Honokiol Inhibits SARS-CoV-2 Replication in Cell Culture at a Post-Entry Step.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019, and the resulting pandemic has already caused the death of over 6 million people. There are currently few antivirals approved for treatment of the 2019 coronavirus disease (COVID-19), and more options would be beneficial, not only now but also to increase our preparedness for future coronavirus outbreaks. Honokiol is a small molecule from magnolia trees for which several biological effects have been reported, including anticancer and anti-inflammatory activities. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we determined that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect, with a 50% effective concentration of 7.8 µM. In viral load reduction assays, honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant human A549 cells expressing angiotensin converting enzyme 2 and transmembrane protease serine 2. Time-of-addition and other assays showed that honokiol inhibited virus replication at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including Omicron, and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to be evaluated further in animal studies and, when successful, maybe even in clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses. IMPORTANCE Honokiol is a compound that shows both anti-inflammatory and antiviral effects, and therefore its effect on SARS-CoV-2 infection was assessed. This small molecule inhibited SARS-CoV-2 replication in various cell-based infection systems, with up to an ~1,000-fold reduction in virus titer. In contrast to earlier reports, our study clearly showed that honokiol acts on a postentry step of the replication cycle. Honokiol also inhibited different recent SARS-CoV-2 variants and other human coronaviruses (Middle East respiratory syndrome CoV and SARS-CoV), demonstrating its broad spectrum of antiviral activity. The anticoronavirus effect, combined with its anti-inflammatory properties, make honokiol an interesting compound to be further explored in animal coronavirus infection models.

R-Propranolol Has Broad-Spectrum Anti-Coronavirus Activity and Suppresses Factors Involved in Pathogenic Angiogenesis

The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals to increase our preparedness. Patients often require treatment by the time that blocking virus replication is less effective. Therefore, therapy should not only aim to inhibit the virus, but also to suppress pathogenic host responses, e.g., leading to microvascular changes and pulmonary damage. Clinical studies have previously linked SARS-CoV-2 infection to pathogenic intussusceptive angiogenesis in the lungs, involving the upregulation of angiogenic factors such as ANGPTL4. The β-blocker propranolol is used to suppress aberrant ANGPTL4 expression in the treatment of hemangiomas. Therefore, we investigated the effect of propranolol on SARS-CoV-2 infection and the expression of ANGPTL4. SARS-CoV-2 upregulated ANGPTL4 in endothelial and other cells, which could be suppressed with R-propranolol. The compound also inhibited the replication of SARS-CoV-2 in Vero-E6 cells and reduced the viral load by up to ~2 logs in various cell lines and primary human airway epithelial cultures. R-propranolol was as effective as S-propranolol but lacks the latter's undesired β-blocker activity. R-propranolol also inhibited SARS-CoV and MERS-CoV. It inhibited a post-entry step of the replication cycle, likely via host factors. The broad-spectrum antiviral effect and suppression of factors involved in pathogenic angiogenesis make R-propranolol an interesting molecule to further explore for the treatment of coronavirus infections.

Impact of changes in human airway epithelial cellular composition and differentiation on SARS-CoV-2 infection biology.

The consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can range from asymptomatic to fatal disease. Variations in epithelial susceptibility to SARS-CoV-2 infection depend on the anatomical location from the proximal to distal respiratory tract. However, the cellular biology underlying these variations is not completely understood. Thus, air-liquid interface (ALI) cultures of well-differentiated primary human tracheal and bronchial epithelial cells were employed to study the impact of epithelial cellular composition and differentiation on SARS-CoV-2 infection by transcriptional (RNA sequencing) and immunofluorescent analyses. Changes of cellular composition were investigated by varying time of differentiation or by using specific compounds. We found that SARS-CoV-2 primarily infected ciliated cells but also goblet cells and transient secretory cells. Viral replication was impacted by differences in cellular composition, which depended on culturing time and anatomical origin. A higher percentage of ciliated cells correlated with a higher viral load. However, DAPT-treatment, which increased number of ciliated cells and reduced goblet cells, decreased viral load, indicating the contribution of goblet cells to infection. Cell-entry factors, especially cathepsin L and transmembrane protease serine 2, were also affected by differentiation time. In conclusion, our study demonstrates that viral replication is affected by changes in cellular composition, especially in cells related to the mucociliary system. This could explain in part the variable susceptibility to SARS-CoV-2 infection between individuals and between anatomical locations in the respiratory tract.

R-Propranolol Has Broad-Spectrum Anti-Coronavirus Activity and Suppresses Factors Involved in Pathogenic Angiogenesis.

The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals to increase our preparedness. Patients often require treatment by the time that blocking virus replication is less effective. Therefore, therapy should not only aim to inhibit the virus, but also to suppress pathogenic host responses, e.g., leading to microvascular changes and pulmonary damage. Clinical studies have previously linked SARS-CoV-2 infection to pathogenic intussusceptive angiogenesis in the lungs, involving the upregulation of angiogenic factors such as ANGPTL4. The ß-blocker propranolol is used to suppress aberrant ANGPTL4 expression in the treatment of hemangiomas. Therefore, we investigated the effect of propranolol on SARS-CoV-2 infection and the expression of ANGPTL4. SARS-CoV-2 upregulated ANGPTL4 in endothelial and other cells, which could be suppressed with R-propranolol. The compound also inhibited the replication of SARS-CoV-2 in Vero-E6 cells and reduced the viral load by up to ~2 logs in various cell lines and primary human airway epithelial cultures. R-propranolol was as effective as S-propranolol but lacks the latter's undesired ß-blocker activity. R-propranolol also inhibited SARS-CoV and MERS-CoV. It inhibited a post-entry step of the replication cycle, likely via host factors. The broad-spectrum antiviral effect and suppression of factors involved in pathogenic angiogenesis make R-propranolol an interesting molecule to further explore for the treatment of coronavirus infections.

Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase.

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naïve animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.

The Cyclophilin-Dependent Calcineurin Inhibitor Voclosporin Inhibits SARS-CoV-2 Replication in Cell Culture.

Kidney transplant recipients (KTRs) are at increased risk for a more severe course of COVID-19, due to their pre-existing comorbidity and immunosuppression. Consensus protocols recommend lowering immunosuppression in KTRs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but the optimal combination remains unclear. Calcineurin inhibitors (CNIs) are cornerstone immunosuppressants used in KTRs and some have been reported to possess antiviral activity against RNA viruses, including coronaviruses. Here, we evaluated the effect of the CNIs tacrolimus, cyclosporin A, and voclosporin (VCS), as well as other immunosuppressants, on SARS-CoV-2 replication in cell-based assays. Unexpected, loss of compound due to plastic binding and interference of excipients in pharmaceutical formulations (false-positive results) complicated the determination of EC50 values of cyclophilin-dependent CNI's in our antiviral assays. Some issues could be circumvented by using exclusively glass lab ware with pure compounds. In these experiments, VCS reduced viral progeny yields in human Calu-3 cells at low micromolar concentrations and did so more effectively than cyclosporin A, tacrolimus or other immunosuppressants. Although, we cannot recommend a particular immunosuppressive regimen in KTRs with COVID-19, our data suggest a potential benefit of cyclophilin-dependent CNIs, in particular VCS in reducing viral progeny, which warrants further clinical evaluation in SARS-CoV-2-infected KTRs.

Potent Inhibition of SARS-CoV-2 nsp14 N7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues.

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.

A Yellow Fever 17D Virus Replicon-Based Vaccine Platform for Emerging Coronaviruses.

The tremendous global impact of the current SARS-CoV-2 pandemic, as well as other current and recent outbreaks of (re)emerging viruses, emphasize the need for fast-track development of effective vaccines. Yellow fever virus 17D (YF17D) is a live-attenuated virus vaccine with an impressive efficacy record in humans, and therefore, it is a very attractive platform for the development of novel chimeric vaccines against various pathogens. In the present study, we generated a YF17D-based replicon vaccine platform by replacing the prM and E surface proteins of YF17D with antigenic subdomains from the spike (S) proteins of three different betacoronaviruses: MERS-CoV, SARS-CoV and MHV. The prM and E proteins were provided in trans for the packaging of these RNA replicons into single-round infectious particles capable of expressing coronavirus antigens in infected cells. YF17D replicon particles expressing the S1 regions of the MERS-CoV and SARS-CoV spike proteins were immunogenic in mice and elicited (neutralizing) antibody responses against both the YF17D vector and the coronavirus inserts. Thus, YF17D replicon-based vaccines, and their potential DNA- or mRNA-based derivatives, may constitute a promising and particularly safe vaccine platform for current and future emerging coronaviruses.

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to cause massive global upheaval. Coronaviruses are positive-strand RNA viruses with an unusually large genome of ~30 kb. They express an RNA-dependent RNA polymerase and a cohort of other replication enzymes and supporting factors to transcribe and replicate their genomes. The proteins performing these essential processes are prime antiviral drug targets, but drug discovery is hindered by our incomplete understanding of coronavirus RNA synthesis and processing. In infected cells, the RNA-dependent RNA polymerase must coordinate with other viral and host factors to produce both viral mRNAs and new genomes. Recent research aiming to decipher and contextualize the structures, functions and interplay of the subunits of the SARS-CoV-2 replication and transcription complex proteins has burgeoned. In this Review, we discuss recent advancements in our understanding of the molecular basis and complexity of the coronavirus RNA-synthesizing machinery. Specifically, we outline the mechanisms and regulation of RNA translation, replication and transcription. We also discuss the composition of the replication and transcription complexes and their suitability as targets for antiviral therapy.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Ad26.COV2.S protects Syrian hamsters against G614 spike variant SARS-CoV-2 and does not enhance respiratory disease.

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy, and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1 × 109 and 1 × 1010 VP elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers that was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these preclinical data confirm efficacy of a one-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy. METHODS: This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course. FINDINGS: Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41-78). Median disease course (time from onset of symptoms to death) was 22 days (range 5-44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5-44]; ten patients with neutrophilic plugs, 21 days [5-44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets.. INTERPRETATION: In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19. FUNDING: Amsterdam UMC Corona Research Fund.

The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2.

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless-as happened occasionally-reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity.IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements.

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5' UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness.

INTRODUCTION: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness. AIM: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness. METHODS: The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery. RESULTS: Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%. CONCLUSION: The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples.

A molecular pore spans the double membrane of the coronavirus replication organelle.

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.