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BACKGROUND: The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. RESULTS: Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. CONCLUSIONS: These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19. Accumulation of mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp may facilitate antigenic drift, generating favipiravir resistance. Focussing on the chain-termination mechanism utilized by favipiravir, we used high-throughput interface-based protein design to generate > 100 000 designs of the favipiravir-binding site of RdRp and identify mutational hotspots. We identified several single-point mutants and designs having a sequence identity of 97%-98% with wild-type RdRp, suggesting that SARS-CoV-2 can develop favipiravir resistance with few mutations. Out of 134 mutations documented in the CoV-GLUE database, 63 specific mutations were already predicted as resistant in our calculations, thus attaining Eœ 47% correlation with the sequencing data. These findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir.

Lung inflammation is a hallmark of coronavirus disease 2019 (COVID-19). In this study, we show that mice develop inflamed lung tissue after being administered exosomes released from the lung epithelial cells exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp12 and Nsp13 (exosomesNsp12Nsp13). Mechanistically, we show that exosomesNsp12Nsp13 are taken up by lung macrophages, leading to activation of nuclear factor κB (NF-κB) and the subsequent induction of an array of inflammatory cytokines. Induction of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β from exosomesNsp12Nsp13-activated lung macrophages contributes to inducing apoptosis in lung epithelial cells. Induction of exosomesNsp12Nsp13-mediated lung inflammation was abolished with ginger exosome-like nanoparticle (GELN) microRNA (miRNA aly-miR396a-5p. The role of GELNs in inhibition of the SARS-CoV-2-induced cytopathic effect (CPE) was further demonstrated via GELN aly-miR396a-5p- and rlcv-miR-rL1-28-3p-mediated inhibition of expression of Nsp12 and spike genes, respectively. Taken together, our results reveal exosomesNsp12Nsp13 as potentially important contributors to the development of lung inflammation, and GELNs are a potential therapeutic agent to treat COVID-19., Graphical Abstract, COVID-19 is a disease affecting the pulmonary system with devastating inflammation. Teng et al. have shown that exosomes released from SARS-CoV-2-infected lung epithelia cells are preferentially taken up by lung macrophages that subsequently induce lung inflammation. Ginger exosome-like nanoparticle miRNA inhibits SARS-CoV-2-induced lung inflammation and viral replication.

COVID-19 is the causative agent for the ongoing pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

Markov models from Folding@home simulations of COVID19 related proteins. Systems include, SARS-CoV-2: Spike, NSP3, NSP5, NSP7, NSP8, NSP9, NSP10, NSP12, NSP13, NSP14, NSP15, NSP16, Nucleoprotein Receptor Binding Domain, Nucleoprotein Dimerization Domain, SARS-CoV-1: Spike, HCoV-NL63: Spike, Human: ACE2, IL6, IL6-R

SARS‐CoV‐2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive‐sense single‐stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA‐dependent‐RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or Food and Drug Administration‐approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

In recent years, humanity has had to face a critical pandemic due to SARS-CoV-2. In the rapid search for effective drugs against this RNA-positive virus, the repurposing of already existing nucleotide/nucleoside analogs able to stop RNA replication by inhibiting the RNA-dependent RNA polymerase enzyme has been evaluated. In this process, a valid contribution has been the use of in silico experiments, which allow for a rapid evaluation of the possible effectiveness of the proposed drugs. Here we propose a molecular dynamic study to provide insight into the inhibition mechanism of Penciclovir, a nucleotide analog on the RNA-dependent RNA polymerase enzyme. Besides the presented results, in this article, for the first time, molecular dynamic simulations have been performed considering not only the RNA-dependent RNA polymerase protein, but also its cofactors (fundamental for RNA replication) and double-strand RNA.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-β (IFN-β) activation in IFN-β promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-β promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-β production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-β promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-β antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-β activation. However, most of these results were generated from IFN-β promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-β promoter luciferase activity, it showed no inhibitory effect on IFN-β production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-β signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

The SARS-CoV-2 belongs to Coronaviridae family infects host cells by the interaction of its spike glycoprotein and angiotensin-converting enzyme 2 (ACE 2) of host cells. Upon entry, the virus uses its RNA dependent RNA polymerase (NSP12) for transcribing its genome to survive in the cell and spread its infection. The protein sequences of receptor-binding domain (RBD) of spike glycoprotein, and NSP12 exhibits high homology in the family of Coronoviridae and are ideal candidates for the development of anti-coronaviral drugs. In the quest to identify inhibitory molecules against these proteins, we searched several molecules that are present in naturally occurring medicinal plants database. Andrographolide which is largely present in the leaf extracts of Andrographis paniculata (AP) and is known to exhibit antiviral, antibacterial, and stabilizes Th1/Th2/Th17 responses; taking this clue, we used in silico approaches to see the binding of andrographolide to RBD and NSP12 molecules. Our docking results showed very strong affinity of andrographolide to RBD and NSP12 of the SARS-CoV-2 virus with dock scores of −10.3460 for RBD and −10.7313 for NSP12 indicating andrographolide acts as an inhibitor of RBD and NSP12. These unique properties of andrographolide, AP extract, can be tested as anti-coronaviral drug.

As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

Coronavirus Disease 2019 (COVID-19) is a deadly emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 is easily transmitted through the air and has a relatively long incubation time, COVID-19 has rapidly developed into a global pandemic. As there are no antiviral agents for the prevention and treatment of this severe pathogen except for remdesivir, development of antiviral therapies to treat infected individuals remains highly urgent. Here, we showed that baicalein and baicalin exhibited significant antiviral activity against SARS-CoV-2, the causative agent of COVID-19 through in vitro studies. Our data through cell-based and biochemical studies showed that both compounds act as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors directly and inhibit the activity of the SARS-CoV-2 RdRp, but baicalein was more potent. We also showed specific binding of baicalein to the SARS-CoV-2 RdRp, making it a potential candidate for further studies towards therapeutic development for COVID-19 as a selective non-nucleoside polymerase inhibitor.

Corona Virus 2019 Disease (COVID-19) is a rapidly emerging pandemic caused by a newly discovered beta coronavirus, called Sever Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2). SARS CoV-2 is an enveloped, single stranded RNA virus that depends on RNA-dependent RNA polymerase (RdRp) to replicate. Therefore, SARS CoV-2 RdRp is considered as a promising target to cease virus replication. SARS CoV-2 polymerase shows high structural similarity to Hepatitis C Virus-1b genotype (HCV-1b) polymerase. Arising from the high similarity between SARS CoV-2 RdRp and HCV NS5B, we utilized the reported small-molecule binders to the palm subdomain of HCV NS5B (genotype 1b) to generate a high-quality DEKOIS 2.0 benchmark set and conducted a benchmarking analysis against HCV NS5B. The three highly cited and publicly available docking tools AutoDock Vina, FRED and PLANTS were benchmarked. Based on the benchmarking results and analysis via pROC-Chemotype plot, PLANTS showed the best screening performance and can recognize potent binders at the early enrichment. Accordingly, we used PLANTS in a prospective virtual screening to repurpose both the FDA-approved drugs (DrugBank) and the HCV-NS5B palm subdomain binders (BindingDB) for SARS CoV-2 RdRp palm subdomain. Further assessment by molecular dynamics simulations for 50 ns recommended diosmin (from DrugBank) and compound 3 (from BindingDB) to be the best potential binders to SARS CoV-2 RdRp palm subdomain. The best predicted compounds are recommended to be biologically investigated against COVID-19. In conclusion, this work provides in-silico analysis to propose possible SARS CoV-2 RdRp palm subdomain binders recommended as a remedy for COVID-19. Up-to-our knowledge, this study is the first to propose binders at the palm subdomain of SARS CoV2 RdRp. Furthermore, this study delivers an example of how to make use of a high quality custom-made DEKOIS 2.0 benchmark set as a procedure to elevate the virtual screening success rate against a vital target of the rapidly emerging pandemic., Graphical abstract Image 1

COVID-19 has emerged as a rapidly escalating serious global health issue, affecting every section of population in a detrimental way. Present situation invigorated researchers to look for potent targets, development as well as repurposing of conventional therapeutic drugs. NSP12, a RNA polymerase, is key player in viral RNA replication and, hence, viral multiplication. In our study, we have screened a battery of FDA-approved drugs against SARS-CoV-2 RNA polymerase using in silico molecular docking approach. Identification of potent inhibitors against SARS-CoV-2 NSP12 (RNA polymerase) were screeened from FDA approved drugs by virtual screening for therapeutic applications in treatment of COVID-19. In this study, virtual screening of 1749 antiviral drugs was executed using AutoDock Vina in PyRx software. Binding affinities between NSP12 and drug molecules were determined using Ligplot+ and PyMOL was used for visualization of docking between interacting residues. Screening of 1749 compounds resulted in 14 compounds that rendered high binding affinity for NSP12 target molecule. Out of 14 compounds, 5 compounds which include 3a (Paritaprevir), 3d (Glecaprevir), 3h (Velpatasvir), 3j (Remdesivir) and 3l (Ribavirin) had a binding affinity of − 10.2 kcal/mol, −9.6 kcal/mol, − 8.5 kcal/mol, − 8.0 kcal/mol and − 6.8 kcal/mol, respectively. Moreover, a number of hydrophobic interactions and hydrogen bonding between these 5 compounds and NSP12 active site were observed. Further, 3l (Ribavirin) was docked with 6M71 and molecular dynamic simulation of the complex was also performed to check the stability of the conformation. In silico analysis postulated the potential of conventional antiviral drugs in treatment of COVID-19. However, these finding may be further supported by experimental data for its possible clinical application in present scenario.

In this study, the Nsp12–Nsp8 complex of SARS-CoV-2 was targeted with structure-based and computer-aided drug design approach because of its vital role in viral replication. Sequence analysis of RNA-dependent RNA polymerase (Nsp12) sequences from 30,366 different isolates were analysed for possible mutations. FDA-approved and investigational drugs were screened for interaction with both mutant and wild-type Nsp12–Nsp8 interfaces. Sequence analysis revealed that 70.42% of Nsp12 sequences showed conserved P323L mutation, located in the Nsp8 binding cleft. Compounds were screened for interface interaction, any with XP GScores lower than −7.0 kcal/mol were considered as possible interface inhibitors. RX-3117 (fluorocyclopentenyl cytosine) and Nebivolol had the highest binding affinities in both mutant and wild-type enzymes, therefore they were selected and resultant protein–ligand complexes were simulated for analysis of stability over 100 ns. Although the selected ligands had partial mobility in the binding cavity, they were not removed from the binding pocket after 100 ns. The ligand RX-3117 remained in the same position in the binding pocket of the mutant and wild-type enzyme after 100 ns MD simulation. However, the ligand Nebivolol folded and embedded in the binding pocket of mutant Nsp12 protein. Overall, FDA-approved and investigational drugs are able to bind to the Nsp12–Nsp8 interaction interface and prevent the formation of the Nsp12–Nsp8 complex. Interruption of viral replication by drugs proposed in this study should be further tested to pave the way for in vivo studies towards the treatment of COVID-19. Communicated by Ramaswamy H. Sarma

SARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-β promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.

Patients with signs of COVID-19 were tested with CDC approved diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from nasopharyngeal/nasal swabs. In order to determine the variants of SARS-CoV-2 circulating in the state of Nevada, 200 patient specimens from positively identified cases were sequenced through our robust protocol for sequencing SARS-CoV-2 genomes from the nasopharyngeal or nasal swabs. This protocol enabled the identification of specific nucleotide variants including those coding for D614G and clades defining mutations. Additionally, these sequences were used for determining the phylogenetic relationships of SARS-CoV-2 genomes of public health importance occurring in the state of Nevada. Our study reports the occurrence of a novel variant in the nsp12 (RdRp-RNA dependent RNA Polymerase) protein at residue 323 (314aa of orf1b) to Phenylalanine (F) from Proline (P), present in the original isolate of SARS-CoV-2 (Wuhan-Hu-1). This 323F variant is found at a very high frequency (46% of the tested specimen) in Northern Nevada, possibly because the virus accumulated this mutation while circulating in the community and the shelter in place orders restricted the introduction and spread of other variants into this region. Structural modeling of the RdRp with P323F variant did not show any significant difference in protein conformation, but the phenotypic effect is unknown and an area of active investigation. In conclusion, our results highlight the introduction and spread of specific SARS-CoV-2 variants at very high frequency within a distinct geographic location that is important for clinical and public health perspectives in understanding the evolution and transmission of SARS-CoV-2.IMPORTANCESARS-COV-2 genomes accumulate nucleotide mutations while passing in the human population and these mutations may confer phenotypic differences including altered immune response and anti-viral drug resistance. We developed a robust workflow to sequence SARS-CoV-2 directly from the nasal/nasopharyngeal swabs containing even a very low viral loads (>35 Ct value samples). Our protocol does not rely on amplicon based sequencing strategies nor the need of passing the virus into tissue culture thus reduces the possibility of an introduction of laboratory-adapted mutations. Sequences of SARS-CoV-2 from the patients of the state of Nevada during early months of the pandemic identified a rare mutation in the RdRp protein (P323F). This mutation occurred at a very high frequency in the variants of SARS-CoV-2 circulating Northern Nevada. Identification of such variants is important for clinical and public health perspectives in understanding transmission mediated evolution of SARS-CoV-2 variants and their implications on therapeutics and diagnostics.

The rapid development of the SARS-CoV-2 mediated COVID-19 pandemic has been the cause of significant health concern, highlighting the immediate need for effective antivirals. SARS-CoV-2 is an RNA virus that has an inherently high mutation rate. These mutations drive viral evolution and genome variability, thereby facilitating viruses to have rapid antigenic shifting to evade host immunity and to develop drug resistance. Viral RNA-dependent RNA polymerases (RdRp) perform viral genome duplication and RNA synthesis. Therefore, we compared the available RdRp sequences of SARS-CoV-2 from Indian isolates and the ‘Wuhan wet sea food market virus’ sequence to identify, if any, variation between them. Our data revealed the occurrence of seven mutations in Indian isolates of SARS-CoV-2. The secondary structure prediction analysis of these seven mutations shows that three of them cause alteration in the structure of RdRp. Furthermore, we did protein modelling studies to show that these mutations can potentially alter the stability of the RdRp protein. Therefore, we propose that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.