Biology subjects

Martinez-Sobrido, L.

Publications and source records attributed to Martinez-Sobrido, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Visualization of SARS-CoV-2 infection dynamic

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

microbiology↗

bioRxiv2021

4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4-fluorouridine (4-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. One-Sentence Summary4-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo.

microbiology↗

bioRxiv2021

Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.

microbiology↗

bioRxiv2021

SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

microbiology↗

bioRxiv2021

Amino acid residues involved in inhibition of host gene expression by influenza A/Brevig Mission/1/1918 PA-X

Influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and a plasmid encoding the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-Xs shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

microbiology↗

bioRxiv2021

Rapid and Efficient Inactivation of SARS-CoV-2 from Surfaces using UVC Light Emitting Diode Device

Efforts are underway to develop countermeasures to prevent the environmental spread of COVID-19 pandemic caused by SARS-CoV-2. Physical decontamination methods like Ultraviolet radiation has shown to be promising. Here, we describe a novel device emitting ultraviolet C radiation (UVC), called NuvaWave, to rapidly and efficiently inactivate SARS-CoV-2. SARS-CoV-2 was dried on a chambered glass slides and introduced in a NuvaWave robotic testing unit. The robot simulated waving NuvaWave over the virus at a pre-determined UVC radiation dose of 1, 2, 4 and 8 seconds. Post-UVC exposure, virus was recovered and titered by plaque assay in Vero E6 cells. We observed that relative control (no UVC exposure), exposure of the virus to UVC for one or two seconds resulted in a >2.9 and 3.8 log10 reduction in viral titers, respectively. Exposure of the virus to UVC for four or eight seconds resulted in a reduction of greater than 4.7-log10 reduction in viral titers. The NuvaWave device inactivates SARS-CoV-2 on surfaces to below the limit of detection within one to four seconds of UVC irradiation. This device can be deployed to rapidly disinfect surfaces from SARS-CoV-2, and to assist in mitigating its spread in a variety of settings.

microbiology↗

bioRxiv2021

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling

Summary/AbstractFACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

molecular biology↗

bioRxiv2021

Epitope classification and RBD binding properties of neutralizing antibodies against SARS-CoV-2 variants of concern

Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.

immunology↗

bioRxiv2021

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient ({Delta}ORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of {Delta}ORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the {Delta}ORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while {Delta}ORF7a, {Delta}ORF7b and {Delta}ORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCEDespite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

microbiology↗

bioRxiv2021

A metal ion orients mRNA to ensure accurate 2'-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

biochemistry↗

bioRxiv2021

Immunogenicity and Protective Efficacy of an Intranasal Live-attenuated Vaccine Against SARS-CoV-2 in Preclinical Animal Models

The global deployment of an effective and safe vaccine is currently a public health priority to curtail the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based intranasal vectored-vaccine in mice and hamsters for its immunogenicity, safety and protective efficacy in challenge studies with SARS-CoV-2. The recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 administrated via intranasal route in mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T cell-mediated immunity. Hamsters vaccinated with two doses of vaccine showed complete protection from clinical disease including lung infection, inflammation, and pathological lesions after SARS-CoV-2 challenge. Importantly, a single or double dose of intranasal rNDV-S vaccine completely blocked SARS-CoV-2 shedding in nasal turbinate and lungs within 4 days of vaccine administration in hamsters. Taken together, intranasal administration of rNDV-S has the potential to control infection at the site of inoculation, which should prevent both the clinical disease and transmission to halt the spread of the COVID-19 pandemic.

immunology↗

bioRxiv2020

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

microbiology↗

bioRxiv2020

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

SARS-CoV-2 infection results in viral burden in the upper and lower respiratory tract, enabling transmission and often leading to substantial lung pathology. Delivering the antiviral treatment directly to the lungs has the potential to improve lung bioavailability and dosing efficiency. As the SARS-CoV-2 Receptor Binding Domain (RBD) of the Spike (S) is increasingly deemed to be a clinically validated target, RBD-specific B cells were isolated from patients following SARS-CoV-2 infection to derive a panel of fully human monoclonal antibodies (hmAbs) that potently neutralize SARS-CoV-2. The most potent hmAb, 1212C2 was derived from an IgM memory B cell, has high affinity for SARS-CoV-2 RBD which enables its direct inhibition of RBD binding to ACE2. The 1212C2 hmAb exhibits in vivo prophylactic and therapeutic activity against SARS-CoV-2 in hamsters when delivered intraperitoneally, achieving a meaningful reduction in upper and lower respiratory viral burden and lung pathology. Furthermore, liquid nebulized inhale treatment of SARS-CoV-2 infected hamsters with as low as 0.6 mg/kg of inhaled dose, corresponding to approximately 0.03 mg/kg of lung deposited dose, mediated a reduction in respiratory viral burden that is below the detection limit, and mitigated lung pathology. The therapeutic efficacy achieved at an exceedingly low-dose of inhaled 1212C2 supports the rationale for local lung delivery and achieving dose-sparing benefits as compared to the conventional parenteral route of administration. Taken together, these results warrant an accelerated clinical development of 1212C2 hmAb formulated and delivered via inhalation for the prevention and treatment of SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

microbiology↗

bioRxiv2020

Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

microbiology↗

bioRxiv2020

Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice

ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

microbiology↗

bioRxiv2020

Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus

The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.

biophysics↗

bioRxiv2020

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

microbiology↗