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

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.

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 [14-16]. 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

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

Figure 1

Selection pressures on 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) and synonymous (d) substitutions per site 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 codons in bats

Codon positiona	Posterior probabilityb	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

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

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

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.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JC and LFW designed the research. JC and JSE analysed the data. JC and ECH drafted the manuscript. All authors read and approved the final manuscript.