Veterinary Immunology and Immunopathology 131 (2009) 190199

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Veterinary Immunology and Immunopathology

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Research paper

Bactrian camel (Camelus bactrianus) integrins avb3 and avb6 as FMDV receptors: Molecular cloning, sequence analysis and 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

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 September 2008 Received in revised form 3 April 2009 Accepted 14 April 2009 Keywords: Bactrian camel FMDV receptors Integrin av family Molecular characteristics Phylogenetic tree Tropism

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 the av integrin family of cellular receptors, avb3, avb6, avb1 and avb8, have been identied 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 analyse the roles of the av integrins from a susceptible species as viral receptors, we have cloned Bactrian camel av, b3 and b6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin av, b3 and b6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel av, b3 and b6 subunits share many structural features with homologues 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 distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species. 2009 Elsevier B.V. All rights reserved.

1. Introduction Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animal species (Thomson et al., 2003; Alexandersen et al., 2003). The Ofce 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

* Corresponding authors. E-mail addresses: changhuiyun@126.com (H. Chang), Caixp@public.lz.gs.cn (X. Cai). 0165-2427/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.04.008

desert and play a major role in the lives of people. The Camelidae inhabit countries in North and East Africa, Middle and East Asia as well as South America where FMD is endemic, 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, Asia1 and South African territories 1, 2 and 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)

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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 articial single-chain antibody fused to intercellular adhesion molecule 1, but these receptors do not require the RGD 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 initiate infection in vitro, and integrins are believed to be the receptors used in the infected animals (McKenna et al., 1995; Neff et al., 1998). Integrins are a large family of heterodimeric transmembrane glycoproteins composed of two subunits (a and b) that interact non-covalently at the cell surface. They mediate cellcell interactions and the binding of cells to the extracellular matrix, and they play a crucial role in cell division, differentiation, migration and survival (GonzalezAmaro 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 are avb1, avb3, avb6, avb8, avb5, a5b1, a8b1 and ajjbb3 (Ruoslahti, 1996; Plow et al., 2000). FMDV utilizes four members of the av subgroup of integrins (avb1, avb3, avb6 and avb8) as receptors to initiate 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 FMDV infection (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 no susceptibility (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 infection. In this study, as the rst 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 to those of other species including the orders Artiodactyla, Primates, Perissodactyla, Carnivora, Rodentia, Galliformes and 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 lung tissues were collected from these Bactrian camels immediately 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 (5 mg) was reverse transcribed using AMV reverse transcriptase (20 U/ ml, Takara, Japan), the oligo-dT18 primer (20 pmol/ml) and the random hexamer primers (20 pmol/ml) in a total volume of 40 ml, according to the manufacturers instructions. The av, b3 and b6 cDNAs were amplied from the cDNA 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 of the 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 amplied (Table 1) and 3 min at 72 8C, followed by a nal 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, b3 and b6 cDNAs The amplied bands corresponding to integrin cDNAs were excised from the 1% agarose gel and puried using the Gel extraction kit (Qiagen, Germany). The puried 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 Biotechnology Information. The sequence homology and divergence were

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Table 1 Primer sequences used for the cloning of integrin cDNAs from Bactrian camels. Primers AlphavF1 AlphavR1 AlphavF2 AlphavR2 Beta3F Beta3R Beta6F Beta6R
a b

Sequence (50 to 30 ) 5 -TCGGCGATGGCTTTTCCGCCGCG-3 50 -GTTTGTCTCTAAATTCAGATTCATCCC-30 50 -AATGGATATCCAGACTTAATTGTAGG-30 50 -CAGTTAAGTTTCTGAGTTTCCTTC-30 50 -GGGCCCAACATCTGTACCACGCGTGG-30 50 -TTAAGTGCCCCGGTACGTGATATTGGTG-30 50 -CTGAGACCGATGGCGATTGATCT-30 50 -ATGTTCTGTCCTTCGGAAAG-30
0 0

Target gene 5 part of integrin av

0
a

Predicted size of PCR products 1.8 kb

Annealing temperature 55.6 8C

30 part of integrin avb Mature integrin b3 Integrin b6

1.7 kb

54.4 8C

2.2 kb

57.8 8C

2.4 kb

56.8 8C

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

Table 2 Accession numbers of integrin sequences used for alignments and phylogenetic analysis. Common name Cattle Pig Human Monkey Chimpanzee Horse Dog Rat Mouse Guinea pig Chicken : no data available. Species Bos taurus Sus scrafa Homo sapiens Macaca mulatta Pan troglodytes Equus caballus Canis familiaris Rattus norvegicus Mus musculus Cavia. Gallus gallus Integrin av DQ871215 EF474019 M14648 XM001104012 XM515969 XM001498530 XM845896 NM001106549 AK149984 M60517 Integrin b3 AF239959 NM214002 M35999 XM001116013 XM523684 NM001081802 NM001003162 NM153720 AK157958 NM204315 Integrin b6 DQ867017 EF432729 NM000888 XM001094740 XM001149234 XM852055 NM001004263 AK036439 M35197

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 software package (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 b6 cDNA and EU867790 for mature Bactrian camel b3 cDNA. The reference sequences included in the analysis were taken from GenBank (Table 2). 3. Results 3.1. Cloning and sequence analysis of Bactrian camel av subunit The complete coding sequence of the Bactrian camel av subunit 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 located between amino acid residues 896 and 897 (KR-D). The

ligand binding domain contains three divalent cationbinding sites (DX[D/N]X[D/N]GXXD). Between the bpropeller domain and the transmembrane domain are the thigh domain (residues C469Q622), the genu domain (residues L623V631) and calf domain (residues C632Q992). The cytoplasmic portion contains a conserved G1025FFKR motif, which normally xes 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 of Bactrian camel integrin av subunit and its comparison to pig, bovine, human, horse and mouse av integrins are shown in Fig. 1A. The nucleotide and predicted amino acid sequence similarities within the different subunit functional regions among the Bactrian camel and several other species of av subunits are shown in Table 3. Overall, the transmembrane and cytoplasmic domains exhibited the highest degree of conservation between Bactrian camel and other species, and the Bactrian camel av subunit displayed a high level of similarity to its bovine and porcine homologues. The similarity results (%) were further conrmed by the phylogenetic analysis (Fig. 1B). The nucleotide sequences of integrin av from several species were classied into six major groups. The Bactrian camel av subunit was clustered into the Artiodactyla group, together with the av subunits of pigs and cattle. It was also shown that av sequences from the orders Rodentia, Primates, Perissodactyla, Carnivora and Galliformes formed separate groups, respectively, which were also distinct from the Artiodactyla group.

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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) and mouse (Mou). Dots indicate the same amino acid residues as the Bactrian camel av subunit. Divalent cation binding sites, potential N-glycosylation sites and cysteines are highlighted in red, yellow and light blue, respectively. The stripes above the sequences represent the deduced different constitutive parts of the protein: the signal peptide ( ), the ligand binding domain ( ), the thigh domain ( ), the genu ( ), the Calf-1 ( ) and Calf-2 ( ) domains, the transmembrane region ( ) and the cytoplasmic tail ( ). The inserted RFVLTC motif in the calf-1 domain 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 gure legend, the reader is referred to the web version of the article.)

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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 Mature subunit Ligand binding domain Signal peptide Ectodomain Transmembrane domain Cytoplasmic domain 93.4/96.6 93.5/97.5 85.6/76.7 95.0/96.8 94.3/100 95.8/100 Pig 93.5/96.0 93.6/97.5 83.3/80.0 93.3/95.4 96.6/100 95.8/100 Human 91.8/95.4 93.5/97.5 70.0/63.3 91.4/94.8 96.6/100 94.8/100 Monkey 91.5/95.1 91.3/96.3 68.9/60.0 91.0/94.5 96.6/100 93.8/100 Horse 92.9/96.4 92.5/97.9 54.3/40.7 92.5/95.7 93.1/96.6 94.6/100 Dog 90.9/95.9 91.3/97.8 91.0/95.8 85.1/96.6 92.7/100 Mouse 86.6/92.1 87.9/94.7 64.4/56.7 86.5/91.5 88.5/100 87.5/100 Chicken 73.6/82.3 74.0/83.8 36.8/21.1 73.5/81.4 74.7/93.1 83.3/93.8

3.2. Cloning and sequence analysis of mature Bactrian camel b3 subunit The 2289-nucleotide cDNA was found to code for the mature Bactrian camel b3 subunit of 762 amino acids with nine 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 glycosylation sites, the total weight of the mature b3 molecule would be 105 kDa. The mature protein consists of a 692residue ectodomain (G1D692) (amino acid 1 is the rst amino acid after cleavage of the signal sequence) with a total of 56 cysteine residues, a single 29-residue transmembrane domain (I693I721) and a 41-residue cytoplasmic 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 domain of 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 et al., 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 b3 protein shares common structural and functional elements with b3 molecules from the other species, and the amino acid sequence of Bactrian camel b3 was aligned with those of pigs, cattle, humans, horses and mice b3 (Fig. 2A). The nucleotide and deduced amino acid sequences within the different functional regions of Bactrian camel b3 showed higher similarity to those of porcine and bovine b3 than to human, equine, murine, canine, chicken and monkey b3 (Table 4). These results were also conrmed by phylogenetic analysis as Bactrian camel b3 was clustered into a group 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 b6 was 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) and a 52-residue cytoplasmic tail (H737G788). The deduced amino acid sequence includes 10 possible N-linked glycosylation sites, one of which is located in the cytoplasmic tail. The protein possesses 58 cysteine

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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 of Bactrian camel and other species are quite similar. The amino acid sequence of Bactrian camel b6 was aligned with 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 ( ) and the cytoplasmic tail ( ). NPXY motif in the cytoplasmic tail is boxed. (B) Phylogenetic relationship of integrin b3 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 gure legend, the reader is referred to the web version of the article.)

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Fig. 2. (Continued ).

Table 4 Nucleotide and encoded amino acid sequence similarities of integrin b3 between Bactrian camels and other species. Function domain % Nucleotide similarity/% amino acid similarity between Bactrian camels and other species Cattle Mature subunit Ligand binding domain Ectodomain Transmembrane domain Cytoplasmic domain 92.5/94.7 92.9/95.1 92.4/94.6 82.6/91.3 98.6/100 Pig 92.3/92.7 92.3/95.5 92.2/92.4 88.4/91.3 91.0/97.9 Human 91.3/95.1 94.1/96.3 91.7/95.2 82.6/87.0 90.1/100 Monkey 91.1/94.8 93.7/95.9 91.3/94.8 85.5/91.3 91.7/97.9 Horse 93.5/95.4 93.6/95.9 93.7/95.3 85.5/91.3 94.4/97.9 Dog 91.6/94.6 93.8/95.9 91.6/94.5 88.4/91.3 94.4/97.9 Rat 86.3/90.9 87.9/93.0 85.8/90.4 87.0/91.3 93.1/97.9 Mouse 86.4/91.5 87.5/92.2 86.0/91.1 87.0/91.3 92.4/97.9 Chicken 75.8/81.1 81.3/87.7 75.8/80.5 78.3/87.0 75.7/87.5

Table 5 Nucleotide and encoded amino acid sequence similarities of integrin b6 between Bactrian camels and other species. Function domain % Nucleotide similarity/% amino acid similarity between Bactrian camels and other species Cattle Mature subunit Ligand binding domain Signal peptide Ectodomain Transmembrane domain Cytoplasmic domain 91.0/94.4 94.4/98.3 93.6/92.3 90.7/94.1 90.8/100 95.6/94.3 Pig 91.7/93.4 93.8/98.8 96.2/92.3 91.3/93.2 95.4/96.6 94.3/94.3 Human 90.3/93.7 92.8/97.1 96.2/92.3 90.1/93.4 95.4/100 89.3/94.3 Monkey 89.9/93.2 92.4/97.1 96.2/92.3 89.4/92.7 94.3/100 93.7/96.2 Chimpanzee 90.1/93.4 92.6/97.1 96.2/92.3 89.9/93.1 95.4/100 89.9/94.3 Dog 91.3/93.8 93.3/97.1 96.2/92.3 90.8/93.5 94.3/100 95.0/94.3 Rat 84.1/89.0 87.1/94.6 82.1/76.9 83.9/88.4 88.5/100 84.3/90.6 Mouse 83.7/89.0 87.2/94.6 83.3/80.8 83.7/88.5 89.7/100 80.5/88.7

acid similarities within the different functional regions showed that Bactrian camel b6 was closely related to those of pigs and cattle (Table 5). Phylogenetic analysis showed that the nucleotide sequences of b6 subunits from several mammalian species were classied into four major groups. The Bactrian camel b6 was clustered into the Artiodactyla group, together with b6 of pigs and cattle (Fig. 3B). 4. Discussion It has denitely 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 factors involved 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 integrins to 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 efciently than it utilizes the human homologues (Neff et al., 2000). The fact that the virus can only infect certain species leaves open the question of how

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differences between hosts may determine the susceptibility 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, the av subunit coding sequences were amplied from cDNAs prepared from Bactrian camel lung and tongue tissues, while the sequence

coding for the b6 subunit was amplied only from tongue tissue and the mature b3 subunit was amplied only from lung tissue. We were unable to get a complete coding sequence which included the Bactrian camel b3 signal peptide sequence and, therefore, only obtained the mature b3 sequence without the signal sequence. Analysis of the distribution of the integrin receptors in susceptible species may be necessary to explain viral pathogenesis within

Fig. 3. (A) Alignment of deduced amino acid sequences of the integrin b6 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 b6 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 signal peptide ( ), the ectodomain ( ), the ligand binding domain ( ), the four cysteine-rich repeat ( ), the transmembrane region ( ) and the cytoplasmic tail ( ). NPXY motif in the cytoplasmic tail is boxed. (B) Phylogenetic relationship of integrin b6 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 gure legend, the reader is referred to the web version of the article.)

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Fig. 3. (Continued ).

different species. At present we are focusing on the changes in integrin proles 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 RTPCR 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 makes contacts with both subunits, the arginine tting into a cleft formed primarily by a b-propeller domain from av, with critical residues D180 and D248, and the aspartate coordinating the cation bA domain of b3 integrin with MIDAS (critical residues D119, S121, S123, E220 and D251) (Xiong et al., 2001, 2002). Characteristic of all integrin b subunits is 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 b6 subunits. The very high interspecies conservation of the putative MIDAS and critical residues conrms their essential role in integrin function. Duque and Baxt (2003) showed that different virus serotypes appear to utilize the bovine integrin receptors with varying efciencies and the ligand-binding domain of the bovine b subunit plays a role in the recognition of the different viral serotypes. Alignments of the predicted amino acid sequences of the av, b3 and b6 subunits with those of other 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 efciency 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 those seen in bovine integrin.

Phylogenetic trees and similarity analyses were performed to conrm 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-andmouth 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 affect both 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 nonstructural 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. The pathogenesis 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) are of low susceptibility to inocu-

J. Du et al. / Veterinary Immunology and Immunopathology 131 (2009) 190199 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., Domingo, E., 2000. Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: exibility in aphthovirus receptor 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-andmouth 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 receptor mediated adsorption. Virology 207, 503509. Beard, C.W., Mason, P.W., 2000. Genetic determinants of altered virulence of 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. Antibodies to the vitronectin receptor (integrin avb3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells. J. Virol. 69, 26642666. Colombatti, A., Bonaldo, P., 1991. The superfamily of proteins with von Willebrand factor type A-like domains: one theme common to components of extracellular matrix, hemostasis, cellular adhesion, and defense mechanisms. Blood 77, 23052315. Dedhar, S., Hannigan, G., 1996. Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr. Opin. Cell Biol. 8 (5), 657669. Duque, H., Baxt, B., 2003. Foot-and-mouth disease virus receptors: comparison 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 cloning and characterization of the alphaX subunit from CD11/CD18 horse integrin. 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 comparison with the human and murine glycoproteins. Gene 325, 97 101. 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 includes the amino acid sequence RGD (arginineglycineaspartic acid). 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 a receptor for foot-and-mouth disease virus: role of the b-chain cytodomain 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. Efcient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J. Virol. 70, 52825287. Jackson, T., King, A.M.Q., Stuart, D.I., Fry, E., 2003. Structure and receptor binding. Virus Res. 91, 3346. Jackson, T., Mould, A.P., Sheppard, D., King, A.M.Q., 2002. Integrin avb1 is a 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 characterization and expression analysis of the gene coding for the porcine b3 integrin subunit (CD61). Gene 408, 917. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5, 150163.

199

Larska, M., Wernery, U., Kinne, J., Schuster, R., Alexandersen, G., Alexandersen, S., August 8, 2008. Differences in the susceptibility of dromedary 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 integrin regulation 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 poliovirus, can infect normally insusceptible cells via the Fc receptor. Virology 192, 568577. Mason, P.W., Grubman, M.J., Baxt, B., 2003. Molecular basis of pathogenesis of foot-and-mouth disease virus. Virus Res. 91, 932. McKenna, T.S., Lubroth, J., Rieder, E., Baxt, B., Mason, P.W., 1995. Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD. J. Virol. 69, 57875790. Moyle, M.M., Napier, M.A., Mclean, J.W., 1991. Cloning and expression of a divergent integrin subunit b8. J. Biol. Chem. 266 (29), 1965019658. Neff, S., Mason, P.W., Baxt, B., 2000. High-efciency utilization of the bovine integrin avb3 as a receptor for foot-and-mouth disease virus is 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 utilizes 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. Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A. Virology 287, 151162. Pardi, R., Bossi, G., Inverardi, L., Rovida, E., Bender, J.R., 1995. Conserved regions in the cytoplasmic domains of the leukocyte integrin aLb2 are involved in endoplasmic reticulum retention, dimerization, and cytoskeletal association. J. Immunol. 155, 12521263. Plow, E.F., Haas, T.A., Zhang, L., Loftus, J., Smith, J.W., 2000. Ligand binding to integrins. J. Biol. Chem. 275 (29), 2178521788. Rieder, E., Berinstein, A., Baxt, B., Kang, A.M., Mason, P.W., 1996. Propagation of an attenuated virus by design: engineering a novel receptor for a 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 to tropism 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 receptors for diverse viral pathogens. Trends Microbiol. 15 (11), 500507. Thomson, G.R., Vosloo, W., Bastos, A.D.S., 2003. Foot and mouth disease in wildlife. 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-like domain 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 av related matrix receptors in metanephric development. J. Cell Biol. 132, 11611176. Wernery, U., Kaaden, O.R., 2004. Foot-and-mouth disease in camelids: a review. 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 structure of the extracellular segment of integrin avb3. 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 of integrin avb3 in complex with an ArgGlyAsp ligand. Science 296, 151155.