The Receptor Binding Domain of the New Middle East Respiratory Syndrome Coronavirus Maps to a 231-Residue Region in the Spike Protein That Efficiently Elicits Neutralizing Antibodies

The spike (S) protein of the recently emerged human Middle East respiratory syndrome coronavirus (MERS-CoV) mediates infection by binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Here we mapped the receptor binding domain in the S protein to a 231-amino-acid fragment (residues 358 to 588) by evaluating the interaction of spike truncation variants with receptor-expressing cells and soluble DPP4. Antibodies to this domain—much less so those to the preceding N-terminal region—efficiently neutralize MERS-CoV infection.     

Glycosylation of Mouse DPP4 Plays a Role in Inhibiting Middle East Respiratory Syndrome Coronavirus Infection

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development.     

Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 β-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.     

Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine.     

Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.     

Modulation of the immune response by Middle East respiratory syndrome coronavirus

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. In 2012, a new human disease called Middle East respiratory syndrome (MERS) emerged in the Middle East. MERS was caused by a virus that was originally called human coronavirus-Erasmus Medical Center/2012 but was later renamed as Middle East respiratory syndrome coronavirus (MERS-CoV). MERS-CoV causes high fever, cough, acute respiratory tract infection, and multiorgan dysfunction that may eventually lead to the death of the infected individuals. The exact origin of MERS-CoV remains unknown, but the transmission pattern and evidence from virological studies suggest that dromedary camels are the major reservoir host, from which human infections may sporadically occur through the zoonotic transmission. Human to human transmission also occurs in healthcare facilities and communities. Recent studies on Middle Eastern respiratory continue to highlight the need for further understanding the virus-host interactions that govern disease severity and infection outcome. In this review, we have highlighted the major mechanisms of immune evasion strategies of MERS-CoV. We have demonstrated that M, 4a, 4b proteins and Plppro of MERS-CoV inhibit the type I interferon (IFN) and nuclear factor-κB signaling pathways and therefore facilitate innate immune evasion. In addition, nonstructural protein 4a (NSP4a), NSP4b, and NSP15 inhibit double-stranded RNA sensors. Therefore, the mentioned proteins limit early induction of IFN and cause rapid apoptosis of macrophages. MERS-CoV strongly inhibits the activation of T cells with downregulation of antigen presentation. In addition, uncontrolled secretion of interferon ɣ-induced protein 10 and monocyte chemoattractant protein-1 can suppress proliferation of human myeloid progenitor cells.     

Spiking the MERS-coronavirus receptor

A novel coronavirus, the Middle East respiratory syndrome coronavirus, recently emerged through zoonotic transmission, causing a severe lower respiratory tract infection in humans. In two recent papers, one published in Cell Research, the crystal structure of the viral receptor-binding domain in complex with the host CD26/dipeptidyl peptidase 4 receptor has now been characterized.In mid 2012, a novel coronavirus (CoV) was isolated form the sputum of a patient with acute pneumonia and renal failure¹. As of July 10^th 2013, this virus, named Middle East respiratory syndrome (MERS)-CoV, has caused 80 laboratory-confirmed infections of which 44 were fatal². The limited data available suggest that the virus is introduced into the human population through multiple independent, zoonotic transmission events from a — so far unknown — animal source with subsequent limited human-to-human spread. However, scenarios in which a single zoonotic transmission event has led to sustained, largely asymptomatic and non-detected human-to-human transmission cannot be excluded yet. Genetically, MERS-CoV is related to SARS-CoV, which killed nearly 10% of approximately 8 000 persons that were infected in the 2003 outbreak. It is therefore of utmost importance to better understand the biology and pathogenesis of this virus.Coronaviruses infect mammals and birds, and occasionally cross the species barrier. The primary determinant of coronavirus host and cell tropism is the viral spike (S) entry protein that functions by binding to a cell surface receptor. The MERS-CoV S protein is a type I membrane glycoprotein, assembled as trimers that constitute the typical crown-like peplomers on the surface of the enveloped coronavirus. Functionally, two regions, S1 and S2, can be defined in the S protein, which are involved in binding and fusion with host cells, respectively. Recent studies have mapped the receptor-binding domain (RBD) to a ∼231-amino acid long region within the S1 region of MERS-CoV³.MERS-CoV uses a cell surface amino peptidase, dipeptidyl peptidase 4 (DPP4), also known as CD26, as a functional receptor⁴. The multifunctional DPP4 — highly conserved among mammals — plays a major role in glucose metabolism by its degradation of incretins. It has been further implicated in T-cell activation, chemotaxis modulation, cell adhesion, and apoptosis⁵. In humans, it is primarily expressed on the epithelial cells in the lungs, kidney, small intestine, liver and prostate, and on activated leukocytes, while it also occurs in a soluble form in the circulation^4,5.The spike-receptor binding interface can be seen as a lock-and-key interaction where minor mutations within the interacting domain of the S protein or the receptor can abrogate infection, placing a barrier for cross-species transmission. Zoonotic potential of coronaviruses has been attributed to the adaptability of the S protein to human receptor orthologs. Intriguingly, the MERS-CoV S protein seems promiscuous in binding to orthologous receptors. Whereas coronaviruses generally tend to have a narrow host tropism, MERS-CoV can infect cells of a wide variety of species, at least in vitro^4,6. The broad cell species tropism suggests that MERS-CoV has acquired facile cross-species transmissibility by binding to an evolutionarily conserved receptor.Just four months after the discovery of the receptor, two Chinese research teams have now independently described the MERS-CoV spike-receptor interface. The study by Wang et al.⁷ recently published in Cell Research, and a recent study by Lu et al.⁸ published in Nature, both reveal the crystal structure of the RBD of the MERS-CoV S protein bound to its receptor, human DPP4. DPP4, of which the structure was published before⁹, consists of an N-terminal eight-bladed β-propeller domain and a C-terminal α/β-hydrolase domain. The RBD of the MERS-CoV S protein contains two subdomains: a conserved core subdomain and a receptor-binding subdomain, with the latter contacting blades 4 and 5 of the DPP4 β-propeller domain. Structural comparison with the RBD of the related betacoronavirus SARS-CoV (using the ACE2 peptidase as a receptor) reveals a conserved core domain and highly variable — both in length and in residues — receptor-binding region, explaining the differential receptor usage.Both teams have scrutinized the RBD-receptor interface and identified critical residues within the interacting domain of the S protein or receptor, which allow MERS-CoV to bind to its receptor. Structural analysis and mutational analysis have identified several key residues in the RBD of the S protein shown to be critical for DPP4 binding and viral entry. This information is crucial to understand the adaptation of MERS-CoV to humans. Studies with SARS-CoV isolated from humans and civet cats (which function as the intermediate host) revealed 2 amino acids in the RBD that caused an > 1 000-fold difference in binding affinity to human receptor ACE2¹⁰. Analysis of the MERS-CoV RBD sequences of the isolates characterized thus far shows no sequence variation except that 2 virus samples isolated from patients in the UK (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"KC667074","term_id":"453061240","term_text":"KC667074"}}KC667074 and {"type":"entrez-nucleotide","attrs":{"text":"KC164505","term_id":"471258596","term_text":"KC164505"}}KC164505) had a leucine-to-phenylalanine substitution at position 506 of the S protein (L506F). As shown by Wang et al.⁷, residue L506 contacts DPP4 and its substitution to alanine reduced MERS-CoV S-mediated infectivity by over 50%.With the structure available, the promiscuous binding of MERS-CoV to DPP4 orthologs can now be analyzed at the molecular level. Relevant to functional usage of orthologous receptors by MERS-CoV is the degree of conservation of the amino acid residues in DPP4 that were identified to contact the viral RBD^7,8. DPP4 sequence comparison reveals that mammalian DPP4 orthologs (e.g., of macaque, horse, rabbit and pig) have no or little variation for residues contacting MERS-CoV RBD in human DPP4 (

二肽基肽酶4为人类中东呼吸道综合征冠状病毒MERS-CoV功能性细胞受体

中东呼吸道综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）是继SARS冠状病毒（SARS-CoV）之后新近出现的又一种能够引发严重呼吸道感染的人类新发冠状病毒. MERS-CoV于2012年9月首次在中东一些国家被发现,截至2013年9月7日,MERS-CoV已经引起114例感染病例,其中54人死亡,死亡率约50%. 病毒受体研究为MERS-CoV等人类新发冠状病毒进化和跨种传播机制提供重要依据．最近,Raj等在Nature发表文章,首次报道了二肽基肽酶4（dipeptidyl peptidase 4,DPP4;又名CD26）为MERS-CoV感染细胞的功能性受体．MERS-CoV功能性受体的发现为人类新冠状病毒溯源和跨种进化研究、病毒传染和流行病学特征分析以及抗病毒药物和疫苗研究提供重要基础.     

CD26/DPP4 Cell-Surface Expression in Bat Cells Correlates with Bat Cell Susceptibility to Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection and Evolution of Persistent Infection

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.     

Middle East Respiratory Syndrome Coronavirus Intra-Host Populations Are Characterized by Numerous High Frequency Variants

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater than 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.     

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050)samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1–10 years (78.65%) and B 3 years (58.19%).Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016–2017. Among the sampled population, 840 humans were camel herders.Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.     

Mouse Dipeptidyl Peptidase 4 Is Not a Functional Receptor for Middle East Respiratory Syndrome Coronavirus Infection

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics.     

Laboratory Investigation and Phylogenetic Analysis of an Imported Middle East Respiratory Syndrome Coronavirus Case in Greece

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1^st and 2^nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.     

A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines

An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.     

Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.     

Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County,Kenya

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified virus causing severe viral respiratory illness in people. Little is known about the reservoir in the Horn of Africa. In Kenya, where no human MERS cases have been reported, our survey of 335 dromedary camels, representing nine herds in Laikipia County, showed a high seroprevalence (46.9%) to MERS-CoV antibodies. Between herd differences were present (14.3%– 82.9%), but was not related to management type or herd isolation. Further research should focus on identifying similarity between MERS-CoV viral isolates in Kenya and clinical isolates from the Middle East and elsewhere.     

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8⁺ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.