Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May;88(9):4953-61.
doi: 10.1128/JVI.00161-14. Epub 2014 Feb 19.

Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection

Arlene Barlan  1 , Jincun Zhao, Mayukh K Sarkar, Kun Li, Paul B McCray Jr, Stanley Perlman, Tom Gallagher
Affiliations

Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection

Arlene Barlan et al. J Virol. 2014 May.

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans.

Importance: MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Properties of Hu and Hu(b4–5) DPP4s. (A) DPP4 includes a transmembrane anchor (residues 7 to 28), a propeller ectodomain (54 to 497), and a protease ectodomain (508 to 766). Exchangeable blades 4 and 5 comprise residues 194 to 350. Hu and Ms DPP4s are in black and gray, respectively. (B) Mouse DBT cells were transfected with pcDNA3.1 (empty vector [EV]) Hu, Ms, or Hu(b4–5) DPP4s and then transduced 1 day later with VSVluc-MERS S. At 16 h postransduction, relative luminescence unit (RLU) values were measured and plotted. (C) Mouse DBT cells were transfected and infected 1 day later with MERS-CoV (multiplicity of infection [MOI] = 1). Secreted viruses were collected after 2 days, and infectivities (PFU/ml) were determined by titration on Vero cell indicators. The hatched horizontal line indicates the lower limit of assay sensitivity. GFP, green fluorescent protein. (D) Mouse DBT cells were transfected and, after 1 day, exposed to the indicated doses of MERS S1-Fc. After 1 h at 37°C, VSVluc-MERS S was inoculated, with MERS S1-Fc concentrations remaining the same. At 16 h postransduction, luminescence values were measured and the relative transduction values were quantified. The concentration of MERS S1-Fc at the IC50 reflects the affinity of MERS S for the DPP4 receptors. In all data sets, the error bars indicate the standard errors of the means from triplicate values. Each experiment was repeated twice.
FIG 2
FIG 2
Camel DPP4 sequence and phylogenetic relationships with other animal DPP4s. Camel DPP4 was compared with the indicated animal DPP4 protein sequences using Molecular Evolutionary Genetics Analysis (MEGA; www.megasoftware.net) to generate the neighbor-joined phylogenetic tree. Numbers at each node are bootstrap values calculated from 500 trees and represent the percentages of trees that resolved clades at the indicated endpoints. The scale bar indicates the relationship between line lengths and sequence dissimilarities (0.05 = 5/100 amino acid divergence). Asterisks denote the animal DPP4s evaluated for this report.
FIG 3
FIG 3
Alignment of DPP4 blade 4 and 5 regions. The human and animal DPP4 sequences are arranged in order of increasing divergence from human blades 4 and 5. Light gray highlighting denotes divergent amino acids. Underlined regions denote the conserved terminal residues comprising the junctions between animal blades 4 and 5 and the mouse DPP4. The arrows and associated numbers at the top point to residues that, in human DPP4, interface closely with the MERS S protein.
FIG 4
FIG 4
Transduction and infection of animal DPP4-positive cells. (A) DPP4-transfected DBT cells were transfected with the indicated DPP4s and transduced 1 day later with VSVluc-MERS S, and luciferase accumulations were determined 16 h later. Standard errors are shown; n = 6. Statistical significance tests were performed using the Student t test. Data shown are representative of five independent results. (B) DBT cells were infected with MERS-CoV, and infectivity in media was quantified by a plaque assay on Vero cells. The hatched horizontal line indicates the lower limit of assay sensitivity. Standard errors are shown; n = 3. Statistical significance tests were performed using the Student t test. (C) DPP4-transfected DBT cells were lysed at 1 day posttransfection (the time of pseudovirus and virus inoculation). DPP4 and actin proteins were visualized by immunoblotting.
FIG 5
FIG 5
Relative levels of anti-FLAG antibody and MERS S1-Fc binding to cells. DBT cells transfected with pcDNA3.1 (EV), or with pCMV6-DPP4 chimeras, were incubated with anti-FLAG antibodies (1:500), or with 50 nM S1-Fc, for 1 h at 4°C and analyzed by flow cytometry. The bars in each panel indicate positive cell windows, and the numbers under the bars indicate the percentages of anti-FLAG antibody- and MERS S1-Fc-positive cells. Positive cell percentages, after background subtraction, are listed to the right in the figure.
FIG 6
FIG 6
Transduction blockade by MERS S1-Fc. DBT cells were transfected with DPP4 chimeras, incubated with the indicated concentrations of MERS S1-Fc for 1 h at 37°C, and transduced with VSVluc-MERS-S. Luminescence values were measured 16 h later. Data were normalized to transduction in the absence of S1-Fc. The concentrations of S1-Fc at each IC50 reflect the affinities of MERS spikes for the different DPP4 receptors. The experiment was repeated four times with similar results. Error bars indicate the standard errors of the means from triplicate values. Student's t tests were used to assess the significance of differences between human and animal DPP4 data; n.s. = not significant.
FIG 7
FIG 7
Effect of TMPRSS2 on MERS S-mediated transduction. (A) DBT cells were cotransfected with the indicated DPP4 chimeras and either wild-type (WT) or catalytically inactive (S441A) TMPRSS2 plasmids at 1:0.1 DPP4/TMPRSS2 DNA ratios. After 1 day, cell subsets were transduced with VSVluc-MERS-S, and luciferase values were determined 16 h later. (B) DBT cells were transfected with Hu(b4–5) or Bt(b4–5) DPP4 chimeras, in conjunction with increasing amounts of wild-type (WT) or catalytically inactive (S441A) TMPRSS2 plasmids. After 1 day, cell subsets were transduced with VSVluc-MERS-S, and luciferase values were determined 16 h later. The dotted line designates the basal level of transduction into MsDPP4-transfected cells. Holm-Sidak Student's t tests were used to reveal the significance of the relative differences between transduction levels in the presence of S441A and WT TMPRs; P values are indicated. To obtain the images beneath the graph, the luciferase-containing lysates were subjected to electrophoresis and the DPP4 and actin proteins visualized by Western blotting. (C) 293 cells were transfected with HuDPP4 (1 μg) in conjunction with the indicated TMPRSS2 doses. After 1 day, the HuDPP4, TMPR, and actin proteins in cell lysates were visualized by Western blotting. The uncleaved (inactive) TMPRSS2 zymogen has a molecular mass of ∼60 kDa, and proteolytically active (WT) TMPRSS2 has a molecular mass of ∼25 kDa.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. The WHO MERS-CoV Research Group. 12 November 2013. State of knowledge and data gaps of Middle East respiratory syndrome coronavirus (MERS-CoV) in humans. PLoS Curr. 10.1371/currents.outbreaks.0bf719e352e7478f8ad85fa30127ddb8 - DOI - PMC - PubMed
    1. Assiri A, Al-Tawfiq JA, Al-Rabeeah AA, Al-Rabiah FA, Al-Hajjar S, Al-Barrak A, Flemban H, Al-Nassir WN, Balkhy HH, Al-Hakeem RF, Makhdoom HQ, Zumla AI, Memish ZA. 2013. Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study. Lancet Infect. Dis. 13:752–761. 10.1016/S1473-3099(13)70204-4 - DOI - PMC - PubMed
    1. Guery B, Poissy J, el Mansouf L, Sejourne C, Ettahar N, Lemaire X, Vuotto F, Goffard A, Behillil S, Enouf V, Caro V, Mailles A, Che D, Manuguerra JC, Mathieu D, Fontanet A, van der Werf S. 2013. Clinical features and viral diagnosis of two cases of infection with Middle East respiratory syndrome coronavirus: a report of nosocomial transmission. Lancet 381:2265–2272. 10.1016/S0140-6736(13)60982-4 - DOI - PMC - PubMed
    1. Lim PL, Lee TH, Rowe EK. 2013. Middle East respiratory syndrome coronavirus (MERS CoV): update 2013. Curr. Infect. Dis. Rep. 15:295–298. 10.1007/s11908-013-0344-2 - DOI - PMC - PubMed
    1. Centers for Disease Control and Prevention (CDC). 2013. Updated information on the epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) infection and guidance for the public, clinicians, and public health authorities, 2012–2013. MMWR Morb Mortal. Wkly. Rep. 62:793–796 - PMC - PubMed

Publication types