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. 2014 Aug;88(16):9220-32.
doi: 10.1128/JVI.00676-14. Epub 2014 Jun 4.

Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4

Neeltje van Doremalen  1 , Kerri L Miazgowicz  1 , Shauna Milne-Price  1 , Trenton Bushmaker  1 , Shelly Robertson  1 , Dana Scott  2 , Joerg Kinne  3 , Jason S McLellan  4 , Jiang Zhu  5 , Vincent J Munster  6
Affiliations

Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4

Neeltje van Doremalen et al. J Virol. 2014 Aug.

Abstract

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.

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Figures

FIG 1
FIG 1
Replication kinetics of MERS-CoV in cell lines of human, nonhuman primate, hamster, mouse, and ferret origin. (A) Huh-7 (red circles), Vero (red squares), BHK (blue circles), 3T3 (blue squares), MEF C57Bl6 (blue triangles), and primary ferret (blue inverted triangles) cell lines were inoculated with MERS-CoV using an MOI of 0.01 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 h postinoculation (hpi), and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Red lines indicate cell lines originating from species known to be susceptible to MERS-CoV infection; blue lines indicate cell lines originating from species nonsusceptible to MERS-CoV infection. (B) Western blots of cellular lysates of Huh-7, Vero, BHK, primary ferret, 3T3, and MEF C57Bl6 cells probed with anti-DPP4 or anti-actin antibodies. (C) Cells were stained using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo and GraphPad software. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations.
FIG 2
FIG 2
DPP4 in rhesus macaque, hamster, mouse, and ferret lung and kidney tissues. IHC was performed on lung and kidney tissues from rhesus macaque, hamster, mouse, and ferret tissues using an anti-DPP4 antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. IHC images, lung: closed arrow, bronchiolar epithelium; open arrow, smooth muscle; asterisk, alveolar macrophage; closed arrowhead, alveolar interstitium. IHC images, kidney: closed arrow, renal tubular epithelium; open arrow, glomerular endothelium (magnification, ×200).
FIG 3
FIG 3
Replication kinetics of MERS-CoV on hamster and ferret cell lines expressing human, hamster, or ferret DPP4. (A) Human DPP4 (red), hamster DPP4 (blue), ferret DPP4 (blue), and GFP (green) were expressed in BHK (circles) or primary ferret (squares) cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK or primary ferret cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. (C) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT.
FIG 4
FIG 4
Interaction between MERS-CoV spike protein and DPP4s of different mammalian species. (A) Cartoon representing the binding between human DPP4 or hamster DPP4 and the spike protein of MERS-CoV. DPP4 is depicted in white; the receptor binding domain (RBD) of the spike protein of MERS-CoV is depicted in magenta and cyan. The far right panel is obtained by clockwise rotation of the middle panel along a longitudinal axis. (B) Binding energies between spike protein of MERS-CoV and DPP4 of different species as well as humanized hamster DPP4 and hamsterized human DPP4. Red bars indicate the binding energies of known binders (human and rhesus macaque DPP4), blue bars indicate the binding energies of nonbinders (hamster, mouse, and ferret DPP4), green bars indicate the binding energies of unknown binders (dromedary camel, goat, cow, and sheep), and purple bars indicate the binding energies of the in silico mutagenized hamster and human DPP4s. The DPP4 homology models were constructed using the human DPP4 structure (PDB ID 4KR0, chain A) as a template and subjected to the binding energy calculation using an all-atom distance-dependent pairwise statistical potential, DFIRE.
FIG 5
FIG 5
Replication kinetics of MERS-CoV on BHK cells expressing mutagenized DPP4s. (A) Humanized hamster DPP4 (blue circles) or hamsterized human DPP4 (red squares) was expressed on BHK cells. As a control, human DPP4 (red circles) was expressed on BHK cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software.
FIG 6
FIG 6
Replication kinetics of MERS-CoV on BHK cells expressing DPP4 of livestock species. Camel (green circles), cow (green squares), goat (green triangles), or sheep (green inverted triangles) DPP4 and rhesus macaque (red squares), ferret (blue squares), or mouse (blue triangles) DPP4 were expressed on BHK cells. As a control, human (red) or hamster (blue) DPP4 was expressed on BHK cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. (C) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT.
FIG 7
FIG 7
DPP4 in camel, goat, cow, and sheep lung and kidney tissue. IHC was performed on lung and kidney tissues from camel, goat, cow, and sheep using an anti-DPP4 antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. IHC images, lung: closed arrow, bronchiolar epithelium; open arrow, smooth muscle; asterisk, alveolar macrophage; closed arrowhead, alveolar interstitium. IHC images, kidney: closed arrow, renal tubular epithelium; open arrow, glomerular endothelium (magnification, ×200).
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