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Four members of the αv integrin family of cellular receptors, can serve as receptors for the foot-and-mouth disease virus (FMDV) in vitro, and these integrins are considered to be the receptors used to target epithelial cells in infected animals.
We analyzed the roles of αv, β1, and β6 integrins as viral receptors in a susceptible species by cloning goat αv, β1, and β6 integrin cDNAs and comparing them with homologues. The coding sequences for goat αv, β1, and β6 integrins were 3147, 2397, and 2367 nucleotides in length, which encoded 1048, 798, and 788 amino acids, respectively. The goat αv, β1 andβ6 subunits shared many structural features with homologues from other species. Phylogenetic trees and similarity analyses indicated the close relationship among integrin genes from sheep, pigs, cattle, and Bactrian camels susceptible to FMDV infection.
This study is important for understanding the differences in integrins as FMDV receptors in other species.
Foot-and-mouth disease (FMD), also known as aphthous fever, is a highly contagious viral disease that affects wild and domestic cloven-hoofed animals. Foot-and-mouth disease virus (FMDV) initiates infection by binding to its cellular receptor via an arginine-glycine-aspartic acid (RGD) sequence found in a surface protrusion that consists of a loop between the bG and bH strands (G-H loop) in the capsid protein VP1 [1.2.3], where a conserved Arg-Gly-Asp (RGD) motif is characteristic of the ligands of several members of the integrin family . This loop (the G-H loop) forms a major antigenic site on the virion where its apex includes an Arg-Gly-Asp (RGD) motif [5,6]. FMDV enters the cell via receptor-mediated endocytosis during a process that begins with the initial attachment of the virus to cell-surface receptors. The G-H loop-containing RGD is highly conserved among FMDV serotypes, showing that viruses can enter cells by means of an RGD-binding integrin. Integrins are heterodimeric molecules that consist of α and β subunits, which interact noncovalently at the cell surface and have a wide species distribution . They are involved in extracellular matrix and cell-cell interactions, while they also serve as signal transducing receptors . FMD is frequently mild or asymptomatic in adult goats, but it can cause
high mortality in young animals. Understanding the mechanism of infection and replication of this virus is important for the control of this worldwide threat. However, an important question that has yet to be addressed in this respect is the role of viral receptors in the pathogenesis of
FMD. In this report, we present an initial step in analyzing the pathogenesisof FMDV in goats, which should help us to understand the reasons for its mild clinical signs and persistent infection.
RNA isolation and amplification of cDNA Tongue and lung tissues were collected from healthy domestic goat (1-2 years of age) immediately after slaughter. All animal experiments were performed according to protocols approved by the institutional committee for use and care of animals. Total RNA from each tissue was extracted with RNeasy Mini Kit (Qiagen, Germany) as described by the manufacturer. The procedures for One Step RT-PCR Kit Ver.2 (Takera, Japan) recommended by the manufacturer were used, with the following cycling parameters: 30 min at 50â„ƒ reverse transcription, 2min at 94â„ƒ for predenaturation, 30 cycles of 1 min at 94â„ƒ, 30s at annealing temperatures depending on integrins to be amplified (Table 1) and 1kb/min at 72â„ƒ, followed by the final extension for 10 min at 72â„ƒ. The resulting PCR products were run on 1% agarose gel containing ethidium bromide and the DNA bands were visualized using a UV transilluminator.
Cloning and sequencing
The amplified products corresponding to integrin cDNAs were gel purified using the Qiaquick Gel Extraction Kit (Qiagen, Germany). The purified products were ligated into the pMD-18 T vector (Takera, Japan), and the resultant recombinant plasmids were transformed into competent Escherichia coli strain JM109 (Promega, USA). Positive clones were selected on LBA/ampicillin/IPTG/X-Gal plates, and plasmid DNA was isolated and purified with Plasmid Miniprep Kit (Takara, Japan) by the manufacturer's protocol. In each cDNA, 3 plasmid clones containing integrand cDNAs were sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, USA). Primers design was performed with Primer Premier 5.0 and oligo 6.0 software (Table 1).
Sequence data analyses were performed using the BLAST search of the National Center for Biotechnology Information. The sequence homology and divergence were calculated using the Laser-gene analysis software package (DNASTAR, USA). The sequences were aligned using Clustal W program available in the BioEdit v7.0.5 software package (Ibis therapeutics, Carlsbad, CA). Phylogenetic tree were constructed using MEGA version 5.1. The sequence data herein have been submitted to GenBank dated April 2012, including accession number [GenBank: JQ965818] for goat αv cDNA, [GenBank: JQ 965819] for goat β1 cDNA, [GenBank: JQ 965817] for goat β6 cDNA. The reference sequences included in the analysis were taken from GenBank (Table 2).
Results and Discussion
The goat αv, β1, and β6 subunits cDNA contained ORFs of 3147, 2397, and 2367 nucleotides, which encoded 1048, 798, and 788 amino-acid residue proteins, respectively, with all the typical features of integrin subunits. The amino acid sequences of goat integrin αv, β1, and β6 subunits shared common structural and functional elements with the αv, β1, and β6 molecules from other species. The displayed sequence of goat integrins αv (Table 3A), β1 (Table 3B) and β6 (Table 3C) were further processed into a mature form, respectively. The goat αv (Figure 1A), β1 (Figure 1B), and β6 (Figure 1C) subunits shared a high level of identity with bovine, porcine, and Bactrian camel homologues. The identity results were further confirmed by phylogenetic analysis (Figure 1A) A, B, and C), respectively. The nucleotide sequences of integrin αv from these species were classified into six major groups. The goat αv, β1, and β6 sequences were clustered into the Artiodactyla group together with those of pig, cattle, and Bactrian camel, whereas the αv, β1, and β6 sequences from the horse, dog, monkey, human, chimpanzee, mouse, rat, and chicken formed separate groups, which were distinct from the Artiodactyla group.
FMD is one of the most highly regulated livestock diseases in the world and extensive international efforts have been undertaken to control the spread of FMDV through restrictions on the movement of animal products and live animals. Goats are one of the oldest domesticated species and they have been used for their milk, meat, hair, and skins throughout much of the world. FMD causes significant economic losses due to high morbidity and a loss of production in infected animals. Thus, it is a major hindrance to the international trade in animals and animal products [9, 10]. Integrins are proteins with crucial biological importance because they are used by cells for binding and responding to the extracellular matrix. Functional integrins consist of two noncovalently bound transmembrane glycoprotein subunits, which are designated as alpha (α) and beta (β) . Integrins are heterodimeric transmembrane glycoproteins formed of noncovalently bound α and β subunits at the cell surface. Eighteen differentα subunits and eight different β subunits have been reported in vertebrates, which can produce at least 24 αβ heterodimers. This may make the integrins the most structurally and functionally diverse family of cell adhesion molecules [12,13 ]. One subgroup within the integrin family is theαv integrins, which includes four heterodimers, i.e., αvβ1, αvβ3, αvβ6, and αvβ8, that recognize the RGD as a binding motif for their natural ligands . FMDV is dependent on integrin receptors for infection in vitro andintegrins are considered to be the receptors used in animals . The development of therapeutic tools based on integrins as targets may facilitate the development of a possible strategy for controlling FMDV infection in vivo, or at least help to control the virus spread during an outbreak . Cattle and swine are seriously affected by FMD infections with high morbidity and overt clinical disease. However, FMD typically has a subclinical form in the goat, although clinical cases can and do occur. Goats may become carriers after exposure to FMDV even if they are immune due to previous vaccinations or recovery from infection. The maximum duration of the carrier state in goats is reported to be four months, compared with 9 months in sheep and up to three and a half years in cattle . The roles that the various integrin receptors play in FMDV tropism and pathogenesis remains unclear [4, 18]. For the first time, we present the sequences of goat integrins αvβ1 and αvβ6 associated with FMDV infection and we demonstrate that, despite some local differences, they share all the common structural and functional elements with integrin molecules from other species. We postulate that FMDV evolved into a disease of cloven-hoofed livestock because the structure of their integrin receptors resulted in a more advantageous ''fit'' with the viral surface, which may have led to higher viral replication and disease incidence within these species . Our phylogenetic analysis also indicated that goat integrin genes are closely related to those of other species in the order Artiodactyla, including cattle, sheep, pig, and Bactrian camels [19, 20, 21, 22]. It is interesting to speculate that FMD is limited to cloven-hoofed animals because of similar integrin receptor characteristics.
We molecularly cloned the goat αv [GenBank: JQ965818], β1 [GenBank: JQ965819], and β6 [GenBank: JQ965817] subunit cDNAs for the first time and compared them with those from other species. Phylogenetic analyses of integrin genes may be useful for studying the evolution relationship among different species. It is also important to note that the receptors alone may not necessarily determine the FMDV species tropism, because other viral and cellular factors may also affect the host range and virulence [4, 19, 23].