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. IMPORTANCEThe novel emerging coronavirus MERS-CoV has infected >1,600 people worldwide, and the case fatality rate is ϳ36%. 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. Middle East respiratory syndrome coronavirus (MERS-CoV) has been detected in Ͼ1,600 patients, and the case fatality rate is 36%. Although the majority of cases have occurred in Saudi Arabia (80%), an outbreak in South Korea sparked by a patient with a history of travel in the Middle East highlights the potential for MERS-CoV to be transmitted via the nosocomial route if no appropriate measures are taken (1). MERS-CoV has an unusual broad host tropism that includes humans and dromedary camels. A better understanding of the molecular basis of the host tropism will help determine the restrictions of potential host species and improve the functional design of animal models and the development of medical countermeasures. Several animal models for MERS-CoV have been developed. Nonhuman primates (NHPs) (2-4) and dromedary camels (5) are naturally susceptible. In addition, several mouse models have been developed, and expression of the human variant of the receptor of MERS-CoV, dipeptidyl peptidase 4 (DPP4), in mice allows viral replication (6)(7)(8).No other small-animal models have been developed. Therefore, if a treatment against MERS-CoV is shown to be successful in the mouse model, further characterization of the treatment needs to be performed in NHPs, a relatively expensive animal model to which access is limited. The availability of a second small-animal model (such as hamsters with a modified DPP4 [9,10]) to confirm results obtained with the mouse model would ensure that only treatments with a high likelihood of succeeding would be investigated in NHPs.Fourteen amino acids are important in the interaction between blades IV and V of human DPP4 (hDPP4) and the receptor binding domain (RBD) of the MERS-CoV spike protein (11,12). We previously showed that hamster DPP4 (haDPP4) does not function as a receptor for MERS-CoV. This restriction is caused by 5 out of 14 interacting amino acids which differ between hDPP4 and haDPP4 ( Fig. 1) (13). In the study described here, we analyzed the minimal combination of these 5 amino acids (aa) allowing haDPP4 to function as a receptor for MERS-CoV. Cells and virus....

Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants

“…In the case of haDPP4, the L288 amino acid identity is conserved between the hamster and human sequences, suggesting that different determinants in haDPP4 and mDPP4 are responsible for blocking MERS-CoV infection. This is consistent with prior studies which identified several amino acid differences between hDPP4 and haDPP4 in blade V that were required to support MERS-CoV infection (17). Therefore, we asked whether specific changes in blade V of haDPP4 could allow it to act as a functional MERS-CoV receptor in combination with the blade IV glycosylation knockout mutation.…”

“…These values can be compared to amino acid sequence identity values of 85%, 88%, 85%, and 87%, respectively. The high sequence similarities and predicted structural similarities between hDPP4 and these orthologs suggest that they can likely act as backbones to support MERS-CoV infection, consistent with previous DPP4 ortholog work (15,17).…”

“…Glycosylation sites that are upstream of (column 1), at the same site as (column 2), or downstream of (column 3) the glycosylation site that is present in mDPP4 (residues 328 to 330) are plotted adjacent to the phylogenetic tree. Permissivity data are indicated in the far right column, with green squares indicating permissive species and red squares indicating nonpermissive species, based on either in vitro or in vivo data (10)(11)(12)(13)(14)(15)(16)(17).…”

“…For MERS-CoV, the host cell receptor is dipeptidyl peptidase IV (DPPIV) (9), a ubiquitously expressed cell surface protein that functions in immune homeostasis. Interestingly, whereas MERS-CoV can utilize bat DPP4 (bDPP4), camel DPP4 (cDPP4), and human DPP4 (hDPP4) molecules for entry (10)(11)(12), it is unable to infect cells using the DPP4 molecules from traditional smallanimal models, including mice, ferrets, guinea pigs, and hamsters (13)(14)(15)(16)(17). The inability of MERS-CoV to infect these species in vivo and in vitro is primarily due to spike-receptor incompatibilities and not due to other species-specific host cell factors (13,18,19).…”

Adaptive Evolution of MERS-CoV to Species Variation in DPP4

“…Structural studies have shown that this interaction relies on multiple contact points (Lu et al, 2013;Song et al, 2014;Wang et al, 2013). Our group has previously demonstrated that hamster DPP4 contains variation in these contact points, which prevents MERS-CoV replication (van van Doremalen et al, 2016). Other genetic variation responsible for the glycosylation of DPP4 in mice, hamsters, ferrets, and guinea pigs forms an additional block to the interaction with MERS-CoV spike (Cockrell et al, 2014;Peck et al, 2017).…”