Mapping the Specific Amino Acid Residues That Make Hamster DPP4 Functional as a Receptor for Middle East Respiratory Syndrome Coronavirus.
ABSTRACT: The novel emerging coronavirus Middle East respiratory syndrome coronavirus (MERS-CoV) binds to its receptor, dipeptidyl peptidase 4 (DPP4), via 14 interacting amino acids. We previously showed that if the five interacting amino acids which differ between hamster and human DPP4 are changed to the residues found in human DPP4, hamster DPP4 does act as a receptor. Here, we show that the functionality of hamster DPP4 as a receptor is severely decreased if less than 4 out of 5 amino acids are changed.The novel emerging coronavirus MERS-CoV has infected >1,600 people worldwide, and the case fatality rate is ?36%. In this study, we show that by changing 4 amino acids in hamster DPP4, this protein functions as a receptor for MERS-CoV. This work is vital in the development of new small-animal models, which will broaden our understanding of MERS-CoV and be instrumental in the development of countermeasures.
Project description:UNLABELLED:Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir. IMPORTANCE:The ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) has caused 701 laboratory-confirmed cases to date, with 249 fatalities. Although bats and dromedary camels have been identified as potential MERS-CoV hosts, the virus has so far not been isolated from any species other than humans. The inability of MERS-CoV to infect commonly used animal models, such as hamster, mice, and ferrets, indicates the presence of a species barrier. We show that the MERS-CoV receptor DPP4 plays a pivotal role in the observed species tropism of MERS-CoV and subsequently identified the amino acids in DPP4 responsible for this restriction. Using a combined modeling and experimental approach, we predict that, based on the ability of MERS-CoV to utilize the DPP4 of common Middle East livestock species, such as camels, goats, sheep, and cows, these form a potential MERS-CoV intermediate host reservoir species.
Project description:Middle East respiratory syndrome (MERS) coronavirus (Co-V) contains a single spike (S) protein, which binds to a receptor molecule, dipeptidyl peptidase 4 (DPP4; also known as CD26), and serves as a neutralizing antigen. Pseudotyped viruses are useful for measuring neutralization titers against highly infectious viruses as well as for studying their mechanism of entry. In this study, we constructed a series of cytoplasmic deletion mutants of MERS-CoV S and compared the efficiency with which they formed pseudotypes with vesicular stomatitis virus. A pseudotype bearing an S protein with the C-terminal 16 amino acids deleted (MERSpv-St16) reached a maximum titer that was approximately tenfold higher than that of a pseudotype bearing a non-truncated full-length S protein. Using MERSpv-St16, we demonstrated the inability of rat DPP4 to serve as a functional receptor for MERS-CoV, suggesting that rats are not susceptible to MERS-CoV infection. This study provides novel information that enhances our understanding of the host range of MERS-CoV.
Project description: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.
Project description: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 ?-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.
Project description: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.
Project description:Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4.
Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. While bat, camel, and human DPP4 support MERS-CoV infection, several DPP4 orthologs, including mouse, ferret, hamster, and guinea pig DPP4, do not. Previous work revealed that glycosylation of mouse DPP4 plays a role in blocking MERS-CoV infection. Here, we tested whether glycosylation also acts as a determinant of permissivity for ferret, hamster, and guinea pig DPP4. We found that, while glycosylation plays an important role in these orthologs, additional sequence and structural determinants impact their ability to act as functional receptors for MERS-CoV. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and better inform our understanding of virus-receptor interactions associated with disease emergence and host susceptibility.IMPORTANCE MERS-CoV is a recently emerged zoonotic virus that is still circulating in the human population with an ?35% mortality rate. With no available vaccines or therapeutics, the study of MERS-CoV pathogenesis is crucial for its control and prevention. However, in vivo studies are limited because MERS-CoV cannot infect wild-type mice due to incompatibilities between the virus spike and the mouse host cell receptor, mouse DPP4 (mDPP4). Specifically, mDPP4 has a nonconserved glycosylation site that acts as a barrier to MERS-CoV infection. Thus, one mouse model strategy has been to modify the mouse genome to remove this glycosylation site. Here, we investigated whether glycosylation acts as a barrier to infection for other nonpermissive small-animal species, namely, ferret, guinea pig, and hamster. Understanding the virus-receptor interactions for these DPP4 orthologs will help in the development of additional animal models while also revealing species-specific differences impacting MERS-CoV host range.
Project description: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.
Project description:BACKGROUND: The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) that first appeared in Saudi Arabia during the summer of 2012 has to date (20th September 2013) caused 58 human deaths. MERS-CoV utilizes the dipeptidyl peptidase 4 (DPP4) host cell receptor, and analysis of the long-term interaction between virus and receptor provides key information on the evolutionary events that lead to the viral emergence. FINDINGS: We show that bat DPP4 genes have been subject to significant adaptive evolution, suggestive of a long-term arms-race between bats and MERS related CoVs. In particular, we identify three positively selected residues in DPP4 that directly interact with the viral surface glycoprotein. CONCLUSIONS: Our study suggests that the evolutionary lineage leading to MERS-CoV may have circulated in bats for a substantial time period.
Project description: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.