The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.     

D614G substitution at the hinge region enhances the stability of trimeric SARS-CoV-2 spike protein

Mutations in the spike protein of SARS-CoV-2 are the major causes for the modulation of ongoing COVID-19 infection. Currently, the D614G substitution in the spike protein has become dominant worldwide. It is associated with higher infectivity than the ancestral (D614)variant. We demonstrate using Gaussian network model-based normal mode analysis that the D614G substitution occurs at the hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Computer-aided mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated. However, contacts involving G614 are energetically favourable, implying the substitution strengthens residue contacts that are formed within as well as between protomers. We also find that the free energy difference (ΔΔG) between two variants is -2.6 kcal/mol for closed and -2.0 kcal/mol for 1-RBD up conformation. Thus, the thermodynamic stability has increased upon D614G substitution. Whereas the reverse mutation in spike protein structures having G614 substitution has resulted in the free energy differences of 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations, respectively, indicating that the overall thermodynamic stability has decreased. These results suggest that the D614G substitution modulates the flexibility of spike protein and confers enhanced thermodynamic stability irrespective of conformational states. This data concurs with the known information demonstrating increased availability of the functional form of spikeprotein trimer upon D614G substitution.     

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.     

Structural effects of spike protein D614G mutation in SARS-CoV-2

A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2. D614G mutation induces structural changes in the spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on S-protein structure. The introduction of the mutation orders the 630-loop structure and thereby induces global structural changes toward the cryoelectron microscopy structure of the D614G S-protein. The ordered 630-loop weakens local interactions between the 614^th residue and others in contrast to disordered structures in the wild-type protein. The mutation allosterically alters global interactions between receptor-binding domains, forming an asymmetric and mobile down conformation and facilitating transitions toward up conformation. The loss of salt bridge between D614 and K854 upon the mutation generally stabilizes S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding the molecular basis of D614G mutation is crucial as it dominates in all variants of concern, including Delta and Omicron.     

Making sense of spike D614G in SARS-CoV-2 transmission

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the etiologic agent of the current coronavirus disease 2019(COVID-19) pandemic, has evolved to adapt to human host and transmission over the past 12 months. One prominent adaptive mutation is the asparagine-to-glycine substitution at amino acid position 614 in the viral spike protein(D614G), which has become dominant in the currently circulating virus strains. Since spike protein determines host ranges, tissue tropism, and pathogenesis through binding to the cellular receptor of angiotensin converting enzyme 2(ACE2), the D614G mutation is hypothesized to enhance viral fitness in human host, leading to increased transmission during the global pandemic. Here we summarize the recent progress on the role of the D614G mutation in viral replication, pathogenesis, transmission, and vaccine and therapeutic antibody development. These findings underscore the importance in closely monitoring viral evolution and defining their functions to ensure countermeasure efficacy against newly emerging variants.     

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline “S2P” spike—widely employed for laboratory work and clinical studies—unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.     

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.     

Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA

Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called “synthetic mismatch integrated crRNA guided Cas12a detection” (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.     

Mutational sensitivity of D614G in spike protein of SARS-CoV-2 in Jordan

BackgroundSpike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human respiratory cells causing COVID-19 disease.AimHere, we aimed to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 from 20 Jordanian nasopharyngeal samples and to determine the percentage of single amino acid variants (SAV) in the spike protein of SARS-CoV-2.MethodsThe output of the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) found four single amino acid variants in the spike gene.ResultsThe first variant represented by 5% of samples that showed tyrosine deletion at Y144 located in the N terminal domain. The second and the dominant variant, represented by 62%, showed aspartate a coil amino acid substitution to glycine an extracellular amino acid at D614G located in the spike recognition binding site. The third variant, represented by 5%, showed aspartate substitution to tyrosine at D1139Y, and the fourth variant, represented by 5% glycine substitution to serine at G1167S.ConclusionOur results have shown low mutational sensitivity in all variants except to D614G the one with the most likely neutral mutational sensitivity that all variants might not explicitly affect the function of spike glycoprotein. However, D614G might change the viral conformational plasticity and hence a potential viral fitness gain but one must be cautious about drawing any concrete conclusions about the severity of symptoms and viral transmission from genomic data only.General significanceStudying mutations such as D614G in deep is essential to control the pandemic in terms of immune systems, antibodies, or even vaccines.     

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203–205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral ‘RG’ motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2’s continued adaptation to human infection.     

Mechanical activation of spike fosters SARS-CoV-2 viral infection

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike’s S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike’s receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~10³ times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 10⁶ times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.Subject terms: Molecular biology, Structural biology     

The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD’s class 1 and class 2 epitopes, including sites 417, 478, and 484–486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.     

Prefusion spike protein conformational changes are slower in SARS-CoV-2 than in SARS-CoV-1

Within the last 2 decades, severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) have caused two major outbreaks; yet, for reasons not fully understood, the coronavirus disease 2019 pandemic caused by SARS-CoV-2 has been significantly more widespread than the 2003 SARS epidemic caused by SARS-CoV-1, despite striking similarities between these two viruses. The SARS-CoV-1 and SARS-CoV-2 spike proteins, both of which bind to host cell angiotensin-converting enzyme 2, have been implied to be a potential source of their differential transmissibility. However, the mechanistic details of prefusion spike protein binding to angiotensin-converting enzyme 2 remain elusive at the molecular level. Here, we performed an extensive set of equilibrium and nonequilibrium microsecond-level all-atom molecular dynamics simulations of SARS-CoV-1 and SARS-CoV-2 prefusion spike proteins to determine their differential dynamic behavior. Our results indicate that the active form of the SARS-CoV-2 spike protein is more stable than that of SARS-CoV-1 and the energy barrier associated with the activation is higher in SARS-CoV-2. These results suggest that not only the receptor-binding domain but also other domains such as the N-terminal domain could play a crucial role in the differential binding behavior of SARS-CoV-1 and SARS-CoV-2 spike proteins.     

Non-synonymous mutations of SARS-CoV-2 leads epitope loss and segregates its variants

The non-synonymous mutations of SARS-CoV-2 isolated from across the world have been identified during the last few months. The surface glycoprotein spike of SARS-CoV-2 forms the most important hotspot for amino acid alterations followed by the ORF1a/ORF1ab poly-proteins. It is evident that the D614G mutation in spike glycoprotein and P4715L in RdRp is the important determinant of SARS-CoV-2 evolution since its emergence. P4715L in RdRp, G251V in ORF3a and S1498F of Nsp3 is associated with the epitope loss that may influence pathogenesis caused by antibody escape variants. The phylogenomics distinguished the ancestral viral samples from China and most part of Asia, isolated since the initial outbreak and the later evolved variants isolated from Europe and Americas. The evolved variants have been found to predominant globally with the loss of epitopes from its proteins. These have implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.     

SARS‐CoV‐2 Alpha,Beta, and Delta variants display enhanced Spike‐mediated syncytia formation

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation.     

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.     

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Modeling the onset of symptoms of COVID-19: Effects of SARS-CoV-2 variant

Identifying order of symptom onset of infectious diseases might aid in differentiating symptomatic infections earlier in a population thereby enabling non-pharmaceutical interventions and reducing disease spread. Previously, we developed a mathematical model predicting the order of symptoms based on data from the initial outbreak of SARS-CoV-2 in China using symptom occurrence at diagnosis and found that the order of COVID-19 symptoms differed from that of other infectious diseases including influenza. Whether this order of COVID-19 symptoms holds in the USA under changing conditions is unclear. Here, we use modeling to predict the order of symptoms using data from both the initial outbreaks in China and in the USA. Whereas patients in China were more likely to have fever before cough and then nausea/vomiting before diarrhea, patients in the USA were more likely to have cough before fever and then diarrhea before nausea/vomiting. Given that the D614G SARS-CoV-2 variant that rapidly spread from Europe to predominate in the USA during the first wave of the outbreak was not present in the initial China outbreak, we hypothesized that this mutation might affect symptom order. Supporting this notion, we found that as SARS-CoV-2 in Japan shifted from the original Wuhan reference strain to the D614G variant, symptom order shifted to the USA pattern. Google Trends analyses supported these findings, while weather, age, and comorbidities did not affect our model’s predictions of symptom order. These findings indicate that symptom order can change with mutation in viral disease and raise the possibility that D614G variant is more transmissible because infected people are more likely to cough in public before being incapacitated with fever.     

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.