Convergent Structures Illuminate Features for Germline Antibody Binding and Pan-Lassa Virus Neutralization.

Lassa virus (LASV) causes hemorrhagic fever and is endemic in West Africa. Protective antibody responses primarily target the LASV surface glycoprotein (GPC), and GPC-B competition group antibodies often show potent neutralizing activity in humans. However, which features confer potent and broadly neutralizing antibody responses is unclear. Here, we compared three crystal structures of LASV GPC complexed with GPC-B antibodies of varying neutralization potency. Each GPC-B antibody recognized an overlapping epitope involved in binding of two adjacent GPC monomers and preserved the prefusion trimeric conformation. Differences among GPC-antibody interactions highlighted specific residues that enhance neutralization. Using structure-guided amino acid substitutions, we increased the neutralization potency and breadth of these antibodies to include all major LASV lineages. The ability to define antibody residues that allow potent and broad neutralizing activity, together with findings from analyses of inferred germline precursors, is critical to develop potent therapeutics and for vaccine design and assessment.

Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

A human monoclonal antibody combination rescues nonhuman primates from advanced disease caused by the major lineages of Lassa virus.

There are no approved treatments for Lassa fever (LF), which is responsible for thousands of deaths each year in West Africa. A major challenge in developing effective medical countermeasures against LF is the high diversity of circulating Lassa virus (LASV) strains with four recognized lineages and four proposed lineages. The recent resurgence of LASV in Nigeria caused by genetically distinct strains underscores this concern. Two LASV lineages (II and III) are dominant in Nigeria. Here, we show that combinations of two or three pan-lineage neutralizing human monoclonal antibodies (8.9F, 12.1F, 37.D) known as Arevirumab-2 or Arevirumab-3 can protect up to 100% of cynomolgus macaques against challenge with both lineage II and III LASV isolates when treatment is initiated at advanced stages of disease on day 8 after LASV exposure. This work demonstrates that it may be possible to develop postexposure interventions that can broadly protect against most strains of LASV.

Hexestrol, an estrogen receptor agonist, inhibits Lassa virus entry.

Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV.

An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice.

The mammarenavirus Lassa virus (LASV) causes the life-threatening hemorrhagic fever disease, Lassa fever. The lack of licensed medical countermeasures against LASV underscores the urgent need for the development of novel LASV vaccines, which has been hampered by the requirement for a biosafety level 4 facility to handle live LASV. Here, we investigated the efficacy of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in mice. Two doses of LASgpc- or LCMnp-mRNA-LNP administered intravenously (i.v.) protected C57BL/6 mice from a lethal challenge with a recombinant (r) LCMV expressing a modified LASgpc (rLCMV/LASgpc2m) inoculated intracranially. Intramuscular (i.m.) immunization with two doses of LASgpc- or LCMnp-mRNA-LNP significantly reduced the viral load in C57BL/6 mice inoculated i.v. with rLCMV/LASgpc2m. High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc2m, which were abrogated by i.m. immunization with two doses of LASgpc-mRNA-LNP. The protective efficacy of two i.m. doses of LCMnp-mRNA-LNP was confirmed in a lethal hemorrhagic disease model of FVB mice i.v. inoculated with wild-type rLCMV. In all conditions tested, negligible and high levels of LASgpc- and LCMnp-specific antibodies were detected in mRNA-LNP-immunized mice, respectively, but robust LASgpc- and LCMnp-specific CD8+ T cell responses were induced. Accordingly, plasma from LASgpc-mRNA-LNP-immunized mice did not exhibit neutralizing activity. Our findings and surrogate mouse models of LASV infection, which can be studied at a reduced biocontainment level, provide a critical foundation for the rapid development of mRNA-LNP-based LASV vaccines.IMPORTANCELassa virus (LASV) is a highly pathogenic mammarenavirus responsible for several hundred thousand infections annually in West African countries, causing a high number of lethal Lassa fever (LF) cases. Despite its significant impact on human health, clinically approved, safe, and effective medical countermeasures against LF are not available. The requirement of a biosafety level 4 facility to handle live LASV has been one of the main obstacles to the research and development of LASV countermeasures. Here, we report that two doses of mRNA-lipid nanoparticle-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of lymphocytic choriomeningitis virus (LCMV), a mammarenavirus genetically closely related to LASV, conferred protection to recombinant LCMV-based surrogate mouse models of lethal LASV infection. Notably, robust LASgpc- and LCMnp-specific CD8+ T cell responses were detected in mRNA-LNP-immunized mice, whereas no virus-neutralizing activity was observed.

Protective Efficacy of Lyophilized Vesicular Stomatitis Virus-Based Vaccines in Animal Model.

We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

Spatio-temporal spread and evolution of Lassa virus in West Africa.

BACKGROUND: Lassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited. METHODS: To enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences. RESULTS: The results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV. CONCLUSION: This study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.

Predicting the fine-scale spatial distribution of zoonotic reservoirs using computer vision.

Zoonotic diseases threaten human health worldwide and are often associated with anthropogenic disturbance. Predicting how disturbance influences spillover risk is critical for effective disease intervention but difficult to achieve at fine spatial scales. Here, we develop a method that learns the spatial distribution of a reservoir species from aerial imagery. Our approach uses neural networks to extract features of known or hypothesized importance from images. The spatial distribution of these features is then summarized and linked to spatially explicit reservoir presence/absence data using boosted regression trees. We demonstrate the utility of our method by applying it to the reservoir of Lassa virus, Mastomys natalensis, within the West African nations of Sierra Leone and Guinea. We show that, when trained using reservoir trapping data and publicly available aerial imagery, our framework learns relationships between environmental features and reservoir occurrence and accurately ranks areas according to the likelihood of reservoir presence.

Environmental Persistence and Disinfection of Lassa Virus.

Lassa fever, caused by Lassa virus (LASV), is endemic to West Africa, where ≈300,000 illnesses and ≈5,000 deaths occur annually. LASV is primarily spread by infected multimammate rats via urine and fomites, highlighting the need to understand the environmental fate of LASV. We evaluated persistence of LASV Josiah and Sauerwald strains on surfaces, in aqueous solutions, and with sodium hypochlorite disinfection. Tested strains were more stable in deionized water (first-order rate constant [k] for Josiah, 0.23 days; for Sauerwald, k = 0.34 days) than primary influent wastewater (Josiah, k = 1.3 days; Sauerwald, k = 1.9 days). Both strains had similar decay rates on high-density polyethylene (Josiah, k = 4.3 days; Sauerwald, k = 2.3 days) and stainless steel (Josiah, k = 5.3 days; Sauerwald, k = 2.7 days). Sodium hypochlorite was highly effective at inactivating both strains. Our findings can inform future risk assessment and management efforts for Lassa fever.

Influence of Landscape Patterns on Exposure to Lassa Fever Virus, Guinea.

Lassa fever virus (LASV) is the causative agent of Lassa fever, a disease endemic in West Africa. Exploring the relationships between environmental factors and LASV transmission across ecologically diverse regions can provide crucial information for the design of appropriate interventions and disease monitoring. We investigated LASV exposure in 2 ecologically diverse regions of Guinea. Our results showed that exposure to LASV was heterogenous between and within sites. LASV IgG seropositivity was 11.9% (95% CI 9.7%-14.5%) in a coastal study site in Basse-Guinée, but it was 59.6% (95% CI 55.5%-63.5%) in a forested study site located in Guinée Forestière. Seropositivity increased with age in the coastal site. We also found significant associations between exposure risk for LASV and landscape fragmentation in coastal and forested regions. Our study highlights the potential link between environmental change and LASV emergence and the urgent need for research on land management practices that reduce disease risks.

Evaluating the Biological Role of Lassa Viral Z Protein-Mediated RIG-I Inhibition Using a Replication-Competent Trisegmented Pichinde Virus System in an Inducible RIG-IN Expression Cell Line.

Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.

Structure-activity relationship studies of anti-bunyaviral cap-dependent endonuclease inhibitors.

Bunyaviruses, including the Lassa virus (LASV), are known to cause hemorrhagic fever and have a high fatality rate among hospitalized patients, as there are few effective treatments. We focused on the fact that bunyaviruses use cap-dependent endonuclease (CEN) for viral replication, which is similar to influenza viruses. This led us to screen carbamoyl pyridone bicycle (CAB) compounds, which compose a series of baloxavir acid (BXA) derivatives, against lymphocytic choriomeningitis virus (LCMV) and Junin virus (JUNV) among the bunyaviruses. This led to the discovery of 1c, which has potent anti-bunyaviral activities. In SAR studies, we found that a large lipophilic side chain is preferred for the 1-position of the CAB scaffold, similar to the influenza CEN inhibitor, and that a small alkyl group for the 3-position shows high activity. Moreover, the 7­carboxyl group of the scaffold is essential for anti-bunyaviral activities, and the antiviral activity is reduced by conversion to various carboxylic acid bioisosteres. The SAR results are discussed using a binding model of 9d in the active center of the known LCMV CEN crystal structure. These compounds show promise as broad-spectrum anti-bunyavirus therapeutics, given their relatively favorable metabolic stability and PK profiles.

Biodistribution and toxicology evaluation of a recombinant measles Schwarz-based Lassa vaccine in cynomolgus macaques.

MV-LASV is an investigational measles Schwarz-based vaccine for the prevention of Lassa fever. A repeated-dose toxicity study in cynomolgus macaques was performed to assess the biodistribution and local and systemic toxicological effects. Monkeys received three immunizations of MV-LASV or saline intramuscularly with a 2-week interval. An increase in anti-measles antibodies confirmed the reaction of the immune system to the vaccine backbone. Clinical observations, body weight, body temperature, local tolerance, electrocardiogram parameters, various clinical pathology parameters (hematology, coagulation urinalysis, serum chemistry, and C-reactive protein) were monitored. Gross pathology and histopathology of various tissues were evaluated. MV-LASV induced a mild increase in fibrinogen and C-reactive protein concentrations. This coincided with microscopic inflammation at the injection sites which partially or fully resolved following a 3-week recovery period. Viral RNA was found in secondary lymphoid organs and injection sites and gall bladder. No viral shedding to the environment was observed. Overall, the vaccine was locally and systemically well tolerated, supporting a first-in-human study.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Design, Synthesis, and Biological Evaluation of Benzimidazole Derivatives as Potential Lassa Virus Inhibitors.

The Lassa virus (LASV) causes Lassa fever, a highly infectious and lethal agent of acute viral hemorrhagic fever. At present, there are still no effective treatments available, creating an urgent need to develop novel therapeutics. Some benzimidazole compounds targeting the arenavirus envelope glycoprotein complex (GPC) are promising inhibitors of LASV. In this study, we synthesized two series of LASV inhibitors based on the benzimidazole structure. Lentiviral pseudotypes bearing the LASV GPC were established to identify virus entry inhibitors. Surface plasmon resonance (SPR) was further used to verify the binding activities of the potential compounds. Compounds 7d-Z, 7h-Z, 13c, 13d, and 13f showed relatively excellent antiviral activities with IC50 values ranging from 7.58 to 15.46 nM and their SI values above 1251. These five representative compounds exhibited stronger binding affinity with low equilibrium dissociation constants (KD < 8.25 × 10-7 M) in SPR study. The compound 7h-Z displayed the most potent antiviral activity (IC50 = 7.58 nM) with a relatively high SI value (2496), which could be further studied as a lead compound. The structure-activity relationship indicated that the compounds with lipophilic and spatially larger substituents might possess higher antiviral activity and a much larger safety margin. This study will provide some good guidance for the development of highly active compounds with a novel skeleton against LASV.

Two Cases of Lassa Fever Successfully Treated with Ribavirin and Adjunct Dexamethasone for Concomitant Infections.

Lassa fever is a viral hemorrhagic fever treated with supportive care and the broad-spectrum antiviral drug ribavirin. The pathophysiology, especially the role of hyperinflammation, of this disease is unknown. We report successful remission of complicated Lassa fever in 2 patients in Nigeria who received the antiinflammatory agent dexamethasone and standard ribavirin.

Pathogens that Cause Illness Clinically Indistinguishable from Lassa Fever, Nigeria, 2018.

During the 2018 Lassa fever outbreak in Nigeria, samples from patients with suspected Lassa fever but negative Lassa virus PCR results were processed through custom gene expression array cards and metagenomic sequencing. Results demonstrated no single etiology, but bacterial and viral pathogens (including mixed co-infections) were detected.

miR-142 Targets TIM-1 in Human Endothelial Cells: Potential Implications for Stroke, COVID-19, Zika, Ebola, Dengue, and Other Viral Infections.

T-cell immunoglobulin and mucin domain 1 (TIM-1) has been recently identified as one of the factors involved in the internalization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells, in addition to angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), neuropilin-1, and others. We hypothesized that specific microRNAs could target TIM-1, with potential implications for the management of patients suffering from coronavirus disease 2019 (COVID-19). By combining bioinformatic analyses and functional assays, we identified miR-142 as a specific regulator of TIM-1 transcription. Since TIM-1 has been implicated in the regulation of endothelial function at the level of the blood-brain barrier (BBB) and its levels have been shown to be associated with stroke and cerebral ischemia-reperfusion injury, we validated miR-142 as a functional modulator of TIM-1 in human brain microvascular endothelial cells (hBMECs). Taken together, our results indicate that miR-142 targets TIM-1, representing a novel strategy against cerebrovascular disorders, as well as systemic complications of SARS-CoV-2 and other viral infections.

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.