Open Access

Adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of Middle East respiratory syndrome coronavirus

  • Jie Cui1Email author,
  • John-Sebastian Eden1,
  • Edward C Holmes1 and
  • Lin-Fa Wang2, 3
Virology Journal201310:304

DOI: 10.1186/1743-422X-10-304

Received: 3 September 2013

Accepted: 3 October 2013

Published: 10 October 2013

Abstract

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.

Keywords

MERS-CoV Bats Arms-race Adaptive evolution Emergence

Main text

Middle East respiratory syndrome coronavirus (MERS-CoV) [1], first described by the World Health Organization (WHO) on 23rd September 2012 [2, 3], has to date (20th September 2013) caused 130 laboratory-confirmed human infections with 58 deaths (http://www.who.int/csr/don/2013_09_20/en/index.html). MERS-CoV belongs to lineage C of the genus Betacoronavirus in the family Coronaviridae, and is closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (Bt-HKU5) [4, 5] and CoVs in Nycteris bats [6], suggestive of a bat-origin [6]. Unlike severe acute respiratory syndrome (SARS) CoV which uses the angiotensin-converting enzyme 2 (ACE2) receptor for cell entry [7], MERS-CoV employs the dipeptidyl peptidase 4 receptor (DPP4; also known as CD26), and recent work has demonstrated that expression of both human and bat DPP4 in non-susceptible cells enabled viral entry [8].

Cell-surface receptors such as DPP4 play a key role in facilitating viral invasion and tropism. As a consequence, the long-term co-evolutionary dynamics between hosts and viruses often leave evolutionary footprints in both receptor-encoding genes of hosts and the receptor-binding domains (RBDs) of viruses in the form of positively selected amino acid residues (i.e. adaptive evolution). For example, signatures of recurrent positive selection have been observed in ACE2 genes in bats [9], supporting the past circulation of SARS related CoVs in bats. To better understand the origins of MERS-CoV, as well as their potentially long-term (compared to short-term which lacks virus-host interaction) evolutionary dynamics with bat hosts [5, 10], we studied the molecular evolution of DPP4 across the mammalian phylogeny.

We first analyzed the selection pressures acting on bat DPP4 genes using the ratio of nonsynonymous (dN) to synonymous (dS) nucleotide substitutions per site (ratio dN/dS), with dN > dS indicative of adaptive evolution. The complete DPP4 mRNA sequence of the common pipistrelle (Pipistrellus pipistrellus) was downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) along with that of the common vampire bat (Desmodus rotundus) from one transcriptome database (http://www.ncbi.nlm.nih.gov/bioproject/178123). These sequences were then used to mine and extract DPP4 mRNA transcripts from a further five bat genomes (Table 1) using tBLASTn and GeneWise [11]. The complete DPP4 genes of bats and non-bat reference genomes from a range of mammalian species (Table 1) were aligned using MUSCLE [12] guided by translated amino acid sequences (n = 32; 727 amino acids). We then compared a series of models within a maximum likelihood framework [13], incorporating the published mammalian species tree [1416]. This analysis (the Free Ratio model) revealed that the dN/dS value on the bat lineage (0.96) was four times greater than the mammalian average (Figure 1). The higher dN/dS ratios leading to bats (Table 2) during mammalian evolution accord with the growing body of data [5, 6, 17, 18] that the newly emerged MERS-CoV ultimately has a bat-origin.
Table 1

Sequences used in the evolutionary analysis of DDP4

Common name

Species name

Family

Accession no.

Sheep

Ovis aries

Bovidae

XM_004004660

Killer whale

Orcinus orca

Delphinidae

XM_004283621

Cow

Bos taurus

Bovidae

NM_174039

Pig

Sus scrofa

Suidae

NM_214257

Pacific walrus

Odobenus rosmarus divergens

Odobenidae

XM_004410199

Ferret

Mustela putorius furo

Mustelidae

DQ266376

Cat

Felis catus

Felidae

NM_001009838

Horse

Equus caballus

Equidae

XM_001493999

Rhinoceros

Ceratotherium simum

Rhinocerotidae

XM_004428264

Large flying fox

Pteropus vampyrus

Pteropodidae

ENSPVAG00000002634

Black flying fox

Pteropus alecto

Pteropodidae

KB031068

Common vampire bat

Desmodus rotundus

Phyllostomidae

GABZ01004546

Brandt’s bat

Myotis brandtii

Vespertilionidae

KE161360

David’s myotis

Myotis davidii

Vespertilionidae

KB109552

Little brown bat

Myotis lucifugus

Vespertilionidae

GL429772

Common pipistrelle

Pipistrellus pipistrellus

Vespertilionidae

KC249974

Guinea pig

Cavia porcellus

Caviidae

XM_003478564

Degu

Octodon degus

Octodontidae

XM_004629976

Lesser Egyptian jerboa

Jaculus jaculus

Dipodidae

XM_004651712

Mouse

Mus musculus

Muridae

BC022183

Rat

Rattus norvegicus

Muridae

NM_012789

Human

Homo sapiens

Hominidae

NM_001935

Chimpanzee

Pan troglodytes

Hominidae

GABE01002695

Pygmy chimpanzee

Pan paniscus

Hominidae

XM_003820939

Gorilla

Gorilla gorilla gorilla

Hominidae

XM_004032706

Orangutan

Pongo abelii

Hominidae

NM_001132869

Gibbon

Nomascus leucogenys

Hylobatidae

XM_003266171

Olive baboon

Papio anubis

Cercopithecidae

XM_003907539

Rhesus monkey

Macaca mulatta

Cercopithecidae

JU474559

Galago

Otolemur garnettii

Galagidae

XM_003795172

Marmoset

Callithrix jacchus

Cebidae

XM_002749392

American pika

Ochotona princeps

Ochotonidae

XM_004577330

https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-304/MediaObjects/12985_2013_Article_2574_Fig1_HTML.jpg
Figure 1

Selection pressures on DPP4 during mammalian evolution. Ratios of nonsynonymous (dN) to synonymous (dS) nucleotide substitutions per site (dN/dS) are shown on four major ancestral branches; dN and dS numbers are also given in parentheses. Values for individual lineages are given in Table 2. DPP4 sequences of bat origin are shaded.

Table 2

Numbers of nonsynonymous (d N ) and synonymous (d S ) substitutions per site DPP4 genes in different mammals

Common name

dN

dS

dN/dS

Sheep

0.004

0.013

0.280

Killer whale

0.023

0.039

0.595

Cow

0.003

0.016

0.157

Pig

0.027

0.109

0.246

Pacific walrus

0.014

0.053

0.260

Ferret

0.015

0.064

0.235

Cat

0.021

0.081

0.258

Horse

0.016

0.055

0.290

Rhinoceros

0.017

0.044

0.385

Large flying fox

0.005

0.001

3.561

Black flying fox

0.004

0.008

0.487

Common vampire bat

0.042

0.125

0.500

Brandt’s bat

0.006

0.012

0.463

David’s myotis

0.010

0.028

0.380

Little brown bat

0.007

0.007

0.943

Common pipistrelle

0.031

0.066

0.470

Guinea pig

0.018

0.078

0.238

Degu

0.016

0.128

0.122

Lesser Egyptian jerboa

0.023

0.179

0.131

Mouse

0.019

0.093

0.206

Rat

0.027

0.110

0.248

Human

0.001

0.007

0.086

Chimpanzee

0.000

0.002

0.000

Pygmy chimpanzee

0.001

0.000

ND

Gorilla

0.003

0.004

0.863

Orangutan

0.002

0.000

ND

Gibbon

0.003

0.009

0.344

Olive baboon

0.000

0.005

0.000

Rhesus monkey

0.000

0.004

0.000

Galago

0.022

0.149

0.149

Marmoset

0.009

0.053

0.160

American pika

0.036

0.229

0.156

ND: Not determined because no synonymous substitutions are present.

We next analysed the selection pressures at individual amino acid sites in bat DPP4. Using the Bayesian FUBAR method [19] in HyPhy package [20], we identified six codons that were assigned dN/dS > 1 with higher posterior probability (a strict cut-off of 95% in this analysis) (Table 3). To identify those sites under positive selection that may interact directly with MERS-CoV-like spike protein, bat DPP4 (from the common pipistrelle) was modelled against the structure of the human DPP4/MERS-CoV spike complex [21] (Figure 2A). This revealed that three of the six positive selected residues (position 187, 288 and 392) were located at the interface between bat DPP4 and MERS-CoV RBD (receptor binding domain) (Figure 2). These residues therefore provide direct evidence of a long-term co-evolutionary history between viruses and their hosts. We also observed several variable regions (Figure 2B) within the bat RBD, that may also have resulted from virally-induced selection pressure and which merit additional investigation in a larger data set.
Table 3

Putatively positive selected DPP4 codons in bats

Codon position a

Posterior probability b

dN/dS

46

0.97

14.95

57

0.97

13.13

112

0.94

10.27

187

0.95

8.55

288

0.98

13.90

392

0.97

14.63

a Codon position corresponding to the human DPP4 (NP_001926) protein sequence.

b Posterior probability of residues assigned a dN/dS ratio greater than 1.

https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-304/MediaObjects/12985_2013_Article_2574_Fig2_HTML.jpg
Figure 2

Interaction of bat DPP4 and MERS-CoV spike protein receptor-binding domain and the location of positively selected sites. The structure was displayed using PyMol v1.6 (http://www.pymol.org/). (A) Homology model showing the structural interactions between bat DPP4 (from common pipistrelle) coloured grey and MERS-CoV spike protein receptor-binding domain coloured blue. The three positively selected residues (positions 187, 288 and 392) located within the interface where the virus-host interact are highlighted as red. (B) Protein alignment of human DPP4 compared to that of seven bat species showing RBD spanning codons 41 – 400. Conserved and variable positions are shown in black and grey text, respectively, and residues under positive selection are coloured red.

Our analysis therefore suggests that the evolutionary lineage leading to current MERS-CoV co-evolved with bat hosts for an extended time period, eventually jumping species boundaries to infect humans and perhaps through an intermediate host. As such, the emergence of MERS-CoV may parallel that of the related SARS-CoV [22]. Although one bat species, Taphozous erforatus, in Saudi Arabia has been found to harbour a small RdRp (RNA-Dependent RNA Polymerase) fragment of MERS-CoV [17], a larger viral sampling of bats and other animals with close exposure to humans, including dromedary camels were serological evidence for MERS-CoV has been identified [23], are clearly needed to better understand the viral transmission route. Alternatively, it is possible that the adaptive evolution present on the bat DPP4 was due to viruses other than MERS-CoVs, and which will need to be better assessed when a larger number of viruses are available for analysis. Overall, our study provides evidence that a long-term evolutionary arms race likely occurred between MERS related CoVs and bats.

Declarations

Acknowledgements

We thank Christopher Cowled at CSIRO Australian Animal Health Laboratory for annotating the Pterous aleco DPP4. This word was supported in part by a grant from the National Research Foundation, Singapore (NRF2012NRF-CRP-001-056) and the CSIRO Office of the Chief Executive Science Leaders Award. ECH is supported by an NHMRC Australia Fellowship.

Authors’ Affiliations

(1)
Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Biological Sciences and Sydney Medical School, The University of Sydney
(2)
Duke-NUS Graduate Medical School
(3)
CSIRO Livestock Industries, Australian Animal Health Laboratory

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© Cui et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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