Content uploaded by Neeltje van Doremalen
Author content
All content in this area was uploaded by Neeltje van Doremalen on Jun 30, 2014
Content may be subject to copyright.
Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through its Receptor 1
Dipeptidyl Peptidase 4 2
3
Neeltje van Doremalen1, Kerri L. Miazgowicz1, Shauna Milne-Price1, Trenton Bushmaker1, Shelly 4
Robertson1, Dana Scott2, Joerg Kinne3, Jason S. McLellan4, Jiang Zhu5, Vincent J. Munster1# 5
6
7
1. Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and 8
Infectious Diseases, National Institutes of Health, Hamilton, MT, USA 9
2. Rocky Mountain Veterinary Branch, Division of Intramural Research, National Institute of 10
Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA 11
3. Central Veterinary Research Laboratories, Dubai, P.O. Box 597, Dubai, UAE 12
4. Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH, USA 13
5. Department of Immunology and Microbial Science, Department of Integrative Structural and 14
Computational Biology, The Scripps Research Institute, La Jolla, CA, USA 15
16
Running title: Species tropism of MERS-CoV is determined by DPP4 17
18
Word count abstract: 250 19
Word count text: 4670 20
21
#Corresponding author: Vincent Munster, vincent.munster@nih.gov 22
JVI Accepts, published online ahead of print on 4 June 2014
J. Virol. doi:10.1128/JVI.00676-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Abstract 23
Middle East Respiratory Syndrome coronavirus (MERS-CoV) emerged in 2012. Recently the MERS-24
CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-25
binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. 26
Animal studies identified rhesus macaques but not hamsters, ferrets or mice to be susceptible for MERS-27
CoV. Here we investigated the role of DPP4 in this observed species tropism. Cell lines of human and 28
non-human primate origin were permissive of MERS-CoV, whereas hamster, ferret or mouse cell lines 29
were not, despite presence of DPP4. Expression of human DPP4 in non-susceptible BHK and ferret cells 30
enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the 31
binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (non-32
susceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of 33
hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-34
CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using 35
the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock 36
was investigated. Modeling of the DPP4 MERS-CoV RBD interaction predicted the ability of MERS-37
CoV to bind the DPP4s of camel, goat, cow and sheep. Expression of the DPP4s of these species on BHK 38
cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the 39
respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir. 40
41
Importance 42
The ongoing outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV) has caused 184 43
laboratory confirmed cases to date, with 80 fatalities. Although bats and dromedary camels have been 44
identified as potential MERS-CoV hosts, the virus has so far not been isolated from any species other 45
than humans. The inability of MERS-CoV to infect commonly used animal models such as hamster, mice 46
and ferrets, indicates the presence of a species barrier. We show that the MERS-CoV receptor DPP4 plays 47
a pivotal role in the observed species tropism of MERS-CoV and subsequently identified the amino acids 48
in DPP4 responsible for this restriction. Using a combined modeling and experimental approach we 49
predict that based on the ability of MERS-CoV to utilize the DPP4 of common Middle East livestock 50
species, such as camels, goats, sheep and cows, these form a potential MERS-CoV intermediate host 51
reservoir species. 52
53
Introduction 54
55
The Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 in a patient 56
from Saudi-Arabia (1). To date, 496 laboratory confirmed cases have been reported in eight different 57
countries, with an estimated 20-25% case fatality rate (2). MERS-CoV is a positive strand RNA virus 58
belonging to the C lineage within the Betacoronavirus genus and is genetically closely related to 59
coronavirus sequences obtained from insectivorous bats originating from Europe, Asia, Africa and the 60
Middle East (1, 3-5). The detection of MERS-CoV neutralizing antibodies as well as the recovery of viral 61
sequences and virus in dromedary camels across the countries of the Middle East suggests the potential 62
involvement of an intermediate reservoir in the emergence of MERS-CoV in humans (6-10). 63
Phylogenetic analysis of MERS-CoV genomes obtained from 43 human cases in Saudi Arabia suggests 64
the occurrence of multiple zoonotic spill-over events (11, 12). 65
Similarly to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), another Betacoronavirus 66
which caused the SARS pandemic, MERS-CoV appears to primarily target the lower respiratory tract 67
causing acute respiratory distress in severe human cases (2, 13, 14). However, in contrast to SARS-CoV, 68
which uses angiotensin converting enzyme 2 (ACE2) as its cellular host receptor (15), MERS-CoV 69
utilizes dipeptidyl peptidase 4 (DPP4, also known as CD26) (16). Interaction of the receptor binding 70
domain (RBD) of the MERS-CoV spike protein with DPP4 initiates attachment to the host cell and 71
subsequent virus internalization. The RBD was mapped to be a 231 amino acid region in the S1 subunit of 72
the spike protein (17). DPP4 is a type II transmembrane glycoprotein, involved in cleavage of dipeptides 73
and degradation of incretins (18). DPP4 is widely expressed in different tissues, such as lungs and kidney 74
and the cells of the immune system, although a detailed description of DPP4 expression in the human 75
respiratory tract and kidney is currently not available. DPP4 is relatively conserved between mammalian 76
species, allowing the MERS-CoV spike protein to bind to both bat and human DPP4 (16, 18). 77
In vitro studies using a variety of different primary and immortalized cell lines reported a broad tropism 78
of MERS-CoV (19-22). Most cell lines with a human-, bat-, non-human primate- or swine-origin were 79
found to be susceptible to infection with MERS-CoV. In contrast, cell lines originating from mice, 80
hamsters, dogs and cats were not susceptible to MERS-CoV infection (16, 19). In vitro data on the 81
species tropism of MERS-CoV appears to correlate with the in vivo host restriction of MERS-CoV; 82
rhesus macaques can be experimentally infected with MERS-CoV, whereas inoculation of other 83
commonly used animal models such as the Syrian hamster, mouse or ferret did not result in efficient viral 84
replication (23-27). Recent studies suggest that DPP4 plays an important role in the non-susceptibility of 85
the mouse and ferret to MERS-CoV (28-30). 86
Here, we investigated the host species restriction of MERS-CoV and the role of the DPP4 receptor in this 87
observed species tropism. Differences in DPP4 between MERS-CoV permissive and non-permissive 88
species were identified to be responsible for the ability of DPP4 to function as the MERS-CoV receptor. 89
90
Materials and methods 91
92
Biosafety statement 93
All infectious work with MERS-CoV was performed in a high containment facility at the Rocky 94
Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and 95
Infectious Diseases (NIAID), National Institutes of Health (NIH). The work was approved by the RML 96
Institutional Biosafety Committee (IBC) at biosafety level 3 (BSL3). 97
98
Ethics statement 99
Fresh animal tissues were obtained from local slaughter facilities (cow, goat and sheep), from an in-house 100
tissue repository (rhesus macaque and mouse) or collected under a tissue sampling protocol (hamster and 101
ferret) approved by the Institutional Animal Care and Use Committee of the Rocky Mountain 102
Laboratories, and the collection was performed following the guidelines of the Association for 103
Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in 104
an AAALAC approved facility. 105
106
Cells and virus 107
Huh-7 (human carcinoma), Vero (African green monkey kidney), BHK (baby hamster kidney), 3T3 108
(mouse embryonic fibroblast) and MEF C57Bl6 (mouse embryonic fibroblast) cells were maintained in 109
Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovin serum (FBS), 2 mM L-110
Glutamine, 50 U/ml penicillin and 50 ȝg/ml of streptomycin (culture DMEM). Primary ferret kidney cells 111
were generated as follows: within 30 minutes of tissue collection, the fibrous capsule, adjacent medulla 112
and any fat, blood cloths and connective tissue were dissected from the ferret kidney which was 113
subsequently cut into small pieces. The tissue sample was washed with ice-cold Hank’s balanced salt 114
solution (HBSS) containing 10 mM EGTA until the supernatant was clear and further cut into 1 mm3 115
pieces. Hereafter, the tissue sample was incubated at 37°C for 20 minutes whilst rolling in 25 ml of warm 116
non-supplemented DMEM/F12 GlutaMAX media containing 1 mg/ml collagenase (Worthington) and 117
passed through a 100 μm sieve, a 70 μm sieve and a 40 μm sieve. Supernatant was centrifuged at 400 g 118
for 5 min at 4 °C and washed 3 times with HBSS. The pellet was then resuspended in DMEM/F12 119
GlutaMAX media supplemented with 10% FBS, 50 U/ml penicillin, 50 ȝg/ml of streptomycin, 2.5 μg/ml 120
Fungizone and 5 μg/ml human transferrin (Sigma Aldrich) and cells were seeded at a density of 5 x 104 121
cells/cm2. The ferret primary kidney cell line was maintained in DMEM/F12 + GlutaMAX supplemented 122
with 10% FBS, 50 U/ml penicillin, 50 ȝg/ml of streptomycin, 2.5 μg/ml Fungizone and 50 μg/ml human 123
transferrin. All cell lines were maintained at 37°C in 5% CO2. All reagents were purchased from Gibco, 124
unless otherwise specified. MERS-CoV (strain HCoV-EMC/2012) was propagated on VeroE6 cells 125
using DMEM supplemented with 2% FBS, 2 mM L-Glutamine, 50 U/ml penicillin and 50 ȝg/ml of 126
streptomycin (complete DMEM). MERS-CoV was titrated by end-point titration in quadruplicate in 127
VeroE6 cells cultured in complete DMEM as follows: cells were inoculated with ten-fold serial dilutions 128
of virus, and scored for cytopathic effect 5 days later. TCID50 was calculated by the method of Spearman-129
Karber. 130
131
DPP4 western blot analysis 132
Cells were washed in phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation (RIPA) 133
buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium-deoxycholate, 0.1% SDS and 134
protease inhibitor cocktail tablets (Sigma)). Lysates were treated with TURBOTM DNase (Life 135
Technologies). Protein concentrations were determined with the bicinchoninic assay (Thermo Scientific). 136
Cellular lysates were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Life 137
Technologies). After blocking in 5% non-fat milk powder in PBS-0.1% Tween (Fisher Scientific), 138
membranes were incubated overnight with Į-DPP4 rabbit polyclonal antibody (1:700, AbCam, ab28340) 139
or an Į-actin antibody (1:5000, Sigma Aldrich, A5441). Membranes were then incubated with a 140
horseradish peroxidase conjugated Į-rabbit or Į-mouse IgG respectively (1:12,500, Jackson 141
Immunoresearch). Signals were detected with Pierce ECL 2 Western Blotting Substrate (Thermo 142
Scientific) and developed on blue autoradiography film (GeneMate). 143
144
Immunohistochemistry 145
Immunohistochemistry was performed as described previously (24) using an Į-DPP4 rabbit polyclonal 146
antibody (Abcam, ab28340) at a 1:400 dilution for rhesus macaque, mouse, ferret, sheep, goat and cow or 147
a 1:800 dilution for hamster, and biotinylated anti-rabbit SS link (undiluted, Biogenex, HK336-9R) as a 148
secondary antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin and 149
processed for immunohistochemistry using the Discovery XT automated processor (Ventana Medical 150
Systems) with a DapMap kit (Ventana Medical Systems). 151
152
Sequencing and cloning of DPP4 sequences 153
Total RNA from lung and kidney samples from different species was extracted using the RNeasy Mini 154
Kit (Qiagen) and cDNAs were synthesized using random hexamers and SuperScript III Reverse 155
Transcriptase (Applied Biosystems). Complete DPP4 genes were amplified using iProof High-Fidelity 156
DNA Polymerase (BioRad) and in-house designed primers (sequence available upon request). 157
158
Plasmids 159
DPP4 amplicons were sequenced by Sanger sequencing and sequences were aligned using the MEGA5.2 160
software package. DPP4 gene sequences for each species were synthesized in expression plasmid 161
pcDNA3.1(+) (GeneArt). All newly generated DPP4 nucleotide sequences are available from GenBank 162
under accession numbers KF574262–8. Mutagenized DPP4 expression plasmids were generated by 163
synthesizing hamster DPP4 containing five human-specific amino acid residues (Ala291, Ile295, Arg336, 164
Val341 and Ile346, humanized hamster) and human DPP4 containing five hamster-specific amino acid 165
residues (Glu291, Thr295, Thr336, Leu341 and Val346, hamsterized human), flanked by restriction sites 166
BamHI and BsgI (human DPP4) or BamHI and EcoRV (hamster DPP4). Human and hamster DPP4 in 167
pcDNA3.1(+) were restriction digested, purified and ligated with the humanized hamster or hamsterized 168
human DPP4 fragments respectively using T4 DNA ligase (New England Biolabs). Modified DPP4 169
sequences were confirmed by Sanger sequencing. 170
171
Transfection of cells 172
BHK and primary ferret cells were transfected with 3 ȝg pcDNA3.1(+) containing the DPP4 genes from 173
different species or pCAGGS-GFP using 8 ȝl of Lipofectamine 2000 (Life Technologies). DPP4 174
expression was confirmed by qRT-PCR and flow cytometry. 175
176
Replication kinetics 177
Multistep replication kinetics were determined by inoculating cells in triplicate with MERS-CoV with a 178
multiplicity of infection (MOI) of 0.01 (normal cell lines) or 1 (transfected cell lines) 50% tissue culture 179
infectious dose (TCID50) per cell. The lower MOI of 0.01 was chosen for experiments performed to 180
determine the ability of cell lines to support multiple replication cycles of MERS-CoV, whereas the 181
higher MOI of 1 was chosen for cells naturally non-susceptible for MERS-CoV but with the various 182
DPP4s transiently expressed to maximize the likelihood of the transfected cell to encounter MERS-CoV. 183
One hour after inoculation, cells were washed once with DMEM and fresh media was placed on the cells. 184
Supernatants were sampled at 0, 24, 48 and 72 h after inoculation, and virus titers in these supernatants 185
were determined as described. 186
187
Flow Cytometry 188
Cells were washed with PBS and removed with 5 mM EDTA (BHKs and 3T3s) or spun down from 189
suspension (primary ferret cells, huh7 cells, vero cells, and MEF) and then washed twice, resuspended in 190
PBS with 2% FBS and stained at 4°C using Į-human DPP4 antibody (R&D, AF1180), followed by 191
staining with FITC-tagged donkey anti-goat antibody (Life technologies, A11055). As a control, samples 192
of cells were stained with secondary antibody only. After staining, cells were washed, resuspended in 193
PBS with 2% FBS, stained with 7-amino actinomysin-D (Life Technologies) and analyzed immediately. 194
Samples were collected using a LSRII flow cytometer (BD Biosciences). Analysis gates were set on 195
viable cells and 10,000 gated events were analyzed for each sample. Data were analyzed using FlowJo 196
software (Treestar) comparing transfected cells against untransfected cells. 197
198
Binding energy modeling 199
The DPP4 homology models were constructed using the human DPP4 structure (PDB ID: 4KR0, Chain 200
A) as template. The sequence alignment was generated using CLUSTALW2 (31) and the initial model 201
was built using Nest (32) based on the alignment and the human DPP4 structure. The resulting structural 202
model was briefly optimized using the TINKER minimization program "minimize.x" with OPLS all-atom 203
force field and L-BFGS quasi-Newton optimization algorithm (33). For each species, the RBD/DPP4 204
complex model was generated by merging the RBD domain (PDB ID: 4KR0, Chain B) with the DPP4 205
model, which was then subjected to the binding energy calculation using an all-atom distance-dependent 206
pairwise statistical potential, DFIRE (34). The energy difference between the complex and two individual 207
structures - DPP4 and RBD - was taken as the binding energy. 208
209
qRT-PCR of DPP4 mRNA expression 210
Expression of DPP4 mRNA was measured via qRT-PCR. Total RNA was extracted from transfected 211
homogenized cells using the standard TRIzol-chloroform procedure (Life technologies), followed by 212
further extraction using the RNeasy mini kit (Qiagen) combined with a 30 minute on-column DNase I 213
(Qiagen) digestion according to manufacturer’s instructions. mRNA was purified from total RNA via the 214
NucleoTrap mRNA mini kit (Macherey-Nagel). One-step qRT-PCR was performed in three separate 215
experiments on the Rotor-GeneQ (Qiagen) for the detection of DPP4 and HPRT using the Quantifast 216
Probe PCR Master Mix (Qiagen) according to manufacturer’s instructions. Probes for DPP4 (FAM-217
AGCTTTGATGGCAGAGGAAGTGGT-BHQ1) and HPRT (FAM-218
ACTTTGTTGGATTTGAAATTCCAGACAAGTTTG-BHQ1) were designed using a cross-species high 219
conservancy region in the gene. Forward and reverse primer sets were species specific (Sequence 220
available upon request). Relative fold increase was calculated by the comparative CT method (35), where 221
DPP4 expression is normalized to HPRT. 222
223
Results 224
225
Replication kinetics of MERS-CoV in different cell lines 226
The replication kinetics of MERS-CoV was studied in cells of different mammalian origin: Huh-7 227
(human), Vero (African green monkey), BHK (hamster), MEFC57Bl6 and 3T3 (mouse) and ferret 228
primary kidney cells. MERS-CoV replicated efficiently in Huh-7 and Vero cells. In contrast, MERS-CoV 229
did not replicate in BHK, MEFC57Bl6, 3T3 and ferret primary kidney cells (Figure 1A). These data 230
correspond with the current information on the ability of MERS-CoV to infect humans and non-human 231
primates (rhesus macaques (23, 25)) and the inability of MERS-CoV to infect mice, hamsters and ferrets 232
(24, 26, 27). The presence of the MERS-CoV receptor, DPP4, is essential in the initiation of infection. To 233
investigate whether the lack of infection by MERS-CoV of non-susceptible cell lines was due to a lack of 234
expression of the DPP4 receptor we performed western blot analyses. DPP4 protein was detected in cell 235
lines both permissive and non-permissive for MERS-CoV infection although not uniformly found to be 236
expressed on the cell surface (Figure 1B, C). 237
238
Detection of DPP4 in tissues 239
To determine the cell types in which DPP4 was expressed in the lungs and kidney of rhesus macaque, 240
hamster, mouse and ferret, immunohistochemistry (IHC) was performed using an Į-DPP4 antibody. In 241
both the lungs and kidneys of the investigated species, DPP4 was found to be present. In the lungs, DPP4 242
was abundantly present on bronchiolar epithelium cells and occasionally present on alveolar interstitium, 243
or absent in the case of ferrets (Figure 2, Table 1). The intensity of the Į-DPP4 staining of bronchiolar 244
epithelium ranged from weak in ferrets, to moderate in the macaque and hamster and very intense in the 245
mouse. All species tested, except the ferret, also demonstrated weak Į-DPP4 immunoreactivity at the 246
level of alveoli. The kidney was similar to lung tissue in that all species demonstrated Į-DPP4 247
immunoreactivity to epithelial cells. The intensity of staining was again variable, with weak staining in 248
the hamster, moderate staining in the macaque and mouse and intense staining in ferret. DPP4 was present 249
on kidney vascular smooth muscle cells, with weak staining in the macaque, mouse, ferret and moderate 250
staining in the hamster. All species except the mouse displayed presence of DPP4 on either glomerular or 251
vascular endothelium with the strongest staining seen in the glomeruli of hamsters and ferrets. 252
The presence of DPP4 in cell lines non-susceptible to MERS-CoV and on cells in the respiratory tract of 253
non-permissive species (mouse, hamster and ferret) suggests that the inability of MERS-CoV to replicate 254
in these species is either due to an inability of the MERS-CoV spike protein to bind to the respective 255
DPP4s or an incompatibility of MERS-CoV with the cellular machinery of these respective species. 256
257
Specificity of MERS-CoV spike protein for DPP4 258
The DPP4 coding sequences of human, hamster and ferret, obtained from GenBank or by sequencing, 259
were cloned into expression vector pcDNA3.1(+) and transfected into cell lines non-susceptible to 260
MERS-CoV replication. The expression of DPP4 on transfected cells was determined by qRT-PCR and 261
flow cytometry (Figure 3B, C). Transient expression of human DPP4 in BHK and primary ferret kidney 262
cells allowed these previously non-susceptible cells to support MERS-CoV replication, whereas transient 263
expression of hamster DPP4 in BHK cells, ferret DPP4 in ferret primary cells or GFP in either cell type 264
did not render these cells susceptible to MERS-CoV replication (Figure 3A). As surface expression of 265
human DPP4, but not hamster or ferret DPP4, allowed MERS-CoV replication in previously non-266
susceptible cell lines, the observed MERS-CoV species tropism is most likely a result of the inability of 267
its spike protein to bind to hamster or ferret DPP4 rather than the incompatibility of MERS-CoV with the 268
hamster or ferret cellular machinery. 269
270
Structural modeling of MERS-CoV receptor binding domain with multispecies DPP4 271
Recent co-crystallization studies of the MERS-CoV spike protein and the human DPP4 identified 14 272
amino acids in DPP4 important in binding to the MERS-CoV spike protein (36, 37). Alignment of the 273
DPP4 amino acid sequences of human and hamster origin revealed a total of five differences within these 274
14 amino acids (Table 2). To investigate the binding potential of the different DPP4s to MERS-CoV 275
spike protein, DPP4 homology models were built using the human DPP4 structure (PDB ID: 4KR0, 276
Chain A) as a template (36). Of the five amino acid residues at the RBD interface that differ between 277
human and hamster DPP4, the residues at positions 291 and 336 appear to be most critical for the species 278
specificity. In the human DPP4:RBD crystal structure, the small methyl side chain of Ala291 in DPP4 279
nestles into a small pocket in the RBD, which cannot accommodate the size and charge of the 280
corresponding glutamic acid residue found in the hamster DPP4 molecule. This steric clash alone is likely 281
sufficient to abrogate binding. In addition, the side chain of Arg336 in human DPP4 forms hydrogen 282
bonds with RBD residue Tyr499 and a salt bridge to RBD residue Asp455. These interactions would not 283
be formed by the corresponding threonine side chain in hamster DPP4. The remaining three DPP4 284
residues at the RBD interface that differ in humans and hamsters are conserved substitutions and are not 285
predicted to greatly impact binding to the RBD (Figure 4A). Furthermore, these analyses showed that 286
hamster DPP4 has significantly higher binding energy (less favorable interactions) than human DPP4 to 287
MERS-CoV (Figure 4B). When mutant DPP4s were designed in silico by introducing the five human-288
specific amino acid residues (Ala291, Ile295, Arg336, Val341 and Ile346) into the hamster DPP4 289
(humanized hamster DPP4), and the five hamster-specific amino acid residues (Glu291, Thr295, Thr336, 290
Leu341 and Val346 into the human DPP4 (hamsterized human DPP4), a reversion of the binding energies 291
was found; the binding energy associated with the humanized hamster DPP4 and MERS-CoV spike 292
protein complex was lower than that of hamsterized human DPP4 and MERS-CoV spike protein (Figure 293
4B). This suggests that the five DPP4 human-specific amino acid residues are responsible for the ability 294
of MERS-CoV spike protein to bind to DPP4. 295
296
In vitro characterization of mutagenized DPP4s 297
The modeling data suggested that introduction of the five human-specific amino acid residues in hamster 298
DPP4 would allow recognition of this DPP4 by the MERS-CoV spike protein. To test this hypothesis, 299
expression plasmids were synthesized with human DPP4 containing the five hamster-specific amino acid 300
residues (hamsterized human DPP4) and hamster DPP4 containing the five human-specific amino acid 301
residues (humanized hamster DPP4). Inoculation of BHK cells transiently expressing humanized hamster 302
DPP4 with MERS-CoV resulted in virus replication, whereas inoculation of BHK cells transiently 303
expressing hamsterized human DPP4 did not (Figure 5A). Expression of DPP4 on BHK cells was 304
confirmed via flow cytometry (Figure 5B). 305
306
Modeling and in vitro characterization of the DPP4 of putative intermediate host species 307
We determined the binding energy of DPP4s to MERS-CoV spike protein of known binders (rhesus 308
macaque) and non-binders (ferret and mouse). Like hamster DPP4, the binding energy of ferret and 309
mouse DPP4 to MERS-CoV spike protein was found to be relatively high. In contrast, rhesus macaque 310
DPP4 was found to have binding energy levels similar to human DPP4 (Figure 4B). These results were 311
confirmed in vitro by transfecting BHK cells with DPP4 from rhesus macaque and subsequently 312
inoculating these cells with MERS-CoV, resulting in virus replication. In contrast, transfection of the 313
DPP4 of ferret or mouse origin into DPP4 did not render these cells susceptible to MERS-CoV replication 314
(Figure 6). With the identification of dromedary camels as a potential intermediate host species for 315
MERS-CoV in mind, we determined the ability of DPP4 from putative intermediate host species 316
(dromedary camel, cow, sheep and goat) to bind to MERS-CoV spike protein. Like DPP4 from human 317
and rhesus macaque, the binding energy associated with dromedary camel, goat, sheep and cow DPP4 318
was found to be relatively low, suggesting these proteins can function as a receptor for MERS-CoV 319
(Figure 4B). Furthermore, expression of dromedary camel, goat, sheep and cow DPP4 on BHK cells 320
supported replication of MERS-CoV (Figure 6A). Expression of DPP4 on BHK cells was confirmed via 321
flow cytometry and qRT-PCR (Figure 6B, C). DPP4 was detected on cells in lung and kidney tissue of 322
camel, goat, cow, and sheep by IHC. Interestingly, DPP4 was more abundantly expressed on alveolar 323
interstitium in cow, goat and sheep lungs compared to dromedary camel, rhesus macaque, hamster, mouse 324
and ferret lungs (Figure 7, Table 1). Finally, we carried out full-length and partial (DPP4 binding domain, 325
amino acids 220-350) protein sequence alignments between DPP4 of camel, goat, cow and sheep. Camel 326
DPP4 diverged from goat, cow, and sheep DPP4, in particular when comparing partial DPP4 protein 327
sequences (Table 3). 328
329
Discussion 330
331
Surface receptors play an essential role in initiating virus entry into the host cell, thereby playing a major 332
role in the tissue- as well as host species-tropism of viruses. DPP4 was recently identified as the cellular 333
receptor for MERS-CoV (16). Based on the ability of MERS-CoV to replicate in cell lines originating 334
from a wide variety of mammalian species (bats, non-human primates, pigs, and humans), it was 335
speculated to have a broad host tropism (19, 21). However, it is currently unclear whether in vitro results 336
correlate directly with in vivo susceptibility (38). MERS-CoV was found to be unable to infect some of 337
the major respiratory animal models (Syrian hamster, mouse and ferrets (24, 26, 27)) in contrast to the 338
ability of MERS-CoV to replicate efficiently in rhesus macaques (23). This suggests the existence of a 339
host-barrier restriction of MERS-CoV for some species. Using in vitro growth kinetics of MERS-CoV we 340
were able to demonstrate this host-species restriction in cell lines from different mammalian origins. 341
MERS-CoV replicated efficiently in cells of human and non-human primate origin, but was not able to 342
replicate in cells of mouse, hamster or ferret origin, despite the presence of DPP4 (Figure 1). Analysis of 343
the presence of DPP4 in Syrian hamster, mouse and ferret lung and kidney tissues as well as the rhesus 344
macaque lung and kidney tissues suggested that the inability of MERS-CoV to infect Syrian hamster, 345
mouse and ferret is not due to a lack of expression of the DPP4 receptor (Figure 2). Our hypothesis that 346
the host species restriction of MERS-CoV lies on the receptor-binding level was supported by the lack of 347
replication in hamster and ferret cells upon exogenous expression of the hamster or ferret DPP4 on the 348
surface of these cells, whereas expression of the human DPP4 receptor rendered these previously non-349
permissive cell lines permissive for MERS-CoV (Figure 3). We observed a >2 log difference in growth of 350
MERS-CoV between hamster and ferret cells, which may be explained by the difference in transfection 351
efficiency (BHKs, 93.0%; primary ferret cells, 65.8%; Figure 3B). The co-crystallization between the 352
human DPP4 and the MERS-CoV spike protein revealed the receptor binding domain of MERS-CoV and 353
the amino acid residues of human DPP4 interacting with this domain (36, 37). Alignment of DPP4 amino 354
acid residues identified as interacting with the MERS-CoV spike protein between human and rhesus 355
DPP4s (binders) and hamster DPP4 (non-binder) revealed a minimal subset of five amino acid changes 356
between the human and the hamster DPP4 (Table 2). These five differential human amino acid residues 357
were introduced into the hamster DPP4 (humanized hamster DPP4) and the five differential hamster 358
amino acid residues were introduced into the human DPP4 (hamsterized human DPP4). Expression of the 359
humanized hamster DPP4 in BHK cells rendered these cells permissive for MERS-CoV, whereas 360
expression of hamsterized human DPP4 did not change the non-permissiveness of the cells (Figure 5). 361
For ferret DPP4 it was recently shown that exchanging the 246 to 505 amino acid region with that of 362
human DPP4 resulted into the ability of MERS-CoV to utilize this chimeric DPP4 (28), as was the case 363
for mouse DPP4 when exchanging amino acids 279 to 346 with the human DPP4 counterpart (29). In 364
addition, both of these studies found amino acids to be important in spike binding that were identified in 365
this study (295, 336 and 346 for mouse and 295, 336 and 341 for ferret) (28, 29). Interestingly, although 366
the humanized hamster DPP4 was able to facilitate MERS-CoV infection, MERS-CoV titers were 367
considerably lower. This could indicate that although substitution of five human amino acids into hamster 368
DPP4 is sufficient for spike binding, additional amino acid residues in the interface might be required for 369
optimal binding and infectivity. 370
Currently the only available animal disease model for MERS-CoV is the rhesus macaque. Research into 371
therapeutic and prophylactic countermeasures is severely restricted by the absence of a small animal 372
model allowing high-throughput in-vivo screening of antivirals with in-vitro efficacy or candidate 373
vaccines (24, 25, 39-42). Our data indicate that transgenic mice expressing the human DPP4 will likely be 374
susceptible to MERS-CoV and would allow the establishment of a much-needed small animal model. 375
This is supported by recent experimental evidence which showed that transient expression of human 376
DPP4 in the lower respiratory tract of mice supported MERS-CoV replication (43). 377
Subsequent modeling between DPP4s of species known to be able to bind MERS-CoV spike protein 378
(human and rhesus macaque) and species known not to be able to bind MERS-CoV spike protein 379
(hamster, mouse and ferret) displayed a stark difference in binding energies between binders and non-380
binders. Utilizing the same model, DPP4s of species implicated in the emergence of MERS-CoV 381
(dromedary camel, sheep, goat and cow) were predicted to be able to bind to the spike protein of MERS-382
CoV (Figure 4B). DPP4 modeling data was supported by experimental work showing that indeed the 383
DPP4s of dromedary camel, sheep, goat and cow were able to support MERS-CoV replication (Figure 6). 384
MERS-CoV-like antibodies have been found in dromedary camels, but not yet in goat, sheep or cow 385
species (7, 44). However, DPP4 from goat, cow and sheep can function as a receptor for MERS-CoV, 386
although with lesser efficiency (Figure 6). Protein sequence alignments of full-length and partial DPP4 387
revealed that camel DPP4 differs from goat, cow and sheep DPP4, in particular when amino acids 220-388
350 of DPP4, which are of importance in spike binding, were investigated (Table 3). It is possible that 389
subtle differences in binding of spike and DPP4 account for the apparent lack of MERS-CoV circulation 390
in the goat, cow and sheep population. The close evolutionary relationship between MERS-CoV and bat 391
CoVs as well as the detection of a short fragment of viral RNA in a bat in Saudi Arabia suggest that 392
MERS-CoV originates from bats (1, 3). The ability of MERS-CoV to utilize the DPP4 from an 393
insectivorous bat underlines the broad host range (16). Future receptor binding and experimental infection 394
studies are needed to investigate the suitability of different bat species to function as a host for MERS-395
CoV. 396
MERS-CoV was found to replicate predominantly in type I and II pneumocytes in the lower respiratory 397
tract of experimentally infected rhesus macaques. The replication of MERS-CoV correlates with DPP4 398
expression on type I and II pneumocytes in the lungs of rhesus macaques (Figure 2) (25). The presence of 399
DPP4 in the lower respiratory tract of dromedary camel, sheep, goat and cow (Table 1) suggest that the 400
tropism of MERS-CoV for these species could be similar to the respiratory tropism observed in rhesus 401
macaques and humans (13, 14, 25). Since the first emergence of MERS-CoV in 2012, the epidemiology 402
has remained unclear. The recent identification of the circulation of MERS-CoV in dromedary camels (6-403
9) together with the multiple introductions of MERS-CoV into the human population (11, 12) suggests 404
that both zoonotic transmission from an intermediate host and human-to-human transmission occur 405
simultaneously. Our data supports the potential for the existence of one or multiple natural reservoirs for 406
MERS-CoV. Although our data do not provide any formal proof for the existence of such a reservoir, the 407
ability of DPP4s of cows, sheep, goats and dromedary camels (the major Middle East livestock species) 408
to function as a MERS-CoV receptor and the abundant DPP4 presence in the respiratory tract of these 409
species suggest that they would be susceptible for MERS-CoV infection. The combination of modeling 410
the binding energies of the MERS-CoV spike with the DPP4s of different species and a molecular 411
experimental approach could guide field programs focused at identifying the existence of an intermediate 412
host. With the potential susceptibility of major livestock species for MERS-CoV, renewed focus should 413
be aimed at elucidating the existence of an intermediate host and thereby preventing further spread of 414
MERS-CoV. 415
416
417
418
1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of 419
a novel coronavirus from a man with pneumonia in Saudi Arabia. The New England journal of 420
medicine 367:1814-1820. 421
2. WHO 2013, posting date. Coronavirus infections. 422
http://www.who.int/csr/don/archive/disease/coronavirus_infections/en/index.html. [Online.] 423
3. Memish ZA, Mishra N, Olival KJ, Fagbo SF, Kapoor V, Epstein JH, Alhakeem R, 424
Durosinloun A, Al Asmari M, Islam A, Kapoor A, Briese T, Daszak P, Al Rabeeah AA, 425
Lipkin WI. 2013 Nov. Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia. 426
Emerg Infect Dis [Internet]. 427
4. Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards LR, Schoeman MC, Drosten 428
C, Drexler JF, Preiser W. 2013 Oct. Close relative of human Middle East respiratory syndrome 429
coronavirus in bat, South Africa [letter]. Emerg Infect Dis [Internet]. 430
5. Annan A, Baldwin HJ, Corman VM, Klose SM, Owusu M, Nkrumah EE, Badu EK, Anti P, 431
Agbenyega O, Meyer B, Oppong S, Sarkodie YA, Kalko EK, Lina PH, Godlevska EV, 432
Reusken C, Seebens A, Gloza-Rausch F, Vallo P, Tschapka M, Drosten C, Drexler JF. 2013. 433
Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerging 434
infectious diseases 19:456-459. 435
6. Reusken CB, Haagmans BL, Muller MA, Gutierrez C, Godeke GJ, Meyer B, Muth D, Raj 436
VS, Vries LS, Corman VM, Drexler JF, Smits SL, El Tahir YE, De Sousa R, van Beek J, 437
Nowotny N, van Maanen K, Hidalgo-Hermoso E, Bosch BJ, Rottier P, Osterhaus A, 438
Gortazar-Schmidt C, Drosten C, Koopmans MP. 2013. Middle East respiratory syndrome 439
coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. 440
The Lancet infectious diseases. 441
7. Perera RA, Wang P, Gomaa MR, El-Shesheny R, Kandeil A, Bagato O, Siu LY, Shehata 442
MM, Kayed AS, Moatasim Y, Li M, Poon LL, Guan Y, Webby RJ, Ali MA, Peiris JS, 443
Kayali G. 2013. Seroepidemiology for MERS coronavirus using microneutralisation and 444
pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary 445
camels in Egypt, June 2013. Euro Surveill 18. 446
8. Haagmans BL, Al Dhahiry SH, Reusken CB, Raj VS, Galiano M, Myers R, Godeke GJ, 447
Jonges M, Farag E, Diab A, Ghobashy H, Alhajri F, Al-Thani M, Al-Marri SA, Al Romaihi 448
HE, Al Khal A, Bermingham A, Osterhaus AD, Alhajri MM, Koopmans MP. 2014. Middle 449
East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. The 450
Lancet infectious diseases 14:140-145. 451
9. Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC, de Wit E, Munster VJ, Hensley 452
LE, Zalmout IS, Kapoor A, Epstein JH, Karesh WB, Daszak P, Mohammed OB, Lipkin 453
WI. 2014. Middle East respiratory syndrome coronavirus infection in dromedary camels in saudi 454
arabia. mBio 5. 455
10. Briese T, Mishra N, Jain K, Zalmout IS, Jabado OJ, Karesh WB, Daszak P, Mohammed 456
OB, Alagaili AN, Lipkin WI. 2014. Middle East Respiratory Syndrome Coronavirus 457
Quasispecies That Include Homologues of Human Isolates Revealed through Whole-Genome 458
Analysis and Virus Cultured from Dromedary Camels in Saudi Arabia. mBio 5. 459
11. Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, 460
Alhakeem RF, Madani H, Alrabiah FA, Hajjar SA, Al-Nassir WN, Albarrak A, Flemban H, 461
Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI, 462
Memish ZA. 2013. Transmission and evolution of the Middle East respiratory syndrome 463
coronavirus in Saudi Arabia: a descriptive genomic study. Lancet. 464
12. Cotten M, Watson SJ, Zumla AI, Makhdoom HQ, Palser AL, Ong SH, Al Rabeeah AA, 465
Alhakeem RF, Assiri A, Al-Tawfiq JA, Albarrak A, Barry M, Shibl A, Alrabiah FA, Hajjar 466
S, Balkhy HH, Flemban H, Rambaut A, Kellam P, Memish ZA. 2014. Spread, circulation, and 467
evolution of the middle East respiratory syndrome coronavirus. mBio 5. 468
13. Guery B, Poissy J, el Mansouf L, Sejourne C, Ettahar N, Lemaire X, Vuotto F, Goffard A, 469
Behillil S, Enouf V, Caro V, Mailles A, Che D, Manuguerra JC, Mathieu D, Fontanet A, van 470
der Werf S. 2013. Clinical features and viral diagnosis of two cases of infection with Middle 471
East Respiratory Syndrome coronavirus: a report of nosocomial transmission. Lancet 381:2265-472
2272. 473
14. Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, Guggemos W, 474
Kallies R, Muth D, Junglen S, Muller MA, Haas W, Guberina H, Rohnisch T, Schmid-475
Wendtner M, Aldabbagh S, Dittmer U, Gold H, Graf P, Bonin F, Rambaut A, Wendtner 476
CM. 2013. Clinical features and virological analysis of a case of Middle East respiratory 477
syndrome coronavirus infection. The Lancet infectious diseases 13:745-751. 478
15. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, 479
Luzuriaga K, Greenough TC, Choe H, Farzan M. 2003. Angiotensin-converting enzyme 2 is a 480
functional receptor for the SARS coronavirus. Nature 426:450-454. 481
16. Raj VS, Mou H, Smits SL, Dekkers DH, Muller MA, Dijkman R, Muth D, Demmers JA, 482
Zaki A, Fouchier RA, Thiel V, Drosten C, Rottier PJ, Osterhaus AD, Bosch BJ, Haagmans 483
BL. 2013. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-484
EMC. Nature 495:251-254. 485
17. Mou H, Raj VS, van Kuppeveld FJ, Rottier PJ, Haagmans BL, Bosch BJ. 2013. The receptor 486
binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue 487
region in the spike protein that efficiently elicits neutralizing antibodies. Journal of virology 488
87:9379-9383. 489
18. Lambeir AM, Durinx C, Scharpe S, De Meester I. 2003. Dipeptidyl-peptidase IV from bench 490
to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP 491
IV. Critical reviews in clinical laboratory sciences 40:209-294. 492
19. Chan JF, Chan KH, Choi GK, To KK, Tse H, Cai JP, Yeung ML, Cheng VC, Chen H, Che 493
XY, Lau SK, Woo PC, Yuen KY. 2013. Differential Cell Line Susceptibility to the Emerging 494
Novel Human Betacoronavirus 2c EMC/2012: Implications for Disease Pathogenesis and Clinical 495
Manifestation. The Journal of infectious diseases. 496
20. Kindler E, Jonsdottir HR, Muth D, Hamming OJ, Hartmann R, Rodriguez R, Geffers R, 497
Fouchier RA, Drosten C, Muller MA, Dijkman R, Thiel V. 2013. Efficient replication of the 498
novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic 499
potential. mBio 4:e00611-00612. 500
21. Muller MA, Raj VS, Muth D, Meyer B, Kallies S, Smits SL, Wollny R, Bestebroer TM, 501
Specht S, Suliman T, Zimmermann K, Binger T, Eckerle I, Tschapka M, Zaki AM, 502
Osterhaus AD, Fouchier RA, Haagmans BL, Drosten C. 2012. Human coronavirus EMC does 503
not require the SARS-coronavirus receptor and maintains broad replicative capability in 504
mammalian cell lines. mBio 3. 505
22. Chan RW, Chan MC, Agnihothram S, Chan LL, Kuok DI, Fong JH, Guan Y, Poon LL, 506
Baric RS, Nicholls JM, Peiris JS. 2013. Tropism and innate immune responses of the novel 507
human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures. Journal of 508
virology. 509
23. Munster VJ, de Wit E, Feldmann H. 2013. Pneumonia from Human Coronavirus in a Macaque 510
Model. The New England journal of medicine. 511
24. de Wit E, Prescott J, Baseler L, Bushmaker T, Thomas T, Lackemeyer MG, Martellaro C, 512
Milne-Price S, Haddock E, Haagmans BL, Feldmann H, Munster VJ. 2013. The Middle East 513
Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters. PloS 514
one 8:e69127. 515
25. de Wit E, Rasmussen AL, Falzarano D, Bushmaker T, Feldmann F, Brining DL, Fischer 516
ER, Martellaro C, Okumura A, Chang J, Scott D, Benecke AG, Katze MG, Feldmann H, 517
Munster VJ. 2013. Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient 518
lower respiratory tract infection in rhesus macaques. Proceedings of the National Academy of 519
Sciences of the United States of America. 520
26. Scobey T, Yount BL, Sims AC, Donaldson EF, Agnihothram SS, Menachery VD, Graham 521
RL, Swanstrom J, Bove PF, Kim JD, Grego S, Randell SH, Baric RS. 2013. Reverse genetics 522
with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. 523
Proceedings of the National Academy of Sciences of the United States of America. 524
27. Enserink M. 2013. New coronavirus reveals some of its secrets, p. 17-18, Science vol. 340. 525
28. Raj VS, Smits SL, Provacia LB, van den Brand JM, Wiersma L, Ouwendijk WJ, 526
Bestebroer TM, Spronken MI, van Amerongen G, Rottier PJ, Fouchier RA, Bosch BJ, 527
Osterhaus AD, Haagmans BL. 2014. Adenosine deaminase acts as a natural antagonist for 528
dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus. 529
Journal of virology 88:1834-1838. 530
29. Cockrell AS, Peck KM, Yount BL, Agnihothram SS, Scobey T, Curnes NR, Baric RS, Heise 531
MT. 2014. Mouse dipeptidyl peptidase 4 (DPP4) is not a functional receptor for Middle East 532
respiratory syndrome coronavirus (MERS-CoV) infection. Journal of virology. 533
30. Barlan A, Zhao J, Sarkar MK, Li K, McCray PB, Jr., Perlman S, Gallagher T. 2014. 534
Receptor variation and susceptibility to MERS coronavirus infection. Journal of virology. 535
31. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, 536
Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. 537
Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948. 538
32. Petrey D, Xiang Z, Tang CL, Xie L, Gimpelev M, Mitros T, Soto CS, Goldsmith-Fischman 539
S, Kernytsky A, Schlessinger A, Koh IY, Alexov E, Honig B. 2003. Using multiple structure 540
alignments, fast model building, and energetic analysis in fold recognition and homology 541
modeling. Proteins 53 Suppl 6:430-435. 542
33. Ponder JW. 1999. TINKER-software tools for molecular design, version 3.7, Washington 543
University; St. Louis, MO. 544
34. Zhou H, Zhou Y. 2002. Distance-scaled, finite ideal-gas reference state improves structure-545
derived potentials of mean force for structure selection and stability prediction. Protein science : a 546
publication of the Protein Society 11:2714-2726. 547
35. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time 548
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408. 549
36. Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, Zhang Y, Zhang W, Yuan Y, Bao J, Zhang B, Shi 550
Y, Yan J, Gao GF. 2013. Molecular basis of binding between novel human coronavirus MERS-551
CoV and its receptor CD26. Nature 500:227-231. 552
37. Wang N, Shi X, Jiang L, Zhang S, Wang D, Tong P, Guo D, Fu L, Cui Y, Liu X, Arledge 553
KC, Chen YH, Zhang L, Wang X. 2013. Structure of MERS-CoV spike receptor-binding 554
domain complexed with human receptor DPP4. Cell research 23:986-993. 555
38. McIntosh K. 2013. A new virulent human coronavirus: how much does tissue culture tropism tell 556
us? The Journal of infectious diseases 207:1630-1632. 557
39. Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, 558
Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, Feldmann H. 559
2013. Treatment with interferon-alpha2b and ribavirin improves outcome in MERS-CoV-infected 560
rhesus macaques. Nature medicine 19:1313-1317. 561
40. Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, Feldmann H. 2013. Inhibition 562
of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin. 563
Scientific reports 3:1686. 564
41. Song F, Fux R, Provacia LB, Volz A, Eickmann M, Becker S, Osterhaus AD, Haagmans BL, 565
Sutter G. 2013. Middle East respiratory syndrome coronavirus spike protein delivered by 566
modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies. Journal of 567
virology 87:11950-11954. 568
42. Almazan F, DeDiego ML, Sola I, Zuniga S, Nieto-Torres JL, Marquez-Jurado S, Andres G, 569
Enjuanes L. 2013. Engineering a replication-competent, propagation-defective Middle East 570
respiratory syndrome coronavirus as a vaccine candidate. mBio 4:e00650-00613. 571
43. Zhao J, Li K, Wohlford-Lenane C, Agnihothram SS, Fett C, Gale MJ, Jr., Baric RS, 572
Enjuanes L, Gallagher T, McCray PB, Jr., Perlman S. 2014. Rapid generation of a mouse 573
model for Middle East respiratory syndrome. Proceedings of the National Academy of Sciences 574
of the United States of America. 575
44. Reusken CB, Ababneh M, Raj VS, Meyer B, Eljarah A, Abutarbush S, Godeke GJ, 576
Bestebroer TM, Zutt I, Muller MA, Bosch BJ, Rottier PJ, Osterhaus AD, Drosten C, 577
Haagmans BL, Koopmans MP. 2013. Middle East Respiratory Syndrome coronavirus (MERS-578
CoV) serology in major livestock species in an affected region in Jordan, June to September 579
2013. Euro surveillance : bulletin europeen sur les maladies transmissibles = European 580
communicable disease bulletin 18:20662. 581
582
583
Acknowledgements 584
585
The authors would like to thank Drs. Bart Haagmans and Ron Fouchier for providing HCoV-EMC/2012 586
and pcDNA3.1(+) human DPP4, Emmie de Wit and Barney Graham for helpful discussions, Aaron 587
Carmody, Carla Weisend and Kent Barbian for excellent technical assistance, Rachel LaCasse and 588
Richard Bowen for providing mammalian tissues and Anita Mora for assistance with the figures. This 589
research was supported by the Intramural Research Program of the National Institute of Allergy and 590
Infectious Diseases (NIAID), National Institutes of Health (NIH). 591
Table 1. DPP4 expression in cells lung and kidney tissue of different mammalian species. 592
Lung
Rhesus
macaque
Hamster Mouse Ferret Camel Sheep Goat Cow
apical bronchial/bronchiolar
epithelium
II III IIII I III III II III
bronchiolar smooth muscle I II III III I I I I
vascular smooth muscle I III III II II I II II
endothelial cells I I I 0 0 I II I
axonal cells ND IIII IIII III ND ND ND 0
alveolar macrophages II I II 0 I ND II 0
alveolar interstitium I II I 0 0 IIII III III
mesothelium I I 0 0 0 III IIII III
Kidney
cortical apical proximal tubular
epithelium
II I II IIII II II II I
arteriolar smooth muscle I II I I III II II I
endothelial cells I II 0 0 0 II 0 II
glomerular endothelial cells I III 0 III 0 I II I
axonal cells 0 IIII II IIII ND I III III
593
The lungs and kidneys of the various species were examined by IHC using an Į-DPP4 antibody. The 594
tissues were evaluated using a scale of 0 to IIII based on the intensity of the IHC signal and/or the 595
distribution of antigen throughout the tissue. A score of 0 indicates that no anti-DPP4 staining was 596
detected. I was used when the signal was very weak and/or was found in only a few, scattered cells. II 597
demonstrated a moderate IHC signal in multifocal to diffuse areas within the tissue. III was used to score 598
cells that stained in a moderate to intense fashion in coalescing to diffuse areas. IIII indicates intense and 599
diffuse IHC staining in the cells of interest. ND = not detected. 600
601
Table 2. Alignment of DPP4 amino acid residues of different mammalian species interacting with the 602
MERS-CoV spike protein. 603
604
Amino acid residues (Human DPP4 numbering)
Species 229 267 286 288 291 294 295 298 317 322 336 341 344 346
Human N K Q T A L I H R Y R V Q I
Hamster . . . . E . T . . . T L . V
Rhesus Macaque . . . . . . . . . . . . . .
Mouse . . . P . A R . . . T S . V
Ferret . . E . D S T Y . . S E E T
Camel . . . V . . . . . . . . . .
Sheep . . . V G . . . . . . . . .
Cow . . . V G . . . . . . . . .
Goat . . . V G . . . . . . . . .
605
Full DPP4 protein sequences were compared using MegAlign software. 606
607
Table 3. Percent identity between DPP4 protein sequences 608
Full DPP4
Camel Goat Cow Sheep
Partial DPP4 Camel 91.6 91.6 91.5
Goat 88.5 98.4 99.3
Cow 88.5 100 98.2
Sheep 88.5 100 100
609
Full DPP4 and partial (amino acid 220-350) DPP4 protein sequences were compared and percent identity 610
were analyzed using MegAlign software. 611
Figure 1. Replication kinetics of MERS-CoV in cell lines of human, non-human primate, hamster, mouse 612
and ferret origin. 613
A.) Huh7 (red, Ɣ), Vero (red, Ŷ), BHK (blue, Ɣ), 3T3 (blue, Ŷ), MEFC57Bl6 (blue, Ÿ) and primary ferret 614
(blue, ź) cell lines were inoculated with MERS-CoV using an MOI of 0.01 TCID50/cell. Supernatants 615
were harvested at 0, 24, 48 and 72 hours post inoculation (hpi) and viral titers were determined by end-616
point titration in quadruplicate in VeroE6 cells. Red lines indicate cell lines originating from species 617
known to be susceptible to MERS-CoV infection; blue lines indicate cell lines originating from species 618
non-susceptible to MERS-CoV infection. B.) Western blots of cellular lysates of Huh7, Vero, BHK, 619
primary ferret, 3T3 and MEFC57Bl6 cells probed with Į-DPP4 or Į-actin antibodies. C.) Cells were 620
stained using Į-DPP4 (R&D) and a FITC-conjugated secondary antibody (Life Technologies). Samples 621
were collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo and GraphPad 622
software. Mean titers were calculated from three independent experiments. Error bars indicate standard 623
deviations. 624
625
Figure 2. DPP4 in rhesus macaque, hamster, mouse and ferret lung and kidney tissue. 626
IHC was performed on lung and kidney tissues from rhesus macaque, hamster, mouse and ferret tissue 627
using an Į-DPP4 antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. 628
IHC images lung: closed arrow = bronchiolar epithelium; open arrow = smooth muscle; asterisk = 629
alveolar macrophage; closed arrowhead = alveolar interstitium. IHC images kidney: closed arrow = renal 630
tubular epithelium; open arrow = glomerular endothelium (magnification, 200x). 631
632
Figure 3. Replication kinetics of MERS-CoV on hamster and ferret cell lines expressing human, hamster 633
or ferret DPP4. 634
A.) Human DPP4 (red), hamster DPP4 (blue), ferret DPP4 (blue,) and GFP (green) were expressed in 635
BHK (Ɣ) or primary ferret (Ŷ) cells. Twenty-four hours post transfection, cells were inoculated with 636
MERS-CoV using a MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48 and 72 hpi and viral 637
titers were determined by end-point titration in quadruplicate in VeroE6 cells. Geometric mean titers were 638
calculated from three independent experiments. Error bars indicate standard deviations. B.) BHK or 639
primary ferret cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 hours 640
post-transfection using Į-DPP4 (R&D) and a FITC-conjugated secondary antibody (Life Technologies). 641
Samples were collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo 642
software. C.) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was 643
calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT. 644
645
Figure 4. Interaction between MERS-CoV spike protein and DPP4s of different mammalian species 646
A. Cartoon representing the binding between human DPP4 or hamster DPP4 and the spike protein of 647
MERS-CoV. DPP4 is depicted in white; the receptor binding domain (RBD) of the spike protein of 648
MERS-CoV is depicted in magenta and cyan. The far right panel is obtained by clockwise rotation of the 649
middle panel along a longitudinal axis. B. Binding energies between spike protein of MERS-CoV and 650
DPP4 of different species as well as humanized hamster DPP4 and hamsterized human DPP4. Red bars 651
indicate the binding energies of known binders (human and rhesus macaque DPP4), blue bars indicate the 652
binding energies of non-binders (hamster, mouse and ferret DPP4), green bars indicate the binding 653
energies of unknown binders (dromedary camel, goat, cow and sheep) and purple bars indicate the 654
binding energies of the in-silico mutagenized hamster and human DPP4s. The DPP4 homology models 655
were constructed using the human DPP4 structure (PDB ID: 4KR0, Chain A) as a template and subjected 656
to the binding energy calculation using an all-atom distance-dependent pairwise statistical potential, 657
DFIRE. 658
659
Figure 5. Replication kinetics of MERS-CoV on BHK cells expressing mutagenized DPP4s. 660
A.) Humanized hamster DPP4 (blue Ɣ) or hamsterized human DPP4 (red Ŷ) were expressed on BHK 661
cells. As a control, human DPP4 (red Ɣ) was expressed on BHK cells. Twenty-four post transfection, cells 662
were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 663
48 and 72 hpi and viral titers were determined by end-point titration in quadruplicate in VeroE6 cells. 664
Geometric mean titers were calculated from three independent experiments. Error bars indicate standard 665
deviations. B.) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 666
hours post-transfection using Į-DPP4 (R&D) and a FITC-conjugated secondary antibody (Life 667
Technologies). Samples were collected using a LSRII flow cytometer (BD Biosciences) and analyzed 668
using FlowJo software. 669
670
Figure 6. Replication kinetics of MERS-CoV on BHK cells expressing DPP4 of livestock species. 671
Camel (Ɣ), cow (Ŷ), goat (Ÿ) or sheep (ź) DPP4 (green) as well as rhesus macaque (Ŷ, red), ferret (Ŷ, 672
blue), or mouse (Ÿ, blue) DPP4 were expressed on BHK cells. As a control, human (red) or hamster 673
(blue) DPP4 were expressed on BHK cells. Twenty-four hours post transfection, cells were inoculated 674
with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48 and 72 hpi and 675
viral titers were determined by end-point titration in quadruplicate in VeroE6 cells. Geometric mean titers 676
were calculated from three independent experiments. Error bars indicate standard deviations. B.) BHK 677
cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 hours post-transfection 678
using Į-DPP4 (R&D) and a FITC-conjugated secondary antibody (Life Technologies). Samples were 679
collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. C.) 680
Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the 681
comparative CT method (35), where DPP4 expression is normalized to HPRT. 682
683
Figure 7. DPP4 in camel, goat, cow and sheep lung and kidney tissue. 684
IHC was performed on lung and kidney tissues from camel, goat, cow and sheep using an Į-DPP4 685
antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. IHC images lung: 686
closed arrow = bronchiolar epithelium; open arrow = smooth muscle; asterisk = alveolar macrophage; 687
closed arrowhead = alveolar interstitium. IHC images kidney: closed arrow = renal tubular epithelium; 688
open arrow = glomerular endothelium (magnification, 200x). 689
690
A preview of this full-text is provided by American Society for Microbiology.
Content available from Journal of Virology
This content is subject to copyright. Terms and conditions apply.
