A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

BSL2-compliant lethal mouse model of SARS-CoV-2 and variants of concern to evaluate therapeutics targeting the Spike protein.

Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -ß, ϒ, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-ß, rVSV-SARS2-Spike-ϒ or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.

Detection of Bourbon Virus-Specific Serum Neutralizing Antibodies in Human Serum in Missouri, USA.

Bourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the tick Amblyomma americanum in the states of Missouri and Kansas in the United States. Despite the high prevalence of BRBV in ticks in these states, very few human cases have been reported, and the true infection burden of BRBV in the community is unknown. Here, we developed two virus neutralization assays, a vesicular stomatitis virus (VSV)-BRBV pseudotyped rapid assay and a BRBV focus reduction neutralization assay, to assess the seroprevalence of BRBV neutralizing antibodies in human sera collected in 2020 in St. Louis, MO. Of 440 human serum samples tested, three (0.7%) were able to potently neutralize both VSV-BRBV and wild-type BRBV. These findings suggest that human infections with BRBV are more common than previously recognized. IMPORTANCE Since the discovery of the Bourbon virus (BRBV) in 2014, a total of five human cases have been identified, including two fatal cases. BRBV is thought to be transmitted by the lone star tick, which is prevalent in the eastern, southeastern, and midwestern United States. BRBV has been detected in ticks in Missouri and Kansas, and serological evidence suggests that it is also present in North Carolina. However, the true infection burden of BRBV in humans is not known. In the present study, we developed two virus neutralization assays to assess the seroprevalence of BRBV-specific antibodies in human sera collected in 2020 in St. Louis, MO. We found that a small subset of individuals are seropositive for neutralizing antibodies against BRBV. Our data suggest that BRBV infection in humans is more common than previously thought.

Longitudinal Study after Sputnik V Vaccination Shows Durable SARS-CoV-2 Neutralizing Antibodies and Reduced Viral Variant Escape to Neutralization over Time.

Recent studies have shown a temporal increase in the neutralizing antibody potency and breadth to SARS-CoV-2 variants in coronavirus disease 2019 (COVID-19) convalescent individuals. Here, we examined longitudinal antibody responses and viral neutralizing capacity to the B.1 lineage virus (Wuhan related), to variants of concern (VOC; Alpha, Beta, Gamma, and Delta), and to a local variant of interest (VOI; Lambda) in volunteers receiving the Sputnik V vaccine in Argentina. Longitudinal serum samples (N = 536) collected from 118 volunteers obtained between January and October 2021 were used. The analysis indicates that while anti-spike IgG levels significantly wane over time, the neutralizing capacity for the Wuhan-related lineages of SARS-CoV-2 and VOC is maintained within 6 months of vaccination. In addition, an improved antibody cross-neutralizing ability for circulating variants of concern (Beta and Gamma) was observed over time postvaccination. The viral variants that displayed higher escape to neutralizing antibodies with respect to the original virus (Beta and Gamma variants) were the ones showing the largest increase in susceptibility to neutralization over time after vaccination. Our observations indicate that serum neutralizing antibodies are maintained for at least 6 months and show a reduction of VOC escape to neutralizing antibodies over time after vaccination. IMPORTANCE Vaccines have been produced in record time for SARS-CoV-2, offering the possibility of halting the global pandemic. However, inequalities in vaccine accessibility in different regions of the world create a need to increase international cooperation. Sputnik V is a recombinant adenovirus-based vaccine that has been widely used in Argentina and other developing countries, but limited information is available about its elicited immune responses. Here, we examined longitudinal antibody levels and viral neutralizing capacity elicited by Sputnik V vaccination. Using a cohort of 118 volunteers, we found that while anti-spike antibodies wane over time, the neutralizing capacity to viral variants of concern and local variants of interest is maintained within 4 months of vaccination. In addition, we observed an increased cross-neutralization activity over time for the Beta and Gamma variants. This study provides valuable information about the immune response generated by a vaccine platform used in many parts of the world.

JIB-04 Has Broad-Spectrum Antiviral Activity and Inhibits SARS-CoV-2 Replication and Coronavirus Pathogenesis.

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.

Noninvasive Immuno-PET Imaging of CD8+ T Cell Behavior in Influenza A Virus-Infected Mice.

Immuno-positron emission tomography (immuno-PET) is a noninvasive imaging method that enables tracking of immune cells in living animals. We used a nanobody that recognizes mouse CD8α and labeled it with 89Zr to image mouse CD8+ T cells in the course of an infection with influenza A virus (IAV). The CD8+ signal showed a strong increase in the mediastinal lymph node (MLN) and thymus as early as 4 days post-infection (dpi), and as early as 6 dpi in the lungs. Over the course of the infection, CD8+ T cells were at first distributed diffusely throughout the lungs and then accumulated more selectively in specific regions of the lungs. These distributions correlated with morbidity as mice reached the peak of weight loss over this interval. CD8+ T cells obtained from control or IAV-infected mice showed a difference in their distribution and migration when comparing their fate upon labeling ex vivo with 89Zr-labeled anti-CD8α nanobody and transfer into infected versus control animals. CD8+ T cells from infected mice, upon transfer, appear to be trained to persist in the lungs, even of uninfected mice. Immuno-PET imaging thus allows noninvasive, dynamic monitoring of the immune response to infectious agents in living animals.

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC 50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2.

The SARS-CoV-2 spike (S) glycoprotein contains an immunodominant receptor-binding domain (RBD) targeted by most neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite (designated site i) recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge, albeit selecting escape mutants in some animals. Indeed, several SARS-CoV-2 variants, including the B.1.1.7, B.1.351, and P.1 lineages, harbor frequent mutations within the NTD supersite, suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs for protective immunity and vaccine design.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation.

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2.

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics, viral escape mutants could compromise their efficacy. To define the immune-selected mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) to generate 50 different escape mutants. The variants were mapped onto the RBD structure and evaluated for cross-resistance to mAbs and convalescent human sera. Each mAb had a unique resistance profile, although many shared residues within an epitope. Some variants ( e.g ., S477N) were resistant to neutralization by multiple mAbs, whereas others ( e.g ., E484K) escaped neutralization by convalescent sera, suggesting some humans induce a narrow repertoire of neutralizing antibodies. Comparing the antibody-mediated mutational landscape in S with sequence variation in circulating SARS-CoV-2, we define substitutions that may attenuate neutralizing immune responses in some humans.

JIB-04 has broad-spectrum antiviral activity and inhibits SARS-CoV-2 replication and coronavirus pathogenesis.

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2.

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.