Molecular insights on the coronavirus MERS-CoV interaction with the CD26 receptor.

The Middle East respiratory syndrome (MERS) is a severe respiratory disease with high fatality rates, caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). The virus initiates infection by binding to the CD26 receptor (also known as dipeptidyl peptidase 4 or DPP4) via its spike protein. Although the receptor-binding domain (RBD) of the viral spike protein and the complex between RBD and the extracellular domain of CD26 have been studied using X-ray crystallography, conflicting studies exist regarding the importance of certain amino acids outside the resolved RBD-CD26 complex interaction interface. To gain atomic-level knowledge of the RBD-CD26 complex, we employed computational simulations to study the complex's dynamic behavior as it evolves from its crystal structure to a conformation stable in solution. Our study revealed previously unidentified interaction regions and interacting amino acids within the complex, determined a novel comprehensive RBD-binding domain of CD26, and by that expanded the current understanding of its structure. Additionally, we examined the impact of a single amino acid substitution, E513A, on the complex's stability. We discovered that this substitution disrupts the complex through an allosteric domino-like mechanism that affects other residues. Since MERS-CoV is a zoonotic virus, we evaluated its potential risk of human infection via animals, and suggest a low likelihood for possible infection by cats or dogs. The molecular structural information gleaned from our insights into the RBD-CD26 complex pre-dissociative states may be proved useful not only from a mechanistic view but also in assessing inter-species transmission and in developing anti-MERS-CoV antiviral therapeutics.

Unraveling DPP4 Receptor Interactions with SARS-CoV-2 Variants and MERS-CoV: Insights into Pulmonary Disorders via Immunoinformatics and Molecular Dynamics.

Human coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as a potential co-receptor for viral cell entry. DPP4, the ubiquitous membrane-bound aminopeptidase, is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID-19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between the receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in the experimentally determined structure of the complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2: DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration and number of hydrogen bonding in the interface. Mutations in the delta variant, L452R and T478K directly participate in DPP4 interaction, enhancing DPP4 binding. E484K in alpha and gamma variants of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein becomes more suitable due to mutation, especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K, respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.

SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the global pandemic of COVID-19. SARS-CoV-2 genome encodes a main protease (nsp5, also called Mpro) and a papain-like protease (nsp3, also called PLpro), which are responsible for processing viral polyproteins to assemble a functional replicase complex. In this study, we found that Mpro of SARS-CoV-2 can cleave human MAGED2 and other mammalian orthologs at Gln-263. Moreover, SARS-CoV and MERS-CoV Mpro can also cleave human MAGED2, suggesting MAGED2 cleavage by Mpro is an evolutionarily conserved mechanism of coronavirus infection in mammals. Intriguingly, Mpro from Beta variant cleaves MAGED2 more efficiently than wild type, but Omicron Mpro is opposite. Further studies show that MAGED2 inhibits SARS-CoV-2 infection at viral replication step. Mechanistically, MAGED2 is associated with SARS-CoV-2 nucleocapsid protein through its N-terminal region in an RNA-dependent manner, and this disrupts the interaction between SARS-CoV-2 nucleocapsid protein and viral genome, thus inhibiting viral replication. When MAGED2 is cleaved by Mpro, the N-terminal of MAGED2 will translocate into the nucleus, and the truncated MAGED2 is unable to suppress SARS-CoV-2 replication. This work not only discovers the antiviral function of MAGED2 but also provides new insights into how SARS-CoV-2 Mpro antagonizes host antiviral response. IMPORTANCE Host factors that restrict severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain elusive. Here, we found that MAGED2 can be cleaved by SARS-CoV-2 main protease (Mpro) at Gln-263. SARS-CoV and MERS-CoV Mpro can also cleave MAGED2, and MAGED2 from multiple species can be cleaved by SARS-CoV-2 Mpro. Mpro from Beta variant cleaves MAGED2 more efficiently efficiently than wild type, but Omicron is the opposite. MAGED2 depletion enhances SARS-CoV-2 infection, suggesting its inhibitory role in SARS-CoV-2 infection. Mechanistically, MAGED2 restricts SARS-CoV-2 replication by disrupting the interaction between nucleocapsid and viral genomes. When MAGED2 is cleaved, its N-terminal will translocate into the nucleus. In this way, Mpro relieves MAGED2' inhibition on viral replication. This study improves our understanding of complex viral-host interaction and provides novel targets to treat SARS-CoV-2 infection.

DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1.

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

Natural Products as Potential Therapeutic Agents for SARS-CoV-2: A Medicinal Chemistry Perspective.

Coronavirus is a single-stranded RNA virus discovered by virologist David Tyrrell in 1960. Till now seven human corona viruses have been identified including HCoV-229E, HCoVOC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2. In the present scenario, the SARS-CoV-2 outbreak causing SARS-CoV-2 pandemic, became the most serious public health emergency of the century worldwide. Natural products have long history and advantages for the drug discovery process. Almost 80% of drugs present in market are evolved from the natural resources. With the outbreak of SARS-CoV-2 pandemic, natural product chemists have made significant efforts for the identification of natural molecules which can be effective against the SARSCoV- 2. In current compilation we have discussed in vitro and in vivo anti-viral potential of natural product-based leads for the treatment of SARS-CoV-2. We have classified these leads in different classes of natural products such as alkaloids, terpenoids, flavonoids, polyphenols, quinones, cannabinoids, steroids, glucosinolates, diarylheptanoids, etc. and discussed the efficacy and mode of action of these natural molecules. The present review will surely opens new direction in future for the development of promising drug candidates, particularly from the natural origin against coronaviruses and other viral diseases.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Novel type-I interferons from the dromedary camel: Molecular identification, prokaryotic expression and functional characterization of camelid interferon-delta.

The last two decades have seen the emergence of three highly pathogenic coronaviruses with zoonotic origins, which prompted immediate attention to the underlying cause and prevention of future outbreaks. Intensification of camel husbandry in the Middle East has resulted in increased human-camel interactions, which has led to the spread of potentially zoonotic viruses with human spillover risks like MERS-coronavirus, camelpox virus, etc. Type-I interferons function as the first line of defense against invading viruses and are pivotal for limiting viral replication and immune-mediated pathologies. Seven novel dromedary camel interferon delta genes were identified and cloned. Functional characterization of this novel class of IFNs from the mammalian suborder tylopoda is reported for the first time. The camel interferon-delta proteins resemble the reported mammalian counterparts in sequence similarity, conservation of cysteines, and phylogenetic proximity. Prokaryotically expressed recombinant camel interferon-δ1 induced IFN-stimulated gene expression and also exerted antiviral action against camelpox virus, an endemic zoonotic virus. The pre-treatment of camel kidney cells with recombinant camel IFN-δ1 increased cell survival and reduced camelpox virus in a dose-dependent manner. The identification of novel IFNs from species with zoonotic spillover risk such as camels, and evaluating their antiviral effects in-vitro will play a key role in improving immunotherapies against viruses and expanding the arsenal to combat emerging zoonotic pathogens.

Differential expression of carcinoembryonic antigen-related cell adhesion molecule-5 (CEACAM5) and dipeptidyl peptidase-4 (DPP4) with detection of Middle East respiratory syndrome-coronavirus in peripheral blood.

BACKGROUND: Middle East respiratory syndrome-coronavirus (MERS-CoV) utilizes CD26 (dipeptidyl peptidase-4) and CD66e or CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) receptors for cell infection. Peripheral blood mononuclear cells (PBMCs) play a critical role in mounting adaptive immune response against the virus. This study was performed to assess the expression of CD26 and CD66e on PBMCs and their susceptibility to MERS-CoV infection. METHODS: Surface expression of CD26 and CD66e receptors on PBMCs from MERS-CoV patients (n = 20) and healthy controls (n = 20) was assessed by flow cytometry and the soluble forms were determined by enzyme-linked immunosorbent assay (ELISA). MERS-CoV UpE and Orf1a genes in PBMCs were detected by using Altona diagnostics reverse transcription polymerase chain reaction (RT-PCR) kit. RESULTS: Mean fluorescent intensity (MFI) of CD66e was significantly higher on CD4 + lymphocytes (462.4 ± 64.35 vs 325.1 ± 19.69; p < 0.05) and CD8 + lymphocytes (533.8 ± 55.32 vs 392.4 ± 37.73; p < 0.04) from patients with MERS-CoV infection compared to the normal controls. No difference in MFI for CD66e was observed on monocytes (381.8 ± 40.34 vs 266.8 ± 20.6; p = 0.3) between the patients and controls. Soluble form of CD66e among MERS-CoV patients was also higher than the normal controls (mean= 338.7 ± 58.75 vs 160.7 ± 29.49 ng/mL; p < 0.01). Surface expression of CD26 on PBMCs and its soluble form were no different between the groups. MERS-CoV was detected by RT-PCR in 16/20 (80%) patients from whole blood, among them 8 patients were tested in PBMCs, 4/8 (50%) patients were positive. CONCLUSION: Increased expression levels of CD66e (CEACAM5) may contribute to increased susceptibility of PBMCs to MERS-CoV infection and disease progression.

Beta interferons from the extant camelids: Unique among eutherian mammals.

The COVID-19 pandemic is a wake-up call on the zoonotic viral spillover events and the need to be prepared for future outbreaks. Zoonotic RNA viruses like the Middle East respiratory syndrome coronavirus (MERS-CoV) are potential pathogens that could trigger the next pandemic. Dromedary camels are the only known animal source of MERS-CoV zoonotic infections, but little is known about the molecular antiviral response in this species. IFN-ß and other type-I interferons provide the first line of defense against invading pathogens in the host immune response. We identified the IFNB gene of the dromedary camel and all extant members of the family Camelidae. Camelid IFN-ß is unique with an even number of cysteines in the mature protein compared to other eutherian mammals with an odd number of cysteines. The viral mimetic poly(I:C) strongly induced IFN-ß expression in camel kidney cells. Induction of IFN-ß expression upon infection with camelpox virus was late and subdued when compared to poly(I:C) treatment. Prokaryotically expressed recombinant dromedary IFN-ß induced expression of IFN-responsive genes in camel kidney cells. Further, recombinant IFN-ß conferred antiviral resistance to camel kidney cells against the cytopathic effects of the camelpox virus, an endemic zoonotic pathogen. IFN-ß from this unique group of mammals will offer insights into antiviral immune mechanisms and aid in the development of specific antivirals against pathogens that have the potential to be the next zoonotic pandemic.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Exploring structural dynamics of the MERS-CoV receptor DPP4 and mutant DPP4 receptors.

Mouse DPP4 (mDPP4) receptor is not a functional receptor for MERS-CoV while human DPP4 (hDPP4) is, despite the high similarities between hDPP4 and mDPP4 receptors. The variability of DPP4 receptors against MERS-CoV is not fully investigated, especially conformational and structural differences. Therefore, investigating the conformational differences of the DPP4 receptors can aid in developing new small animal models for MERS-CoV vaccines and antiviral agents evaluation. Here we used MD simulations and docking techniques to investigate these structural differences in DPP4 receptors. The results showed chimeric mouse mDPP4 (cmDPP4) has a similar compact conformation as wild-type hDPP4 based on the structural analysis. Interestingly, a single Thr288Ala mutation induced a relaxed conformation in chimeric 2 hDPP4 (c2hDPP4) and chimeric 2 mDPP4 (c2mDPP4); in addition to its significant effect on the DPP4 flexibility. The Thr288 residue is known for its critical function in MERS-CoV RBD interaction. Moreover, MERS-CoV RBD adopts a "standing" conformation when docked to hDPP4 and cmDPP4 in blade IV and V regions. In conclusion, the results could explain the functionality differences between mouse and human DPP4 receptors against MERS-CoV. However, further structural studies are needed to evaluate how DPP4 conformations affects MERS-CoV RBD binding and affinity.Communicated by Ramaswamy H. Sarma.

Development of a recombinant vaccine containing a spike S1-Fc fusion protein induced protection against MERS-CoV in human DPP4 knockin transgenic mice.

The Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the family Coronaviridae and genus Betacoronavirus, has been recognized as a highly pathogenic virus. Due to the lack of therapeutic or preventive agents against MERS-CoV, developing an effective vaccine is essential for preventing a viral outbreak. To address this, we developed a recombinant S1 subunit of MERS-CoV spike protein fused with the human IgG4 Fc fragment (LV-MS1-Fc) in Chinese hamster ovary (CHO) cells. Thereafter, we identified the baculovirus gp64 signal peptide-directed secretion of LV-MS1-Fc protein in the extracellular fluid. To demonstrate the immunogenicity of the recombinant LV-MS1-Fc proteins, BALB/c mice were inoculated with 2.5 µg of LV-MS1-Fc. The inoculated mice demonstrated a significant humoral immune response, measured via total IgG and neutralizing antibodies. In addition, human dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with LV-MS1-Fc showed the protective capacity of LV-MS1-Fc against MERS-CoV with no inflammatory cell infiltration. These data showed that the S1 and Fc fusion protein induced potent humoral immunity and antigen-specific neutralizing antibodies in mice, and conferred protection against coronavirus viral challenge, indicating that LV-MS1-Fc is an effective vaccine candidate against MERS-CoV infection.

Comparative Study of SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-229E and Influenza Host Gene Expression in Asthma: Importance of Sex, Disease Severity, and Epithelial Heterogeneity.

Epithelial characteristics underlying the differential susceptibility of chronic asthma to SARS-CoV-2 (COVID-19) and other viral infections are currently unclear. By revisiting transcriptomic data from patients with Th2 low versus Th2 high asthma, as well as mild, moderate, and severe asthmatics, we characterized the changes in expression of human coronavirus and influenza viral entry genes relative to sex, airway location, and disease endotype. We found sexual dimorphism in the expression of SARS-CoV-2-related genes ACE2, TMPRSS2, TMPRSS4, and SLC6A19. ACE2 receptor downregulation occurred specifically in females in Th2 high asthma, while proteases broadly assisting coronavirus and influenza viral entry, TMPRSS2, and TMPRSS4, were highly upregulated in both sexes. Overall, changes in SARS-CoV-2-related gene expression were specific to the Th2 high molecular endotype of asthma and different by asthma severity and airway location. The downregulation of ACE2 (COVID-19, SARS) and ANPEP (HCoV-229E) viral receptors wascorrelated with loss of club and ciliated cells in Th2 high asthma. Meanwhile, the increase in DPP4 (MERS-CoV), ST3GAL4, and ST6GAL1 (influenza) was associated with increased goblet and basal activated cells. Overall, this study elucidates sex, airway location, disease endotype, and changes in epithelial heterogeneity as potential factors underlying asthmatic susceptibility, or lack thereof, to SARS-CoV-2.

A Comparative Analysis of Coronavirus Nucleocapsid (N) Proteins Reveals the SADS-CoV N Protein Antagonizes IFN-ß Production by Inducing Ubiquitination of RIG-I.

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Metagenomic analysis of fecal and tissue samples from 18 endemic bat species in Switzerland revealed a diverse virus composition including potentially zoonotic viruses.

Many recent disease outbreaks in humans had a zoonotic virus etiology. Bats in particular have been recognized as reservoirs to a large variety of viruses with the potential to cross-species transmission. In order to assess the risk of bats in Switzerland for such transmissions, we determined the virome of tissue and fecal samples of 14 native and 4 migrating bat species. In total, sequences belonging to 39 different virus families, 16 of which are known to infect vertebrates, were detected. Contigs of coronaviruses, adenoviruses, hepeviruses, rotaviruses A and H, and parvoviruses with potential zoonotic risk were characterized in more detail. Most interestingly, in a ground stool sample of a Vespertilio murinus colony an almost complete genome of a Middle East respiratory syndrome-related coronavirus (MERS-CoV) was detected by Next generation sequencing and confirmed by PCR. In conclusion, bats in Switzerland naturally harbour many different viruses. Metagenomic analyses of non-invasive samples like ground stool may support effective surveillance and early detection of viral zoonoses.

Gene signatures and potential therapeutic targets of Middle East respiratory syndrome coronavirus (MERS-CoV)-infected human lung adenocarcinoma epithelial cells.

BACKGROUND: Pathogenic coronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. These viruses have induced outbreaks worldwide, and there are currently no effective medications against them. Therefore, there is an urgent need to develop potential drugs against coronaviruses. METHODS: High-throughput technology is widely used to explore differences in messenger (m)RNA and micro (mi)RNA expression profiles, especially to investigate protein-protein interactions and search for new therapeutic compounds. We integrated miRNA and mRNA expression profiles in MERS-CoV-infected cells and compared them to mock-infected controls from public databases. RESULTS: Through the bioinformatics analysis, there were 251 upregulated genes and eight highly differentiated miRNAs that overlapped in the two datasets. External validation verified that these genes had high expression in MERS-CoV-infected cells, including RC3H1, NF-κB, CD69, TNFAIP3, LEAP-2, DUSP10, CREB5, CXCL2, etc. We revealed that immune, olfactory or sensory system-related, and signal-transduction networks were discovered from upregulated mRNAs in MERS-CoV-infected cells. In total, 115 genes were predicted to be related to miRNAs, with the intersection of upregulated mRNAs and miRNA-targeting prediction genes such as TCF4, NR3C1, and POU2F2. Through the Connectivity Map (CMap) platform, we suggested potential compounds to use against MERS-CoV infection, including diethylcarbamazine, harpagoside, bumetanide, enalapril, and valproic acid. CONCLUSIONS: The present study illustrates the crucial roles of miRNA-mRNA interacting networks in MERS-CoV-infected cells. The genes we identified are potential targets for treating MERS-CoV infection; however, these could possibly be extended to other coronavirus infections.