BACKGROUND
pattern recognition receptors  are crucial to animal surveillance of pathogen invasion.
the prrs recognise conserved pathogen-associated molecular pattern  motifs, including proteins, lipids and nucleotides, resulting in activation of host innate defences  <cit> .
the prrs comprise three major groups, toll like receptors , retinoic acid induced rig-i like receptors and nucleotide oligomerization domain  containing proteins, sensing pamps extracellularly or within the cytoplasmic region.
the rig-i like receptors are crucial to the rna virus triggered interferon response.
they consist of three members, retinoic acid-inducible gene i  box polypeptide  <dig> ) and melanoma differentiation-associated gene  <dig> ), and laboratory of genetics and physiology  <dig>  box polypeptide  <dig> ), which share a common functional rna helicase domain near the c terminus  specifically binding to the rna molecules with viral origin  <cit> .
two tandem arranged caspase activation and recruitment domains  involved in protein-protein interactions are present at the n terminal region of the rig-i and mda <dig> proteins but not lgp <dig>  triggering the interferon response via activation of interferon regulatory factor  <dig> and nfkb  <cit> .
another distinct core domain is the atp dependent dexd/h domain containing a conserved motif asp-glu-x-asp/his  which is involved in atp-dependent rna or dna unwinding.
rig-i/mda <dig> directed interferon signalling is now known to be controlled by the mitochondria antiviral signalling protein , a card containing protein associated with mitochondria, and negatively regulated by lgp <dig> which lacks a card domain  <cit> .
lgp <dig> has been shown to interfere with the binding process of rig-i/mda <dig> to viral rnas  <cit> .
both rig-i and mda <dig> appear to have overlapping binding properties with viral pamps and share similar signalling pathways leading to activation of the interferon system.
however, evidence of differential recognition of viral pamps by rig-i has begun to emerge recently.
it seems that mda <dig> preferentially binds long, capped di- or mono-5' phosphate double stranded  rnas whilst rig-i has high binding affinity with short dsrnas or 5' ppp uncapped single stranded  rnas  <cit> .
interestingly, neither rig-i nor mda <dig> has a classic rna binding motif.
a zinc-binding domain located at the c terminal region  of human has been shown to specifically bind to viral derived 5'ppp rna  <cit> .
rig-i and mda <dig> respond differently to infection with various viral strains, with rig-i sensitive to paramyxoviruses, orthomyxoviruses, and the rhabdovirus vesicular stomatitis virus whilst mda <dig> reacts to picornaviruses  <cit> .
some viral proteins, such as the v protein of paramyxoviruses, interact with mda <dig>  a mechanism possibly used by viruses as a means to escape host surveillance.
whilst most studies are focused on the rig-i like prrs in mammals, little is known about such molecules in other living organisms.
a recent study surveying the purple sea urchin genome has revealed multiple putative rig-i like homologues that appear to be present in invertebrates  <cit> .
more recently, it has been hypothesised that mda <dig> might have emerged before rig-i and their domain arrangement evolved independently by domain grafting rather than by a simple gene duplication event  <cit> .
in this study, we took a comparative genomics approach by analysing rig-i like prrs in a number of invertebrate and vertebrate genomes, in order to elucidate the origin and evolution of the rig-i like prr family.
bioinformatic analysis of functional domains of rig-i, mda <dig> and lgp <dig> has identified two evolutionary conserved domains in mda <dig> and lgp <dig> which may be critical to the recognition and processing of viral nucleotide pamps.
RESULTS
sequence identification
extensive blast analysis of vertebrate genomes or expressed sequence tag databases using known rig-i protein sequences identified a putative full length rig-i homologue in western clawed xenopus but not in chicken .
the putative xenopus rig-i is  <dig> aa in length, sharing  <dig> % identity with human rig-i and contains conserved domains such as a dexd/h domain in the middle region and a helicase domain at the c terminal region.
a less homologous card domain at the n terminus is also apparent.
in the zebrafish genome, a single rig-i like gene encoding a protein of  <dig> aa was found in chromosome  <dig> , some  <dig> aa shorter than the mammalian and amphibian rig-i proteins.
although it contains a dexd/h domain and a card domain at the n terminus, it lacks a classic helicase c  domain.
it is uncertain whether zebrafish rig-i is produced as a functional protein since no est matching the predicted rig-i exists.
in japanese pufferfish, tetraodon, medaka and stickleback, no rig-i homologues were identified either in the est databases or genome databases.
unlike rig-i, whose presence in chicken and some fish species is uncertain, the mda <dig> homologues can be found throughout vertebrate species including fish, amphibians, birds and mammals .
mda <dig> is encoded by a single copy gene and the putative proteins have comparable length, ranging from  <dig> aa to  <dig> aa .
in addition to the conserved dexd/h domain and helicc domain, two tandem card domains at the n terminal regions are predicted and are well conserved among vertebrate mda <dig> molecules except for the zebrafish mda <dig> that lacks a clear card domain.
the card motifs near the n-terminus  are more diverse than the second card motif .
note: the amino acid position of each domain is listed and the e values against the pfam database are indicated in parentheses.
lgp <dig> is an adaptor protein lacking card domains but containing a dexd/h domain and a helicc domain homologous to their corresponding motifs in the rig-i and mda <dig> protein.
it competes with rig-i and mda <dig> for the ligands, viral derived rna pamps, but is unable to interact with down stream signalling proteins due to the absence of card domains.
thus it acts as a negative regulator of the rig-i/mda <dig> directed antiviral response.
lgp <dig> appears to co-exist with mda <dig> in vertebrates as a single copy gene.
it is located in a different chromosome to mda <dig> in every species analysed.
the putative lgp <dig> proteins from non-mammalian species contain 588– <dig> aa, much shorter than the rig-i and mda <dig> proteins.
the dexd/h domain and the helicc domain in the lgp <dig> protein share higher homology with the corresponding regions in mda <dig> than those in rig-i.
the lgp <dig> dexd/h domains are  <dig> – <dig> % identical to the mda <dig> counterparts compared to  <dig> – <dig> % for the rig-i proteins.
similarly,  <dig> – <dig> % identity is seen between the lgp <dig> helicc domains and the mda <dig> helicc domains, in contrast to  <dig> – <dig> % between lgp <dig> helicc domains and the rig-i helicase domains.
twelve genes coding for rna helicase proteins homologous to rig-i/mda5/lgp <dig> have been reported in a recent survey of the sea urchin genome draft  <cit> .
some of the deduced proteins contain card domains in addition to dexd/h and helicc domains.
using the human mda <dig> protein sequence as a bait, a partial homologue sequence was obtained from the sea anemone nematostella vectensis genome database ,  this partial sequence was then used to search the ncbi database and two contigs  were retrieved, which encoded two putative rig-i/mda5/lgp <dig> homologues.
the putative proteins are  <dig> aa and  <dig> aa in length, similar to that of lgp <dig>  further prediction of functional motifs revealed the presence of a dexd/h domain and a helicc domain but not the n terminal card domain.
the proteins share  <dig> – <dig>  identity with rig-i,  <dig> – <dig> % with mda <dig> and  <dig> – <dig> % with lgp <dig>
gene synteny analysis
to gain an insight into whether the genes surrounding rig-i, mda <dig> and lgp <dig> are evolutionary conserved, we analysed draft genome sequences of invertebrates, fish, amphibians, birds and humans.
the rig-i locus was identifiable in xenopus where the genes neighbouring rig-i were different to those in humans except for the aco <dig> gene .
since the upstream region of the rig-i locus was not available in the xenopus genome database, whether rig-i clustered with topors remains to be determined.
in zebrafish, the rig-i gene was immediately downstream of the topors gene, which is adjacent to the rig-i gene in humans.
in contrast to the observation in zebrafish, the stickleback genome appears to lack rig-i, which is not due to poor genome analysis since the sequence of the rig-i locus is of good quality.
in fugu, medaka and chicken, the rig-i gene could not be identified but was hindered by incomplete sequence data.
the gene synteny of mda5/lgp <dig> is well conserved in vertebrates, from fish to humans .
eight genes surrounding mda <dig> in stickleback, fugu and medaka appear in the genomes of xenopus, chicken and humans, in the same order and the same transcriptional orientation.
less conservation of gene synteny was noted in the zebrafish genome where only  <dig> conserved neighbouring genes were present in the mda <dig> locus.
similarly, the gene composition and arrangement in the lgp <dig> gene locus shows remarkable conservation during vertebrate evolution.
in invertebrates, two loci containing prototype homologues of rig-i/mda <dig> were found downstream of two independent genes coding for two card like molecules in nematostella vectensis .
some  <dig> rig-i/mda like genes have also been predicted from the purple sea urchin genome  <cit> .
whilst it was possible to find appropriate contigs containing rig-i/mda <dig> like genes from the sea urchin genome project, it was not possible to assess gene synteny due to a lack of genome assembly.
phylogenetic analysis
rig-i, mda <dig> and lgp <dig> are believed to bind rna molecules through helicase-c domains.
closely related proteins containing a rna binding helicase-c domain and a dexd/h helicase domain are the double-stranded rna-specific endoribonuclease  and elongation initiation factor  4a.
dicer belongs to the rnase iii family that cleaves double-stranded rna  into short double-stranded rna fragments, called small interfering rna, that is required for specific cleavage of complementary viral rnas, whilst eif4a mainly participates in translation and other processes such as pre-mrna splicing and ribosome biogenesis.
since homology analysis of the rig-i and mda <dig> proteins in the databases using the blastp programme gave top scores to dicer and eif4a in addition to the rig-i, mda <dig> and lgp <dig> proteins, we included dicer and eif4a in the phylogenetic tree analysis using the neighbour joining method within the mega <dig> programme.
as shown in fig.
<dig>  dicer and eif4a formed two distinct groups with a long evolutionary distance to the branch that evolved into invertebrate dicer like helicase , rig-i, mda <dig> and lgp <dig>  apparently, dicers are more distantly related to the ancestor of rig-i, mda <dig> and lgp <dig> than eif4a.
comparing to dicers and eif4as, c. elegans drhs were located close to some of the sea urchin rig-i/mda like proteins, the prototype of the vertebrate counterparts.
within the rig-i branch are two of the rig-i like homologues from nematostella vectensis, two from the sea urchin , and predicted or known rig-i molecules from zebrafish, xenopus, and mammals, suggesting rig-i may have emerged in invertebrates.
the fact that rig-i homologues were absent in most fish species suggests that the rig-i gene may have been lost in some fish lineages.
the tree also indicates that lgp <dig> and mda <dig> proteins from vertebrate species form two clear groups closely neighbouring each other, suggesting they possibly diverged from a common ancestor that originated from a rig-i like molecule in invertebrates or early vertebrates.
in addition, two phylogenetic trees were constructed using helicase c domains and dexd/h domains and a similar grouping of the major branches was observed .
domain analysis
the putative domains were analysed in the pfam database using a cut-off e-value of  <dig>  and selected domains listed in table  <dig>  a comprison of the domains is shown in fig.
<dig>  a classical helicase c domain is present in all  <dig> types of dexd/h helicases, including rig-i, mda <dig>  lgp <dig>  dicer and eif4a.
the dexd/h box responsible for atp binding and hydrolysis was detected in the middle region of rig-i, dicer and eif4a by the pfam hmm search.
similarly, the corresponding region in mda <dig> and lgp <dig> was detected as a conserved restriction domain of bacterial type iii restriction enzymes  , sharing some degree of homology with the rig-i dexd/h box.
two card domains were predicted in the n terminal regions of rig-i and mda <dig> except for xenopus and zebrafish rig-i, which contained a single card domain corresponding to the second card domain of the human molecule .
in general, the n-terminal card domain  was less conserved than the card <dig> domain.
the e-value of card <dig> in rig-i ranges between  <dig>  and  <dig> , significantly higher than that in mda <dig> , indicating significant divergence of the card domains in different vertebrate groups.
in addition, a fragment at the c-terminal region in most mda <dig> proteins of chicken, xenopus and fish, contained a putative domain with moderate e-values  to the  <dig> cysteine  type transcription factor  iis central domain.
the c terminal region of human rig-i comprised a region distantly related to the c <dig> type zinc finger domain, which was shown to bind to dsrna and 5'ppp viral rna with the involvement of zinc ion  <cit> .
the pfam hmm analysis failed to identify this c <dig> tfiis domain in rig-i possibly due to low homology.
the domain sequences were further analysed by multiple alignment and their  <dig> dimensional structures modelled.
it is apparent that the dexd/h and res iii domains comprised a well conserved dech motif .
the overall structures of dexd/h boxes and res iii domains were similar, with β sheets sandwiched by α-helices on each side .
six β strands were arranged in the same orientation in the human rig-i dexd/h box and mda5/lgp <dig> res iii domains except for the human mda <dig> res iii domain, where  <dig> β strands were present.
conversely, numbers of predicted α-helices varied significantly among domains.
although homology analysis showed low sequence similarities between the mda <dig> tfiis domain and the c <dig> type zinc finger nucleotide binding motif within the human tfiis central domain, 3d modelling displayed significant structural similarities .
our modelling data indicated the c terminal region of rig-i/mda <dig> proteins across vertebrates possessed a conserved c <dig> type zinc finger nucleotide binding motif, in agreement with the studies in humans where a putative domain distantly related to the c <dig> type zinc finger protein was shown to bind to viral nucleotide pamps  <cit> .
remarkably, the  <dig> cysteines involved in capturing zinc ion were in close physical contact, forming a conserved pocket on the surface of all the domains analysed.
however, arrangement of the cysteines differed in the human tfiis-c domain and rig-i/mda <dig> domains .
in addition to the c <dig> type zinc finger nucleotide binding motif, the rig-i c terminal region and the mda <dig> tfiis domain contained another noticeable β strand structure which appeared to support the c <dig> type pocket.
in the human rig-i, a single α helix was also detected.
discussion
the rig-i like helicase family members have recently been reported to play pivotal roles in recognising viral nucleotides in mammals.
in this report, the rig-like homologues have been identified in silico in the nucleotide databases of invertebrates and vertebrates and their evolutionary origin discussed.
double stranded rna is the genetic component of viruses with double stranded genomes and part of a single stranded rna with secondary structures.
it can be generated during viral replication and rna metabolism.
this nature of dsrna makes it the prime target for host prrs.
classical double stranded rna binding domains are often used by some cytosolic prrs, such as pkrs, as the detectors to sense viral presence.
dicer proteins also contain two dsrna binding domains  for capturing dsrna molecules.
in the present study, a zinc finger domain similar to that of transcription factor  iis has been found in mda <dig> but not in lgp <dig>  with moderate homology to the rig-i c terminal region.
furthermore, a well conserved type iii restriction enzyme domain responsible for restriction in prokaryotic organisms is identified in the middle of both mda <dig> and the n terminal region of lgp <dig>  this domain was not detected in rig-i molecules by the pfam hmm programme although it shared some degree of homology.
we speculate that these two domains may serve as potential binding domains to interact with viral pamps.
one striking finding is that a well conserved restriction enzyme iii  domain is predicted in all mda <dig> and lgp <dig> proteins .
the res iii domain is structurally similar to the dexd/h domain.
restriction enzymes are important components of prokaryotic dna restriction-modification mechanisms in defence against foreign dna  <cit> .
they function in combination with one or two modification enzymes  that protect the cell's own dna from cleavage by the restriction enzymes.
restriction enzymes consist of four types depending on their recognition sequences and location of cleavage sites.
type iii enzymes recognize short 5– <dig> bp long asymmetric dna sequences and cleave 25– <dig> bp downstream to generate short, single-stranded 5' overhang ends.
type iii enzymes contain two functional subunits res  and mod , specifically for dna cleavage of unmethylated double stranded foreign dna  and protection of self dna from damage by methylation , respectively.
classic strand separation helicase activities have not been detected for type iii restriction enzymes  <cit> .
the res iii domain predicted in mda <dig> and lgp <dig> have significant homology with bacterial res iii domains and multiple alignment reveals significant conservation .
mda5/lgp <dig> are also similar to the rnase iii domains in the rna endonuclease dicer and dicer like helicases which process dsrna into 21– <dig> nt 3' overhang small rnas, with  <dig> nt protrusions, and atp-binding domains in bacterial and yeast dna helicases  <cit> .
integrated nuclease domains with excision activities are seen in the dicer proteins where two ribonuclease iii domains cut double stranded rnas, releasing  <dig> nt 3' end overhang 21– <dig> nt rna molecules which are essential for specific cleavage of viral rnas  <cit> .
another putative important domain, a zinc finger motif similar to that of transcription factor  iis, was identified by homology analysis in the pfam database.
the zinc finger motif can bind a range of targets including dnas, rnas, proteins and even lipids.
it is known that the zinc finger motif at the c-terminus of the tfiis is essential for rna binding and processing  <cit> .
integrated tfiis zinc ribbon c-terminal domains are also found in some viral proteins  <cit> .
the tfiis motif located near the c terminus of the rig-i/mda <dig> proteins was detectable in the pfam database although the e value  is moderate .
structural modelling confirmed remarkable conservation of a c <dig> type zinc finger pocket and a β-strand structure compared to the c <dig> type zinc finger nucleic acid binding domain in the human tfiis.
furthermore, a β-strand motif is also present within this domain in addition to the c <dig> type β-strand zinc finger structure.
whether it is involved in recognition of viral rna pamps remains to be determined.
a recent study has demonstrated that a c terminal domain in human rig-i  was involved in binding dsrna or 5'ppp rna, which was confirmed by magnetic resonance and x-ray crystallography  <cit> .
this region was also shown to suppress rig-i signalling  <cit> .
thus it is possible that viruses could interfere with this host recognition system by their own tfiis-c containing proteins.
the origin and evolution of rig-i, mda <dig> and lgp <dig> were analysed in this study.
our data suggest they evolved from common invertebrate ancestors encoding distinct core domains , which was supported by the presence of the rig-i like genes in sea anemone nematostella vectensis and sea urchin genomes  <cit> .
tandem clustering of two card genes with the rig-i like genes without card domains in the nematostella vectensis genome provides a strong clue as to how rig-i, mda <dig> and lgp <dig> could have evolved during evolution through gene fusion, domain duplication and domain deletion , supporting recent analysis suggesting that card <dig> could have been grafted independently rather than duplicated from card <dig> during evolution  <cit> .
in deuterostome invertebrates, the rig-i/mda <dig> like genes appear to have expanded enormously, as seen in the sea urchin, with some differentiating into molecules with a closer phylogenetic relationship to the vertebrate rig-i molecules .
we speculate that rig-i emerged earlier than mda5/lgp <dig> since vertebrate rig-is grouped with the invertebrate progenitors rather than the mda <dig> and lgp <dig> proteins which are present uniquely in vertebrates, in stark contrast with the evolutionary model proposed by sarkar et al  <cit> , where lgp <dig> preceded both mda <dig> and rig-i in evolution.
moreover, the phylogenetic tree constructed in the present study shows that mda <dig> has a closer relationship with lgp <dig> rather than rig-i, suggesting mda <dig> and lgp <dig> originated from a more recent gene duplication event, unlike the phylogenetic results obtained by sarker et al  <cit> .
if mda5/lgp <dig> did diverge from rig-i more recently, the order of their appearance is not clear.
also, it is uncertain from the present study why rig-i was not found in all teleost fish.
although a putative gene coding for a partial rig-i is predicted in the zebrafish genome, with conserved gene synteny to the xenopus and human rig-i locus , it is absent in the other fish genomes.
poor quality of the genome sequences makes conclusions difficult but as no rig-i sequences were found in the vast number of fish est sequences, perhaps functional rig-i genes have been lost in some teleost fish species.
CONCLUSIONS
the rig-i/mda5/lgp <dig> system is an ancient antiviral system well conserved in vertebrates.
our data suggest that these helicase prrs have evolved from an ancient progenitor originated from genes coding for individual functional domains and expanded by multiple evolutionary events leading to gene and/or domain gain and loss.
the present study provides important clues for further elucidation of rig-i/mda <dig> mediated antiviral defence in vertebrates.

 pattern recognition receptors are crucial to animal surveillance of pathogen invasion. the prrs recognise conserved pathogen-associated molecular pattern motifs, including proteins, lipids and nucleotides, resulting in activation of host innate defences. the prrs comprise three major groups, toll like receptors, retinoic acid induced rig-i like receptors and nucleotide oligomerization domain containing proteins, sensing pamps extracellularly or within the cytoplasmic region. the rig-i like receptors are crucial to the rna virus triggered interferon response. they consist of three members, retinoic acid-inducible gene i box polypeptide ) and melanoma differentiation-associated gene ), and laboratory of genetics and physiology box polypeptide ), which share a common functional rna helicase domain near the c terminus specifically binding to the rna molecules with viral origin. two tandem arranged caspase activation and recruitment domains involved in protein-protein interactions are present at the n terminal region of the rig-i and mda proteins but not lgp triggering the interferon response via activation of interferon regulatory factor and nfkb. another distinct core domain is the atp dependent dexd/h domain containing a conserved motif asp-glu-x-asp/his which is involved in atp-dependent rna or dna unwinding. rig-i/mda directed interferon signalling is now known to be controlled by the mitochondria antiviral signalling protein, a card containing protein associated with mitochondria, and negatively regulated by lgp which lacks a card domain. lgp has been shown to interfere with the binding process of rig-i/mda to viral rnas. both rig-i and mda appear to have overlapping binding properties with viral pamps and share similar signalling pathways leading to activation of the interferon system. however, evidence of differential recognition of viral pamps by rig-i has begun to emerge recently. it seems that mda preferentially binds long, capped di- or mono-5' phosphate double stranded rnas whilst rig-i has high binding affinity with short dsrnas or 5' ppp uncapped single stranded rnas. interestingly, neither rig-i nor mda has a classic rna binding motif. a zinc-binding domain located at the c terminal region of human has been shown to specifically bind to viral derived 5'ppp rna. rig-i and mda respond differently to infection with various viral strains, with rig-i sensitive to paramyxoviruses, orthomyxoviruses, and the rhabdovirus vesicular stomatitis virus whilst mda reacts to picornaviruses. some viral proteins, such as the v protein of paramyxoviruses, interact with mda a mechanism possibly used by viruses as a means to escape host surveillance. whilst most studies are focused on the rig-i like prrs in mammals, little is known about such molecules in other living organisms. a recent study surveying the purple sea urchin genome has revealed multiple putative rig-i like homologues that appear to be present in invertebrates. more recently, it has been hypothesised that mda might have emerged before rig-i and their domain arrangement evolved independently by domain grafting rather than by a simple gene duplication event. in this study, we took a comparative genomics approach by analysing rig-i like prrs in a number of invertebrate and vertebrate genomes, in order to elucidate the origin and evolution of the rig-i like prr family. bioinformatic analysis of functional domains of rig-i, mda and lgp has identified two evolutionary conserved domains in mda and lgp which may be critical to the recognition and processing of viral nucleotide pamps.  sequence identification extensive blast analysis of vertebrate genomes or expressed sequence tag databases using known rig-i protein sequences identified a putative full length rig-i homologue in western clawed xenopus but not in chicken. the putative xenopus rig-i is aa in length, sharing % identity with human rig-i and contains conserved domains such as a dexd/h domain in the middle region and a helicase domain at the c terminal region. a less homologous card domain at the n terminus is also apparent. in the zebrafish genome, a single rig-i like gene encoding a protein of aa was found in chromosome, some aa shorter than the mammalian and amphibian rig-i proteins. although it contains a dexd/h domain and a card domain at the n terminus, it lacks a classic helicase c domain. it is uncertain whether zebrafish rig-i is produced as a functional protein since no est matching the predicted rig-i exists. in japanese pufferfish, tetraodon, medaka and stickleback, no rig-i homologues were identified either in the est databases or genome databases. unlike rig-i, whose presence in chicken and some fish species is uncertain, the mda homologues can be found throughout vertebrate species including fish, amphibians, birds and mammals. mda is encoded by a single copy gene and the putative proteins have comparable length, ranging from aa to aa. in addition to the conserved dexd/h domain and helicc domain, two tandem card domains at the n terminal regions are predicted and are well conserved among vertebrate mda molecules except for the zebrafish mda that lacks a clear card domain. the card motifs near the n-terminus are more diverse than the second card motif. note: the amino acid position of each domain is listed and the e values against the pfam database are indicated in parentheses. lgp is an adaptor protein lacking card domains but containing a dexd/h domain and a helicc domain homologous to their corresponding motifs in the rig-i and mda protein. it competes with rig-i and mda for the ligands, viral derived rna pamps, but is unable to interact with down stream signalling proteins due to the absence of card domains. thus it acts as a negative regulator of the rig-i/mda directed antiviral response. lgp appears to co-exist with mda in vertebrates as a single copy gene. it is located in a different chromosome to mda in every species analysed. the putative lgp proteins from non-mammalian species contain 588– aa, much shorter than the rig-i and mda proteins. the dexd/h domain and the helicc domain in the lgp protein share higher homology with the corresponding regions in mda than those in rig-i. the lgp dexd/h domains are – % identical to the mda counterparts compared to – % for the rig-i proteins. similarly, – % identity is seen between the lgp helicc domains and the mda helicc domains, in contrast to – % between lgp helicc domains and the rig-i helicase domains. twelve genes coding for rna helicase proteins homologous to rig-i/mda5/lgp have been reported in a recent survey of the sea urchin genome draft. some of the deduced proteins contain card domains in addition to dexd/h and helicc domains. using the human mda protein sequence as a bait, a partial homologue sequence was obtained from the sea anemone nematostella vectensis genome database, this partial sequence was then used to search the ncbi database and two contigs were retrieved, which encoded two putative rig-i/mda5/lgp homologues. the putative proteins are aa and aa in length, similar to that of lgp further prediction of functional motifs revealed the presence of a dexd/h domain and a helicc domain but not the n terminal card domain. the proteins share – identity with rig-i, – % with mda and – % with lgp gene synteny analysis to gain an insight into whether the genes surrounding rig-i, mda and lgp are evolutionary conserved, we analysed draft genome sequences of invertebrates, fish, amphibians, birds and humans. the rig-i locus was identifiable in xenopus where the genes neighbouring rig-i were different to those in humans except for the aco gene. since the upstream region of the rig-i locus was not available in the xenopus genome database, whether rig-i clustered with topors remains to be determined. in zebrafish, the rig-i gene was immediately downstream of the topors gene, which is adjacent to the rig-i gene in humans. in contrast to the observation in zebrafish, the stickleback genome appears to lack rig-i, which is not due to poor genome analysis since the sequence of the rig-i locus is of good quality. in fugu, medaka and chicken, the rig-i gene could not be identified but was hindered by incomplete sequence data. the gene synteny of mda5/lgp is well conserved in vertebrates, from fish to humans. eight genes surrounding mda in stickleback, fugu and medaka appear in the genomes of xenopus, chicken and humans, in the same order and the same transcriptional orientation. less conservation of gene synteny was noted in the zebrafish genome where only conserved neighbouring genes were present in the mda locus. similarly, the gene composition and arrangement in the lgp gene locus shows remarkable conservation during vertebrate evolution. in invertebrates, two loci containing prototype homologues of rig-i/mda were found downstream of two independent genes coding for two card like molecules in nematostella vectensis. some rig-i/mda like genes have also been predicted from the purple sea urchin genome. whilst it was possible to find appropriate contigs containing rig-i/mda like genes from the sea urchin genome project, it was not possible to assess gene synteny due to a lack of genome assembly. phylogenetic analysis rig-i, mda and lgp are believed to bind rna molecules through helicase-c domains. closely related proteins containing a rna binding helicase-c domain and a dexd/h helicase domain are the double-stranded rna-specific endoribonuclease and elongation initiation factor 4a. dicer belongs to the rnase iii family that cleaves double-stranded rna into short double-stranded rna fragments, called small interfering rna, that is required for specific cleavage of complementary viral rnas, whilst eif4a mainly participates in translation and other processes such as pre-mrna splicing and ribosome biogenesis. since homology analysis of the rig-i and mda proteins in the databases using the blastp programme gave top scores to dicer and eif4a in addition to the rig-i, mda and lgp proteins, we included dicer and eif4a in the phylogenetic tree analysis using the neighbour joining method within the mega programme. as shown in fig. dicer and eif4a formed two distinct groups with a long evolutionary distance to the branch that evolved into invertebrate dicer like helicase, rig-i, mda and lgp apparently, dicers are more distantly related to the ancestor of rig-i, mda and lgp than eif4a. comparing to dicers and eif4as, c. elegans drhs were located close to some of the sea urchin rig-i/mda like proteins, the prototype of the vertebrate counterparts. within the rig-i branch are two of the rig-i like homologues from nematostella vectensis, two from the sea urchin, and predicted or known rig-i molecules from zebrafish, xenopus, and mammals, suggesting rig-i may have emerged in invertebrates. the fact that rig-i homologues were absent in most fish species suggests that the rig-i gene may have been lost in some fish lineages. the tree also indicates that lgp and mda proteins from vertebrate species form two clear groups closely neighbouring each other, suggesting they possibly diverged from a common ancestor that originated from a rig-i like molecule in invertebrates or early vertebrates. in addition, two phylogenetic trees were constructed using helicase c domains and dexd/h domains and a similar grouping of the major branches was observed. domain analysis the putative domains were analysed in the pfam database using a cut-off e-value of and selected domains listed in table a comprison of the domains is shown in fig. a classical helicase c domain is present in all types of dexd/h helicases, including rig-i, mda lgp dicer and eif4a. the dexd/h box responsible for atp binding and hydrolysis was detected in the middle region of rig-i, dicer and eif4a by the pfam hmm search. similarly, the corresponding region in mda and lgp was detected as a conserved restriction domain of bacterial type iii restriction enzymes, sharing some degree of homology with the rig-i dexd/h box. two card domains were predicted in the n terminal regions of rig-i and mda except for xenopus and zebrafish rig-i, which contained a single card domain corresponding to the second card domain of the human molecule. in general, the n-terminal card domain was less conserved than the card domain. the e-value of card in rig-i ranges between and, significantly higher than that in mda, indicating significant divergence of the card domains in different vertebrate groups. in addition, a fragment at the c-terminal region in most mda proteins of chicken, xenopus and fish, contained a putative domain with moderate e-values to the cysteine type transcription factor iis central domain. the c terminal region of human rig-i comprised a region distantly related to the c type zinc finger domain, which was shown to bind to dsrna and 5'ppp viral rna with the involvement of zinc ion. the pfam hmm analysis failed to identify this c tfiis domain in rig-i possibly due to low homology. the domain sequences were further analysed by multiple alignment and their dimensional structures modelled. it is apparent that the dexd/h and res iii domains comprised a well conserved dech motif. the overall structures of dexd/h boxes and res iii domains were similar, with β sheets sandwiched by α-helices on each side. six β strands were arranged in the same orientation in the human rig-i dexd/h box and mda5/lgp res iii domains except for the human mda res iii domain, where β strands were present. conversely, numbers of predicted α-helices varied significantly among domains. although homology analysis showed low sequence similarities between the mda tfiis domain and the c type zinc finger nucleotide binding motif within the human tfiis central domain, 3d modelling displayed significant structural similarities. our modelling data indicated the c terminal region of rig-i/mda proteins across vertebrates possessed a conserved c type zinc finger nucleotide binding motif, in agreement with the studies in humans where a putative domain distantly related to the c type zinc finger protein was shown to bind to viral nucleotide pamps. remarkably, the cysteines involved in capturing zinc ion were in close physical contact, forming a conserved pocket on the surface of all the domains analysed. however, arrangement of the cysteines differed in the human tfiis-c domain and rig-i/mda domains. in addition to the c type zinc finger nucleotide binding motif, the rig-i c terminal region and the mda tfiis domain contained another noticeable β strand structure which appeared to support the c type pocket. in the human rig-i, a single α helix was also detected. discussion the rig-i like helicase family members have recently been reported to play pivotal roles in recognising viral nucleotides in mammals. in this report, the rig-like homologues have been identified in silico in the nucleotide databases of invertebrates and vertebrates and their evolutionary origin discussed. double stranded rna is the genetic component of viruses with double stranded genomes and part of a single stranded rna with secondary structures. it can be generated during viral replication and rna metabolism. this nature of dsrna makes it the prime target for host prrs. classical double stranded rna binding domains are often used by some cytosolic prrs, such as pkrs, as the detectors to sense viral presence. dicer proteins also contain two dsrna binding domains for capturing dsrna molecules. in the present study, a zinc finger domain similar to that of transcription factor iis has been found in mda but not in lgp with moderate homology to the rig-i c terminal region. furthermore, a well conserved type iii restriction enzyme domain responsible for restriction in prokaryotic organisms is identified in the middle of both mda and the n terminal region of lgp this domain was not detected in rig-i molecules by the pfam hmm programme although it shared some degree of homology. we speculate that these two domains may serve as potential binding domains to interact with viral pamps. one striking finding is that a well conserved restriction enzyme iii domain is predicted in all mda and lgp proteins. the res iii domain is structurally similar to the dexd/h domain. restriction enzymes are important components of prokaryotic dna restriction-modification mechanisms in defence against foreign dna. they function in combination with one or two modification enzymes that protect the cell's own dna from cleavage by the restriction enzymes. restriction enzymes consist of four types depending on their recognition sequences and location of cleavage sites. type iii enzymes recognize short 5– bp long asymmetric dna sequences and cleave 25– bp downstream to generate short, single-stranded 5' overhang ends. type iii enzymes contain two functional subunits res and mod, specifically for dna cleavage of unmethylated double stranded foreign dna and protection of self dna from damage by methylation, respectively. classic strand separation helicase activities have not been detected for type iii restriction enzymes. the res iii domain predicted in mda and lgp have significant homology with bacterial res iii domains and multiple alignment reveals significant conservation. mda5/lgp are also similar to the rnase iii domains in the rna endonuclease dicer and dicer like helicases which process dsrna into 21– nt 3' overhang small rnas, with nt protrusions, and atp-binding domains in bacterial and yeast dna helicases. integrated nuclease domains with excision activities are seen in the dicer proteins where two ribonuclease iii domains cut double stranded rnas, releasing nt 3' end overhang 21– nt rna molecules which are essential for specific cleavage of viral rnas. another putative important domain, a zinc finger motif similar to that of transcription factor iis, was identified by homology analysis in the pfam database. the zinc finger motif can bind a range of targets including dnas, rnas, proteins and even lipids. it is known that the zinc finger motif at the c-terminus of the tfiis is essential for rna binding and processing. integrated tfiis zinc ribbon c-terminal domains are also found in some viral proteins. the tfiis motif located near the c terminus of the rig-i/mda proteins was detectable in the pfam database although the e value is moderate. structural modelling confirmed remarkable conservation of a c type zinc finger pocket and a β-strand structure compared to the c type zinc finger nucleic acid binding domain in the human tfiis. furthermore, a β-strand motif is also present within this domain in addition to the c type β-strand zinc finger structure. whether it is involved in recognition of viral rna pamps remains to be determined. a recent study has demonstrated that a c terminal domain in human rig-i was involved in binding dsrna or 5'ppp rna, which was confirmed by magnetic resonance and x-ray crystallography. this region was also shown to suppress rig-i signalling. thus it is possible that viruses could interfere with this host recognition system by their own tfiis-c containing proteins. the origin and evolution of rig-i, mda and lgp were analysed in this study. our data suggest they evolved from common invertebrate ancestors encoding distinct core domains, which was supported by the presence of the rig-i like genes in sea anemone nematostella vectensis and sea urchin genomes. tandem clustering of two card genes with the rig-i like genes without card domains in the nematostella vectensis genome provides a strong clue as to how rig-i, mda and lgp could have evolved during evolution through gene fusion, domain duplication and domain deletion, supporting recent analysis suggesting that card could have been grafted independently rather than duplicated from card during evolution. in deuterostome invertebrates, the rig-i/mda like genes appear to have expanded enormously, as seen in the sea urchin, with some differentiating into molecules with a closer phylogenetic relationship to the vertebrate rig-i molecules. we speculate that rig-i emerged earlier than mda5/lgp since vertebrate rig-is grouped with the invertebrate progenitors rather than the mda and lgp proteins which are present uniquely in vertebrates, in stark contrast with the evolutionary model proposed by sarkar et al, where lgp preceded both mda and rig-i in evolution. moreover, the phylogenetic tree constructed in the present study shows that mda has a closer relationship with lgp rather than rig-i, suggesting mda and lgp originated from a more recent gene duplication event, unlike the phylogenetic results obtained by sarker et al. if mda5/lgp did diverge from rig-i more recently, the order of their appearance is not clear. also, it is uncertain from the present study why rig-i was not found in all teleost fish. although a putative gene coding for a partial rig-i is predicted in the zebrafish genome, with conserved gene synteny to the xenopus and human rig-i locus, it is absent in the other fish genomes. poor quality of the genome sequences makes conclusions difficult but as no rig-i sequences were found in the vast number of fish est sequences, perhaps functional rig-i genes have been lost in some teleost fish species.  the rig-i/mda5/lgp system is an ancient antiviral system well conserved in vertebrates. our data suggest that these helicase prrs have evolved from an ancient progenitor originated from genes coding for individual functional domains and expanded by multiple evolutionary events leading to gene and/or domain gain and loss. the present study provides important clues for further elucidation of rig-i/mda mediated antiviral defence in vertebrates. 
our data suggest that the rig-i like helicases possibly originated from three distinct genes coding for the core domains including the helicc, card and atp dependent dexd/h domains through gene fusion and gene/domain duplication. they share three core protein domains including a rna helicase domain near the c terminus, one or more caspase activation and recruitment domains and an atp dependent dexd/h domain. the rig-i/mda directed interferon response is negatively regulated by laboratory of genetics and physiology and is believed to be controlled by the mitochondria antiviral signalling protein, a card containing protein associated with mitochondria. invertebrate homologues had a closer phylogenetic relationship with the vertebrate rig-is than the mda5/lgp molecules, suggesting the rig-i homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. the rig-i and mda proteins differentially recognise conserved pamps in double stranded or single stranded viral rna molecules, leading to activation of the interferon system in vertebrates. the dexd/h containing rna helicases including rig-i, mda and lgp were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. the dexd/h domain containing rna helicases such as retinoic acid-inducible gene i and melanoma differentiation-associated gene are key cytosolic pattern recognition receptors for detecting nucleotide pathogen associated molecular patterns of invading viruses. the gene synteny of mda and lgp is well conserved among vertebrates whilst conservation of the gene synteny of rig-i is less apparent. the rig-i like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. furthermore, presence of domains similar to a prokaryotic dna restriction enzyme iii domain, and a zinc finger domain of transcription factor iis have been detected by bioinformatic analysis. diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral pamps.
 the dexd/h domain containing rna helicases such as retinoic acid-inducible gene i and melanoma differentiation-associated gene are key cytosolic pattern recognition receptors for detecting nucleotide pathogen associated molecular patterns of invading viruses. the rig-i and mda proteins differentially recognise conserved pamps in double stranded or single stranded viral rna molecules, leading to activation of the interferon system in vertebrates. they share three core protein domains including a rna helicase domain near the c terminus, one or more caspase activation and recruitment domains and an atp dependent dexd/h domain. the rig-i/mda directed interferon response is negatively regulated by laboratory of genetics and physiology and is believed to be controlled by the mitochondria antiviral signalling protein, a card containing protein associated with mitochondria.  the dexd/h containing rna helicases including rig-i, mda and lgp were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. the gene synteny of mda and lgp is well conserved among vertebrates whilst conservation of the gene synteny of rig-i is less apparent. invertebrate homologues had a closer phylogenetic relationship with the vertebrate rig-is than the mda5/lgp molecules, suggesting the rig-i homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. our data suggest that the rig-i like helicases possibly originated from three distinct genes coding for the core domains including the helicc, card and atp dependent dexd/h domains through gene fusion and gene/domain duplication. furthermore, presence of domains similar to a prokaryotic dna restriction enzyme iii domain, and a zinc finger domain of transcription factor iis have been detected by bioinformatic analysis.  the rig-i/mda viral surveillance system is conserved in vertebrates. the rig-i like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral pamps. 