the latter bound (Tb) to the active site. This can be rewritten as In the above is the relative stoichiometry between T and N, while is the relative effective incorporation rates of N and T at equimolar conditions.
On an infinite construct, polymerization will proceed until the first T is incorporated, after which it terminates. At termination, the product has incorporated − 1 Ns, and finally one T, with probability If the construct only allows for the addition of Ns and Ts, the average number of Ns and Ts in the product will instead be For a genome of length , with the relative abundance of templating bases for N and T, we thus expect there to be at most = Ns and Ts incorporated at termination. At termination the product then has the average length