bactrian camel camelus bactrianus integrins 3 and 6 as fmdv receptors

7/30/2019 Bactrian camel Camelus bactrianus integrins 3 and 6 as FMDV receptors

1/10

Research paper

Bactrian camel (Camelus bactrianus) integrins avb3 and avb6 asFMDV receptors: Molecular cloning, sequence analysisand comparison with other species

Junzheng Du, Shandian Gao, Huiyun Chang *, Guozheng Cong, Tong Lin, Junjun Shao,Zaixin Liu, Xiangtao Liu, Xuepeng Cai *

Key Laboratory of Animal Virology of the Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease

Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

1. Introduction

Foot-and-mouth disease (FMD) is a highly contagiousdisease of cloven-hoofed animal species (Thomson et al.,

2003; Alexandersen et al., 2003). The Office International

des Epizooties (OIE) code chapter on FMD includes the

Camelidae as susceptible species to FMD, similar to cattle,

pigs, sheep and goats. The animals of the Camelidae family

are extremely important in the puna of the Andes and Gobi

desert and play a major role in the lives of people. The

Camelidae inhabitcountries in North andEast Africa, Middle

and East Asia as well as South America where FMD isendemic, and they may play an important role, as FMDV

reservoirs and potential carriers, in the epidemiology of

FMD. Foot-and-mouth disease virus (FMDV) is a member of

the aphthovirus genus of the Picornaviridae family and exists

as many subtypes and variants within seven different

serotypes(A,O,C,Asia1andSouthAfricanterritories1,2and

3). FMDV is a 140S particle consisting of a single-stranded

RNA genome and 60 copies each of four structural proteins

(VP1, VP2, VP3 and VP4) (Belsham, 2005). FMDV initiates

infection by binding to a cellular integrin receptor via a

highly conserved arginineglycineaspartic acid (RGD)

Veterinary Immunology and Immunopathology 131 (2009) 190199

A R T I C L E I N F O

Article history:

Received 26 September 2008

Received in revised form 3 April 2009

Accepted 14 April 2009

Keywords:

Bactrian camel

FMDV receptors

Integrin av familyMolecular characteristics

Phylogenetic treeTropism

A B S T R A C T

Integrins are heterodimeric adhesion receptors that participate in a variety of cellcell and

cellextracellular matrix protein interactions. Many integrins recognize RGD sequences

displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins.

Four members of theav integrin family of cellular receptors,avb3,avb6,avb1 andavb8,have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and

integrins are believed to be the receptors used to target epithelial cells in the infected

animals.To analysethe rolesof theav integrins froma susceptiblespecies as viral receptors,wehave cloned Bactrian camelav,b3andb6 integrincDNAsand comparedthem to those ofother species. Thecodingsequences for Bactrian camel integrinav,b3 andb6 werefound to

be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids,respectively.The Bactriancamelav,b3andb6 subunits share many structural features withhomologues of other species, including the ligand binding domain and cysteine-rich region.

Phylogenetic trees and similarity analyses showed the close relationships of integrin genes

from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that

were distinctfrom the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and

Xenopus. We postulate that hosttropismof FMDV mayin partbe relatedto thedivergencein

integrin subunits among different species.

2009 Elsevier B.V. All rights reserved.

* Corresponding authors.

E-mail addresses: [email protected] (H. Chang),

[email protected] (X. Cai).

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v e t i m m

0165-2427/$ see front matter 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.vetimm.2009.04.008
mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/01652427http://dx.doi.org/10.1016/j.vetimm.2009.04.008http://dx.doi.org/10.1016/j.vetimm.2009.04.008http://www.sciencedirect.com/science/journal/01652427mailto:[email protected]:[email protected]


2/10

sequence motif found within a surface protrusion consisting

of the loop between the bG and bH strands (GH loop,residues 140160) of the capsid protein VP1 (Fox et al.,

1989; Jackson et al., 2003; Grubman and Baxt, 2004 ). In

addition to integrins, the virus can utilize other receptors on

cultured cells, such as the Fc receptor or heparan sulfate or

an artificial single-chain antibody fused to intercellular

adhesion molecule 1, but these receptors do not require theRGD sequence (Baxt and Mason, 1995; Mason et al., 1993;

Baranowski et al., 1998; Jackson et al., 1996; Rieder et al.,

1996). Field viruses are dependent on integrin receptors to

initiateinfectionin vitro, and integrins are believedto be the

receptors used in the infected animals (McKenna et al.,

1995; Neff et al., 1998).

Integrins are a large family of heterodimeric trans-

membrane glycoproteins composed of two subunits (aandb) that interact non-covalently at the cell surface. Theymediate cellcell interactions and the binding of cells to

the extracellular matrix, and they play a crucial role in cell

division, differentiation, migration and survival (Gonzalez-

Amaro and Sanchez-Madrid, 1999; Hynes, 2002; Luo et al.,2007). In addition, a number of viruses, including the

adenovirus, herpesvirus, hantavirus, picornavirus and

rotavirus, utilize integrins for cell invasion and they do

so via a variety of mechanisms (Schneider-Schaulies, 2000;

Stewart and Nemerow, 2007). Of the 24 known integrins,

eight recognize RGD as a binding motif sequence on their

natural ligands: these areavb1,avb3,avb6,avb8,avb5,a5b1, a8b1 and ajjbb3 (Ruoslahti, 1996; Plow et al.,2000). FMDV utilizes four members of the av subgroup ofintegrins (avb1, avb3, avb6 and avb8) as receptors toinitiate infection in vitro (Berinstein et al., 1995; Jackson

et al., 2000, 2002, 2004). Several other integrins (avb5,

a5b1, a8b1 and ajjbb3) appear unable to support FMDVinfection (Baranowski et al., 2000; Duque and Baxt, 2003).The family Camelidae includes two Old World camels

(OWC), the Bactrian camel (Camelus bactrianus) and the

dromedary (Camelus dromedaries), and four New World

camels (NWC), the guanaco (Lama guanicoe), llama (Lama

glama), alpaca (Lama pacos) and vicuna (Lama vicugna) at

the present time (Novoa, 1989; Stanley et al., 1994). All

experimental studies on FMD in NWC have clearly shown

that NWC can be infected with FMDV, and can even

transmit the virus to other susceptible animals (Wernery

and Kaaden, 2004). Recent studies showed convincingly

that Bactrian camels were found to be susceptible to

FMDV, but Dromedary camels showed very low or nosusceptibility (Wernery et al., 2006; Alexandersen et al.,

2008; Larska et al., 2008). Some cases of FMD in Bactrian

camels have been described in Russia and Mongolia

(Wernery and Kaaden, 2004). Thus far, there is no

information about FMDV receptors in the camels, though

integrins are likely to be important molecules in the

susceptibility of cloven-hoofed animals to FMDV infec-

tion. In this study, as the first step towards understanding

the susceptibility of Bactrian camels to FMDV, we

molecularly cloned cDNAs encoding the Bactrian camel

av, b3 and b6 integrin subunits and compared them tothose of other species including the orders Artiodactyla,

Primates, Perissodactyla, Carnivora, Rodentia, Galliformesand Xenopus.

2. Materials and methods

2.1. Animals and tissues

The four Bactrian camels, two females and two males,

selected for the study were 510 years of age and resided in

Alashan county of Inner Mongolia, China, at an altitude of

between 1200 and 1300 m. They grazed on desert and

semi-desert steppe throughout the year. Tongue and lungtissues were collected from these Bactrian camels imme-

diately after slaughter. Approximately 500 mg of each

sample were kept in liquid nitrogen until use. All animal

experiments were performed according to protocols

approved by the institutional committee for the use and

care of animals.

2.2. RNA extraction and RT-PCR

Tissues were ground thoroughly with an RNase-free,

liquid-nitrogen-cooled mortar and pestle. Total RNA was

extracted from each tissue sample using RNeasy Mini Kit

(Qiagen, Germany) as per the recommendations of the

manufacturer. An aliquot of the total RNA (5mg) wasreverse transcribed using AMV reverse transcriptase (20 U/

ml, Takara, Japan), the oligo-dT18 primer (20 pmol/ml) andthe random hexamer primers (20 pmol/ml) in a totalvolume of 40 ml, according to the manufacturers instruc-tions. The av, b3 and b6 cDNAs were amplified from thecDNA preparations of Bactrian camel lung and tongue

tissues by PCR using primers based on the integrin

sequences of bovines and other animals reported in

GenBank (Table 1). PCR was carried out in a total volume

of 100 ml containing 10 mM TrisHCl (pH 9.0), 50 mM KCl,1.25 mM MgCl2, 0.2 mM dNTPs, 5 U of Taq polymerase

(Takara, Japan), 40 pmol each of the primers and 10 ml ofthe cDNA sample. Cycling conditions for PCR were 5 min at

95 8C for predenaturation, 35 cycles of 1 min at 95 8C, 30 s

at annealing temperatures depending on the integrin to be

amplified (Table 1) and 3 min at 72 8C, followed by a final

extension for 10 min at 72 8C. The PCR products were run

on 1% agarose gel containing ethidium bromide and the

DNA bands were visualized using a UV transilluminator.

2.3. Cloning and sequencing of Bactrian camel av, b3and b6 cDNAs

The amplified bands corresponding to integrin cDNAswere excised from the 1% agarose gel and purified using

the Gel extraction kit (Qiagen, Germany). The purified PCR

products were ligated into the pGEM-T Easy vector

(Promega, USA), and the resultant recombinant plasmids

were transformed into competent Escherichia coli strain

JM109. For each cDNA, 46 plasmid clones containing

integrin cDNAs were sequenced using M13+/ universal

primers (Takara, Japan).

2.4. Sequence and phylogenetic analysis

Sequence data analyses were performed using the

BLAST search of the National Center for BiotechnologyInformation. The sequence homology and divergence were

J. Du et al. / Veterinary Immunology and Immunopathology 13 1 (2009) 190199 191


3/10

calculated using the Laser-gene analysis software package

(DNASTAR, USA). The sequences were aligned using the

Clustal W program available in the BioEdit v7.0.5 softwarepackage (Ibis therapeutics, Carlsbad, CA).Phylogenetic trees

were constructed using MEGA version 3.1 (Kumar et al.,

2004). The sequence data herein have been submitted to

GenBank and assigned accession numbers EU367990 for

Bactrian camel av cDNA, EF613220 for Bactrian camel b6cDNA and EU867790 for mature Bactrian camel b3 cDNA.Thereference sequences includedin the analysis weretaken

from GenBank (Table 2).

3. Results

3.1. Cloning and sequence analysis of Bactrian

camel av subunit

The complete coding sequence of the Bactrian camelavsubunit cDNA comprised 3165 nucleotides coding for a

protein with 1054 amino acid residues. The encoded

protein consists of a 30-residue signal peptide (M1A30), a

963-residue ectodomain (F30P993), a single 29-residue

transmembrane domain (A994Y1022) and a 32-residue

cytoplasmic domain (R1023T1054). This protein possesses

20 cysteine residues, one of which is located in the signal

peptide. The ectodomain includes 14 putative N-linked

glycosylation sites (N-X-S/T, where X is not P), a putative

ligand binding domain (b-propeller domain, residues F31

R468) and a known proteolytic cleavage site locatedbetween amino acid residues 896 and 897 (KR-D). The

ligand binding domain contains three divalent cation-

binding sites (DX[D/N]X[D/N]GXXD). Between the b-

propeller domain and the transmembrane domain arethe thigh domain (residues C469Q622), the genu domain

(residues L623V631) and calf domain (residues C632Q992).

The cytoplasmic portion contains a conserved G1025FFKR

motif, which normally fixes the integrin in an inactive state

(Pardi et al., 1995). It is noteworthy that the Calf1 domain

includes an inserted R642FVLTC motif that is absent in the

av subunits of other species. The amino acid sequence ofBactrian camel integrin av subunit and its comparison topig, bovine, human, horse and mouse av integrins areshown in Fig. 1A. The nucleotide and predicted amino acid

sequence similarities within the different subunit func-

tional regions among the Bactrian camel and several other

species ofav subunits are shown in Table 3. Overall, thetransmembrane and cytoplasmic domains exhibited thehighest degree of conservation between Bactrian camel

and other species, and the Bactrian camel av subunitdisplayed a high level of similarity to its bovine and

porcine homologues. The similarity results (%) were

further confirmed by the phylogenetic analysis (Fig. 1B).

The nucleotide sequences of integrin av from severalspecies were classified into six major groups. The Bactrian

camel av subunit was clustered into the Artiodactylagroup, together with the av subunits of pigs and cattle. Itwas also shown that av sequences from the ordersRodentia, Primates, Perissodactyla, Carnivora and Galli-

formes formed separate groups, respectively, which werealso distinct from the Artiodactyla group.

Table 2

Accession numbers of integrin sequences used for alignments and phylogenetic analysis.

Common name Species Integrin av Integrin b3 Integrin b6

Cattle Bos taurus DQ871215 AF239959 DQ867017

Pig Sus scrafa EF474019 NM214002 EF432729

Human Homo sapiens M14648 M35999 NM000888

Monkey Macaca mulatta XM001104012 XM001116013 XM001094740

Chimpanzee Pan troglodytes XM515969 XM523684 XM001149234

Horse Equus caballus XM001498530 NM001081802

Dog Canis familiaris XM845896 NM001003162 XM852055

Rat Rattus norvegicus NM001106549 NM153720 NM001004263

Mouse Mus musculus AK149984 AK157958 AK036439

Guinea pig Cavia. M35197

Chicken Gallus gallus M60517 NM204315

: no data available.

Table 1

Primer sequences used for the cloning of integrin cDNAs from Bactrian camels.

Primers Sequence (50 to 30) Target gene Predicted size of PCR products Annealing temperature

AlphavF1 50-TCGGCGATGGCTTTTCCGCCGCG-30 50 part of integrin ava 1.8 kb 55.6 8CAlphavR1 50-GTTTGTCTCTAAATTCAGATTCATCCC-3 0

AlphavF2 50-AATGGATATCCAGACTTAATTGTAGG-30 30 part of integrin avb 1.7 kb 54.4 8CAlphavR2 50-CAGTTAAGTTTCTGAGTTTCCTTC-30

Beta3F 50-GGGCCCAACATCTGTACCACGCGTGG-3 0 Mature integrin b3 2.2 kb 57.8 8CBeta3R 50-TTAAGTGCCCCGGTACGTGATATTGGTG-30

Beta6F 50-CTGAGACCGATGGCGATTGATCT-30 Integrin b6 2.4 kb 56.8 8CBeta6R 50-ATGTTCTGTCCTTCGGAAAG-30

a The 50 part of integrin a overlap with 390 bp.b The 30 part of integrin a overlap with 390 bp.

J. Du et al. / Veterinary Immunology and Immunopathology 131 (2009) 190199192


4/10

Fig. 1. (A) Alignment of deduced amino acid sequences of the integrin av subunit from the Bactrian camel (Cam), pig (Pig), cattle (Cat), human (Hum), horse(Hor) andmouse (Mou). Dotsindicate the same amino acid residues as theBactrian camelav subunit. Divalent cation bindingsites, potential N-glycosylationsites andcysteines arehighlightedin red,yellow andlightblue,respectively. Thestripes above thesequences representthe deduceddifferent constitutive parts

of the protein: the signal peptide ( ), the ligand binding domain ( ), the thigh domain ( ), the genu ( ), the Calf-1 ( ) and Calf-2

( ) domains,thetransmembraneregion ( ) and the cytoplasmic tail( ).The insertedRFVLTCmotifin the calf-1domain and the important

GFFKR motif in the cytoplasmic tail are boxed. (B) Phylogenetic relationship of integrin av at the nucleotide level from Bactrian camel and other species.Bactrian camel is indicated by (^). The scale bar indicates the genetic distance. Bootstrap resampling was done for 1000 replications. Bootstrap values are

shown along the branches. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)



5/10

3.2. Cloning and sequence analysis of mature Bactrian

camel b3 subunit

The 2289-nucleotide cDNA was found to code for the

mature Bactrian camelb3 subunit of 762 amino acids withnine potential N-linked glycosylation sites (N-X-S/T). If

carbohydrate chains with an average molecular weight of

2.5 kDa are assumed to attach all nine putative glycosyla-

tion sites, the total weight of the mature b3 moleculewould be 105 kDa. The mature protein consists of a 692-

residue ectodomain (G1D692) (amino acid 1 is the first

amino acid after cleavage of the signal sequence) with a

total of 56 cysteine residues, a single 29-residue trans-membrane domain (I693I721) and a 41-residue cytoplas-

mic tail (H722T762). The protein includes 32 cysteine

residues of which are arranged in four cysteine-rich,

tandemly repeated regions of about 4050 residues each

(residues C437T604), located next to the transmembrane

domain. The ectodomain contains an inserted bA domainof 243 amino acids (putative ligand-binding domain,

residues Y110R352), which are homologous to the A

domain of von Willebrand factor, and includes a putative

metal ion-dependent adhesion site (MIDAS) (residues D119,

S121, S123, E220 and D251) (Fig. 2A) that is critical for the

RGD-ligand binding function of the receptor (Colombatti

and Bonaldo, 1991; Tozer et al., 1996; Jimenez-Marn etal.,2008). The cytoplasmic tail contains one NPXY motif at

position 743746 that has been shown to be important in

various assays of integrin function or protein association

(Dedhar and Hannigan, 1996). The Bactrian camel b3protein shares common structural and functional elements

with b3 molecules from the other species, and the aminoacid sequence of Bactrian camelb3 was aligned with thoseof pigs, cattle, humans, horses and mice b3 (Fig. 2A). Thenucleotide and deduced amino acid sequences within the

different functional regions of Bactrian camel b3 showedhigher similarity to those of porcine and bovineb3 than tohuman, equine, murine, canine, chicken and monkey b3(Table 4). These results were also confirmed by phyloge-

netic analysis as Bactrian camel b3 was clustered into agroup together with b3 of pigs and cattle (Fig. 2B).

3.3. Cloning and sequence analysis of Bactrian

camel b6 subunit

The complete coding sequence for Bactrian camel b6was found to be 2367 nuclotides in length, encoding 788

amino acids consisting of a 26-residue putative signal

peptide (M1G26), a 681-residue ectodomain (G27N707), a

single 29-residue transmembrane domain (I708F736)anda

52-residue cytoplasmic tail (H737G788). The deduced

amino acid sequence includes 10 possible N-linked

glycosylation sites, one of which is located in thecytoplasmic tail. The protein possesses 58 cysteine

Fig. 1. (Continued).

Table 3

Nucleotide and encoded amino acid sequence similarities of integrin av between Bactrian camels and other species.

Function domain % Nucleotide similarity/% amino acid similarity between Bactrian camels and other species

Cattle Pig Human Monkey Horse Dog Mouse Chicken

Mature subunit 93.4/96.6 93.5/96.0 91.8/95.4 91.5/95.1 92.9/96.4 90.9/95.9 86.6/92.1 73.6/82.3

Ligand binding domain 93.5/97.5 93.6/97.5 93.5/97.5 91.3/96.3 92.5/97.9 91.3/97.8 87.9/94.7 74.0/83.8

Signal peptide 85.6/76.7 83.3/80.0 70.0/63.3 68.9/60.0 54.3/40.7 64.4/56.7 36.8/21.1

Ectodomain 95.0/96.8 93.3/95.4 91.4/94.8 91.0/94.5 92.5/95.7 91.0/95.8 86.5/91.5 73.5/81.4

Transmembrane domain 94.3/100 96.6/100 96.6/100 96.6/100 93.1/96.6 85.1/96.6 88.5/100 74.7/93.1

Cytoplasmic domain 95.8/100 95.8/100 94.8/100 93.8/100 94.6/100 92.7/100 87.5/100 83.3/93.8



6/10

residues, two located within the signal peptide and 56

located within the ectodomain, which are conserved in the

b6 subunits of other species. Similar to the b3 subunit,most of these cysteines (30 residues) are arranged in a

cysteine-rich region (residues C456T619). The ectodomain

also contains the ligand-binding domain of 242 amino

acids (residues Y131R372) and includes a putative MIDAS

(residues D140, S142, S144, E240 and D271). The cytoplasmic

tail also contains one conserved NPXY motif (residues

759762). The general organizations of the b6 subunits ofBactrian camel and other species are quite similar. The

amino acid sequence of Bactrian camel b6 was alignedwith those of cattle, pigs, humans, dogs and mice b3(Fig. 3A). Comparison of the nucleotide and deduced amino

Fig. 2. (A) Alignment of deduced amino acid sequences of the mature integrin b3 subunit from the Bactrian camel (Cam), pig (Pig), cattle (Cat), human(Hum), horse (Hor) and mouse (Mou). Dots indicate the same amino acid residues as the Bactrian camel b3 subunit. Potential N-glycosylation sites,cysteines and MIDAS sites are highlighted in yellow, light blue and red, respectively. The stripes above the sequences represent the deduced different

constitutive parts of the protein: the ectodomain ( ), the ligand binding domain ( ), the four cysteine-rich repeat ( ), the transmembrane

region ( ) a nd t he c ytoplasmic t ail ( ). NPXY motif in thecytoplasmictail is boxed. (B) Phylogenetic relationshipof integrinb3 atthe nucleotidelevel from Bactrian camel andother species. Bactriancamel is indicated by (^). The scale bar indicatesthe genetic distance. Bootstrapresampling was done

for 1000replications. Bootstrap values are shown alongthe branches.(For interpretation of the references to color in thisfigure legend, thereader is referredto the web version of the article.)



7/10

acid similarities within the different functional regions

showed that Bactrian camelb6 wasclosely related to thoseof pigs and cattle (Table 5). Phylogenetic analysis showedthat the nucleotide sequences ofb6 subunits from severalmammalian species were classified into four major groups.

The Bactrian camel b6 was clustered into the Artiodactylagroup, together with b6 of pigs and cattle (Fig. 3B).

4. Discussion

It has definitely been shown that Bactrian camels can be

infected with FMDV (Wernery and Kaaden, 2004; Wernery

et al., 2006; Larska et al., 2008). The resistances and

susceptibilities to FMD among different animal species

have not been elucidated. Of the possible host factorsinvolved in the pathogenesis of FMDV, the viral receptor

likely plays a major role in both host and tissue tropism

(Schneider-Schaulies, 2000; Stewart and Nemerow, 2007).For this reason, it is necessary to study FMDV receptors.

Interspecies comparisons of the integrin subunits have

shown that there are differences in the deduced amino acid

sequences among the species sequenced to date (Wada

et al., 1996; Neff et al., 2000; Espino-solis et al., 2008; Fett

et al., 2004). FMDV can utilize the human or simian

homologues of the avb1, avb3, avb6 and avb8 integrinsto infect cells; nevertheless, this virus does not cause

disease in humans (Berinstein et al., 1995; Jackson et al.,

2000, 2002, 2004; Neff et al., 1998). Interestingly, some

studies have indicated that FMDV is able to utilize the

bovine integrins more efficiently than it utilizes the human

homologues (Neff et al., 2000). The fact that the virus canonly infect certain species leaves open the question of how


Table 5

Nucleotide and encoded amino acid sequence similarities of integrin b6 between Bactrian camels and other species.


Cattle Pig Human Monkey Chimpanzee Dog Rat Mouse

Mature subunit 91.0/94.4 91.7/93.4 90.3/93.7 89.9/93.2 90.1/93.4 91.3/93.8 84.1/89.0 83.7/89.0

Ligand binding domain 94.4/98.3 93.8/98.8 92.8/97.1 92.4/97.1 92.6/97.1 93.3/97.1 87.1/94.6 87.2/94.6

Signal peptide 93.6/92.3 96.2/92.3 96.2/92.3 96.2/92.3 96.2/92.3 96.2/92.3 82.1/76.9 83.3/80.8

Ectodomain 90.7/94.1 91.3/93.2 90.1/93.4 89.4/92.7 89.9/93.1 90.8/93.5 83.9/88.4 83.7/88.5

Transmembrane domain 90.8/100 95.4/96.6 95.4/100 94.3/100 95.4/100 94.3/100 88.5/100 89.7/100

Cytoplasmic domain 95.6/94.3 94.3/94.3 89.3/94.3 93.7/96.2 89.9/94.3 95.0/94.3 84.3/90.6 80.5/88.7

Table 4

Nucleotide and encoded amino acid sequence similarities of integrin b3 between Bactrian camels and other species.


Cattle Pig Human Monkey Horse Dog Rat Mouse Chicken

Mature subunit 92.5/94.7 92.3/92.7 91.3/95.1 91.1/94.8 93.5/95.4 91.6/94.6 86.3/90.9 86.4/91.5 75.8/81.1

Ligand binding domain 92.9/95.1 92.3/95.5 94.1/96.3 93.7/95.9 93.6/95.9 93.8/95.9 87.9/93.0 87.5/92.2 81.3/87.7

Ectodomain 92.4/94.6 92.2/92.4 91.7/95.2 91.3/94.8 93.7/95.3 91.6/94.5 85.8/90.4 86.0/91.1 75.8/80.5

Transmembrane domain 82.6/91.3 88.4/91.3 82.6/87.0 85.5/91.3 85.5/91.3 88.4/91.3 87.0/91.3 87.0/91.3 78.3/87.0

Cytoplasmic domain 98.6/100 91.0/97.9 90.1/100 91.7/97.9 94.4/97.9 94.4/97 .9 93.1/97.9 92.4/97.9 75.7/87.5



8/10

differences between hosts may determine the suscept-

ibility to FMDV. To begin to answer this question, we

thought it important to obtain cDNAs encoding the

integrins from Bactrian camels, which are susceptible to

FMDV infection, and compare these sequences with those

of other species. In the present study, theav subunit codingsequences were amplified from cDNAs prepared from

Bactrian camel lung and tongue tissues, while the sequence

coding for the b6 subunit was amplified only from tonguetissue and the mature b3 subunit was amplified only fromlung tissue. We were unable to get a complete coding

sequence which included the Bactrian camel b3 signalpeptide sequence and, therefore, only obtained the mature

b3 sequence without the signal sequence. Analysis of thedistribution of the integrin receptors in susceptible species

may be necessary to explain viral pathogenesis within

Fig.3. (A) Alignment of deduced amino acidsequences of the integrinb6 subunit from the Bactrian camel (Cam),pig (Pig),cattle (Cat),human (Hum),horse(Hor) andmouse (Mou).Dots indicatethe same aminoacid residues as theBactrian camelb6 subunit. Potential N-glycosylation sites, cysteines and MIDASsites are highlighted in yellow, light blue and red, respectively. The stripes above the sequences represent the deduced different constitutive parts of the

protein: the signal peptide ( ) , the ectodomain ( ) , the ligand binding domain ( ) , the four cysteine-rich repeat ( ) , the

transmembraneregion( ) andthecytoplasmic tail ( ) . NPXY motif in the cytoplasmic tailis boxed. (B) Phylogenetic relationship of integrinb6at the nucleotide level from Bactrian camel and other species. Bactrian camel is indicated by (^). The scale bar indicates the genetic distance. Bootstrap

resampling was done for 1000 replications. Bootstrap values are shown along the branches. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of the article.)



9/10

different species. At present we are focusing on the changesin integrin profiles associated with FMDV infection.

Investigations of the mRNA expression and the distribution

of integrins which act as FMDV receptors in Bactrian

camels are in progress using the real-time quantitative RT-

PCR technique and confocal microscopy.

The Bactrian camel integrins share common structural

and functional elements with integrin molecules from

other species. The solved crystal structure of the RGD

avb3 complex has shown that the RGD motif makescontacts with both subunits, the arginine fitting into a cleft

formed primarily by a b-propeller domain from av, withcritical residues D180 and D248, and the aspartate coordi-

nating the cation bA domain ofb3 integrin with MIDAS(critical residues D119, S121, S123, E220 and D251) (Xionget al., 2001, 2002). Characteristic of all integrin b subunitsis the high content of cysteine residues and the four

tandem cysteine-rich epidermal growth factor (EGF)-like

domains known as the cysteine-rich repeats (Moyle et al.,

1991; Luo et al., 2007). All of these critical residues are

conserved in Bactrian camel integrin av, b3 and b6subunits. The very high interspecies conservation of the

putative MIDAS and critical residues confirms their

essential role in integrin function. Duque and Baxt

(2003) showed that different virus serotypes appear to

utilize the bovine integrin receptors with varying efficien-

cies and theligand-bindingdomain of the bovineb subunitplays a role in the recognition of the different viralserotypes. Alignments of the predicted amino acid

sequences of the av, b3 and b6 subunits with those ofother species showed that all cysteines were highly

conserved and may therefore play an important role in

determining the tertiary structure and functional integrity

of integrins. Neff et al. (2000) have previously found that

the increased efficiency of the bovine b3 subunit,compared with that of the human homologue, as a

receptor for FMDV appeared to relate to the cysteine-rich

repeat region. It will be interesting to see whether the roles

of the ligand binding domain and cysteine-rich region of

the b subunit from the Bactrian camel are similar to thoseseen in bovine integrin.

Phylogenetic trees and similarity analyses were per-formed to confirm the close relationships among the

integrins of cloven-hoofed animals, including Bactrian

camels, pigs and cattle, that are susceptible to FMDV

infection. It is interesting to speculate why foot-and-

mouth disease is limited to cloven-hoofed animals from

the standpoint of receptors. We postulate that FMDV

evolved into a disease of cloven-hoofed livestock because

the structures of their integrin receptors were more

susceptible to binding with the viral surface, which would

lead to much greater viral replication and disease within

these species. It is also important to note that receptors

alone may not necessarily determine FMDV species

tropism; other viral and cellular factors may also affectboth host range and virulence (Mason et al., 2003;

Alexandersen et al., 2003). It has, for example, been

demonstrated that an attenuation of virulence in cattle and

a reduced ability of the virus to replicate in bovine cells are

associated with a 10-amino acid deletion in the non-

structural protein 3A of FMDV (Beard and Mason, 2000;

ODonnell et al., 2001).

Acknowledgements

This work was supported by the Chinese National

Key Basic Research Program (No. 2005CB523201), the

Chinese National Key Technology R&D Program (No.2006BAD06A03), the National Natural Science Foundation

of China (No. 30800833) and the Chinese High Technology

Research and Development program (No. 2006AA10A204).

We thank Prof. Soren Alexandersen and Dr. Graham

Belsham from National Veterinary Institute, Technical

University of Denmark, for helpful comments and critical

reading of the manuscript.

References

Alexandersen, S., Zhang, Z., Donaldson, A.I., Garland, A.J., 2003. Thepathogenesis and diagnosis of foot-and-mouth disease. J. Comp.Pathol. 129, 136.

Alexandersen, S., Wernery,U., Nagy,P., Frederiksen,T., Normann,P., 2008.Dromedaries (Camelus dromedarius) areof lowsusceptibility to inocu-




10/10

lation with foot-and-mouth disease virus serotype O. J. Comp. Pathol.139 (4), 187193.

Baranowski, E., Ruiz-Jarabo, C.M., Sevilla, N., Andreu, D., Beck, E., Dom-ingo, E., 2000. Cell recognition by foot-and-mouth disease virus thatlacks theRGD integrin-bindingmotif:flexibility in aphthovirus recep-tor usage. J. Virol. 74, 16411647.

Baranowski, E., Sevilla, N., Verdaguer, N., Ruiz-Jarabo, C.M., Beck, E.,Domingo, E., 1998. Multiple virulence determinants of foot-and-mouth disease virus in cell culture. J. Virol. 72, 63626372.

Baxt, B., Mason, P.W., 1995. Foot-and-mouth disease virus undergoes

restricted replication in macrophage cell cultures following Fc recep-tor mediated adsorption. Virology 207, 503509.

Beard, C.W.,Mason, P.W.,2000. Genetic determinants of altered virulenceof Taiwanese foot-and-mouth disease virus. J. Virol. 74, 987991.

Belsham, G.J., 2005. Translation and replication of FMDV RNA. Curr. Top.Microbiol. Immunol. 288, 4370.

Berinstein, A., Roivainen, M., Hovi, T., Mason, P.W., Baxt, B., 1995. Anti-bodies to the vitronectin receptor (integrinavb3) inhibit binding andinfection of foot-and-mouth disease virus to culturedcells. J. Virol. 69,26642666.

Colombatti, A., Bonaldo, P., 1991. The superfamily of proteins with vonWillebrand factor type A-like domains: one theme common to com-ponents of extracellular matrix, hemostasis, cellular adhesion, anddefense mechanisms. Blood 77, 23052315.

Dedhar, S., Hannigan, G., 1996. Integrin cytoplasmic interactions andbidirectional transmembrane signalling. Curr. Opin. Cell Biol. 8 (5),657669.

Duque, H., Baxt, B., 2003. Foot-and-mouth disease virus receptors: com-parison of bovine av integrin utilization by type A and O viruses. J.Virol. 77, 25002511.

Espino-solis, G.P., Osuna-Quintero, J., Possani, L.D., 2008. Molecular clon-ing andcharacterizationof the alphaX subunitfrom CD11/CD18horseintegrin. Vet. Immunol. Immunopathol. 122, 326334.

Fett, T.,Zecchinon,L., Baise,E., Desmecht, D.,2004.The bovine(Bos taurus)CD11a-encoding cDNA: molecular cloning,characterization and com-parison with the human and murine glycoproteins. Gene 325, 97101.

Fox, G., Parry, N.R., Barnett, P.V., McGinn, B., Rowlands, D.J., Brown, F.,1989. Cell attachment site on foot-and-mouth disease virus includesthe amino acidsequence RGD (arginineglycineasparticacid). J. Gen.Virol. 70, 625637.

Gonzalez-Amaro, R., Sanchez-Madrid, F., 1999. Cell adhesion molecules:selectins and integrins. Crit. Rev. Immunol. 19, 389429.

Grubman, M.J., Baxt, B., 2004. Foot-and-mouth disease. Clin. Microbiol.

Rev. 17, 465493.Hynes, R.O., 2002. Integrins: bidirectional, allosteric signaling machines.

Cell 110, 673687.Jackson, T., Clark, S., Berryman, S., Burman, A., Cambier, S., Mu, D.,

Nishimura, S., King, A.M.Q., 2004. Integrin avb8 functions as areceptor for foot-and-mouth disease virus: role of the b-chain cyto-domain in integrin-mediated infection. J. Virol. 78, 45334540.

Jackson, T., Ellard, F.M., Ghazaleh, R.A., Brookes, S.M., Blakemore, W.E.,Corteyn, A.H., Stuart, D.I., Newman, J.W., King, A.M., 1996. Efficientinfection of cells in culture by type O foot-and-mouth disease virusrequires bindingto cell surface heparan sulfate. J. Virol. 70,52825287.

Jackson, T., King, A.M.Q., Stuart, D.I., Fry, E., 2003. Structure and receptorbinding. Virus Res. 91, 3346.

Jackson, T., Mould, A.P., Sheppard, D., King, A.M.Q., 2002. Integrinavb1 isa receptor for foot-and-mouth disease virus. J. Virol. 76, 935941.

Jackson, T., Sheppard, D., Denyer, M., Blakemore, W., King, A.M.Q., 2000.The epithelial integrin avb6 is a receptor for foot-and-mouth disease

virus. J. Virol. 74, 49494956.Jimenez-Marn, A., Yubero, N., Esteso, G., Moreno, A., Mulas, J.M., Morera,

L., Llanes, D., Barbancho, M., Garrido, J.J., 2008. Molecular character-ization and expression analysis of the gene coding for the porcine b3integrin subunit (CD61). Gene 408, 917.

Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software formolecular evolutionary genetics analysis and sequence alignment.Brief Bioinform. 5, 150163.

Larska, M., Wernery, U., Kinne, J., Schuster, R., Alexandersen, G., Alex-andersen, S., August 8, 2008. Differences in the susceptibility ofdromedary and Bactrian camel to foot-and mouth disease virus.Epidemiol. Infect. 16 (Epub ahead of print).

Luo, B.H., Carman, C.V., Springer, T.A., 2007. Structural basis of integrinregulation and signaling. Annu. Rev. Immunol. 25, 619647.

Mason, P.W., Baxt, B., Brown, F., Harber, J., Murdin, A., Wimmer, E., 1993.Antibody-complexed foot-and-mouth disease virus, but not polio-virus, can infect normally insusceptible cells via the Fc receptor.Virology 192, 568577.

Mason, P.W., Grubman, M.J., Baxt, B., 2003. Molecular basis of pathogen-esis of foot-and-mouth disease virus. Virus Res. 91, 932.

McKenna, T.S., Lubroth, J., Rieder, E., Baxt, B., Mason, P.W., 1995. Receptorbinding site-deleted foot-and-mouth disease (FMD) virus protectscattle from FMD. J. Virol. 69, 57875790.

Moyle,M.M.,Napier, M.A., Mclean, J.W., 1991. Cloning and expression of adivergent integrin subunit b8. J. Biol. Chem. 266 (29), 1965019658.

Neff, S., Mason, P.W., Baxt, B., 2000. High-efficiency utilization of thebovine integrin avb3 as a receptor for foot-and-mouth disease virusis dependent on the bovine b3 subunit. J. Virol. 74, 72987306.

Neff, S., Sa-Carvalho, D., Rieder, E., Mason, P.W., Blystone, S.D., Brown, E.J.,Baxt, B., 1998. Foot-and-mouth disease virus virulent for cattle uti-lizes the integrin avb3 as its receptor. J. Virol. 72, 35873594.

Novoa, C.M., 1989. Genetic improvement of South American camelids.Revta. Bras. Genet. 12, 123135.

ODonnell, V.K., Pacheco, J.M., Henry, T.M., Mason, P.W., 2001. Subcellulardistribution of the foot-and-mouth disease virus 3A protein in cells

infected with viruses encoding wild-type and bovine-attenuatedforms of 3A. Virology 287, 151162.

Pardi, R., Bossi, G., Inverardi, L., Rovida, E., Bender, J.R., 1995. Conservedregions in the cytoplasmic domains of the leukocyte integrin aLb2are involved in endoplasmic reticulum retention, dimerization, andcytoskeletal association. J. Immunol. 155, 12521263.

Plow, E.F., Haas, T.A.,Zhang, L., Loftus, J., Smith, J.W., 2000. Ligand bindingto integrins. J. Biol. Chem. 275 (29), 2178521788.

Rieder, E., Berinstein, A., Baxt, B., Kang, A.M., Mason, P.W., 1996. Propaga-tion of an attenuated virus by design: engineeringa novel receptor fora noninfectious foot-and-mouth disease virus. Proc. Natl. Acad. Sci.U.S.A. 93, 1042810433.

Ruoslahti, E., 1996. RGD and other recognition sequences for integrins.Annu. Rev. Cell Dev. Biol. 12, 697715.

Schneider-Schaulies, J., 2000. Cellular receptors for viruses: links totropism and pathogenesis. J. Gen. Virol. 81, 14131429.

Stanley, H.F., Kadwell, M., Wheeler, J.C., 1994. Molecular evolution of the

family Camelidae: a mitochondrial DNA study. Proc. Biol. Sci. 256(1345), 16.

Stewart, P.L., Nemerow, G.R., 2007. Cell integrins: commonly used recep-tors for diverse viral pathogens. Trends Microbiol. 15 (11), 500507.

Thomson,G.R., Vosloo, W.,Bastos,A.D.S., 2003. Foot andmouth disease inwildlife. Virus Res. 91, 145161.

Tozer, E.C.,Liddington, R.C.,Sutcliffe, M.J.,Smeeton,A.H., Loftus, J.C.,1996.Ligand binding to integrin aIIbb3 is dependent on a MIDAS-likedomain in the b3 subunit. J. Biol. Chem. 271 (36), 2197821984.

Wada, J.,Kumar, A.,Liu,Z., Ruoslahti, E.,Reichardt, L.,Marvaldi, J.,Kanwar,Y.S., 1996. Cloning of mouse integrin av cDNA and role of the avrelated matrix receptors in metanephric development. J. Cell Biol.132, 11611176.

Wernery, U., Kaaden, O.R., 2004. Foot-and-mouth disease in camelids: areview. Vet. J. 168, 134142.

Wernery,U., Nagy, P., Amaral-Doel,C.M., Zhang, Z., Alexandersen, S., 2006.Lack of susceptibility of dromedary camel (Camelus dromedarius) to

foot-and-mouth disease serotype O. Vet. Rec. 158, 201203.Xiong, J.P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D.L.,

Joachimiak, A., Goodman, S.L., Arnaout, M.A., 2001. Crystal structureof the extracellular segment of integrinavb3. Science 294, 339345.

Xiong, J.P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L.,Arnaout, M.A., 2002. Crystal structure of the extracellular segment ofintegrin avb3 in complex with an ArgGlyAsp ligand. Science 296,151155.


bactrian camel camelus bactrianus integrins 3 and 6 as fmdv receptors

Documents