innate immunity mediated through pamp recognition by prrs is the earliest stage of immunity against viral infection .
the subsequent modulation of the adaptive immune response by prr signaling has been studied using cells and mice deficient in specific tlrs , rlrs , or their associated signaling adaptor proteins .
recent studies demonstrated , for example , that the absence of specific tlr pathways impairs adaptive immune responses against a variety of viruses ( 18 , 19 ) .
rlr signaling also seems to be critical for the outcome of japanese encephalitis virus ( jev ) , vesicular stomatitis virus ( vsv ) , influenza virus , and encephalomyocarditis virus ( emcv ) infection ( 11 , 20 , 21 ) . in mice lacking rig - i ,
stimulator 1 , jev , vsv , influenza virus , and ecmv were more virulent and replicated to higher levels than in wild - type mice , suggesting that rlr pathways are essential for controlling infection by these viruses ( 11 , 21 , 22 ) .
for example , irf3 target genes induced by rlr signaling directly control viral replication in infected tissues ( 23 , 24 ) .
thus , depending on the nature of virus infection , tlrs and rlrs may work together or independently to mount an efficient immune response .
it is thought that the innate immune system protects the host from infection in a nonspecific way .
this , along with differences in cellular location likely serve to distinguish self - rna from nonself pamp rna , thus avoiding type i ifn induction in response to components of host nucleic acids . for example
, tlr7 recognizes a specific motif within uridine - rich ribonucleotide sequences ( 25 , 26 ) , which are hypothesized to be unique to rna viruses .
tlr9 recognizes dna pamp ligands and triggers signaling through the myd88 adaptor protein to induce type i ifn production ( 27 ) .
the recognition of nonself dna ligands by tlr9 might also be sequence dependent , and some studies have implicated the sugar - base - backbone sequence of pamp dna as a recognition factor ( 2830 ) .
unlike tlrs , which are found either on the cell surface or within membrane - bound vesicles , rlrs are found in the cytoplasm where cellular rna is also present ( 1 ) .
rig - i preferentially recognizes single - stranded rna ( ssrna ) over dsrna ( 15 , 31 ) .
ssrnas containing a terminal 5 triphosphate ( ppp ) , but not 5oh or a 5-methylguanosine cap , bind to the rig - i repressor domain and promote a conformational change that activates rig - i signaling ( 10 , 1316 ) .
rna ligands of rig - i are longer than 23 nucleotides , have a linear structure , and contain a uridine- or adenosine - rich ribonucleotide sequence ( 3133 ) .
host mrna , trna , mirna , snrna , and rrna are not ideal rig - i ligands because their length , structure , 5 end modifications , and interactions with ribonucleoproteins limit their recognition .
self - rnas therefore do not typically trigger innate immune programs or type i ifn expression ( 34 ) .
mda5 triggers innate immune signaling in response to emcv infection and synthetic dsrna , such as poly i : c ( 11 , 20 ) .
but ssrna viruses such as dengue virus and west nile virus , as well as reovirus , a dsrna virus , have also been shown to trigger signaling partially via mda5 ( 12 , 35 ) .
however , in these studies , mda5 appeared only to amplify type i ifn production as compared with rig - i , whose actions were essential to initiate innate immunity ( 12 ) .
( 17 ) in this issue describes a plausible mechanism of rna ligand recognition by mda5 , providing new insights into the differential roles of rlrs .
the study reveals that mda5 binding to dsrna does not depend on its 5 end modification , but rather on its length .
dsrnas with lengths of 2 , 3 , and 4 kb were shown to increasingly activate mda5 signaling , whereas rig - i recognized ssrna and , to a lesser extent , short dsrna motifs .
these results help explain how reovirus can trigger both rig - i and mda5 signaling .
the genome of reovirus comprises at least 10 segments of dsrna that vary in length , including long ( 3.9 kbp ) , medium ( 2.2 kbp ) , and short ( 1.2 kbp ) rna segments .
show that rig - i and mda5 recognize the short and long dsrna segments of the reovirus genome , respectively . in the case of emcv infection , the authors show that the viral genome forms long dsrna via its antisense replication intermediate , and this dsrna is recognized by mda5 but not by rig - i . on the other hand , vsv , which triggers rig - i  dependent immunity , forms medium - length dsrna replication intermediates that are not recognized by mda5 .
propose a model for pamp recognition by mda5 in which only long dsrna viral genomes or stable long duplex viral rnas formed during virus replication serve as mda ligands .
this model might explain why mda5 ignores self - dsrna , which is typically not present as long duplex rnas .
the current observations advance our understanding of how mda5 discriminates between potential rna ligands , but several questions about recognition of rna by rig - i and mda5 still remain .
it is widely accepted that ssrna viruses generate dsrna during their replication process ( 36 , 37 ) . however , only mda5 recognizes emcv ( 8 kb ) , whereas rig - i recognizes other viruses with lengthy rna genomes , such as vsv ( 11 kb ) , jev ( 11 kb ) , and hepatitis c virus ( 9.6 kb ) .
mda5 's failure to recognize these latter viruses may be due to their inability to form long or perhaps stable dsrna .
it is also possible that recognition by mda5 depends on specific compartmentalization of the pamp rna with mda5 , as differences in the subcellular site of viral genome replication may influence the pamp 
the mechanism by which mda5 discriminates between pamp ligands in terms of their length is also unclear .
demonstrate that mda5 binds long , capped , di- or mono-5 phosphate dsrna , whereas rig - i binds to short dsrna or 5ppp uncapped ssrna .
when rig - i binds to 5ppp ssrna , it changes conformation , which disrupts the inhibitory interaction between the rig - i repressor domain and the cards .
this alteration results in the initiation of downstream signaling by the cards and exposes a 17- or 30-kd trypsin - resistant fragment corresponding to the rig - i repressor domain , thereby marking the activation of rig - i ( 10 , 15 , 16 ) . however , poly i : c binding to rig - i promotes a distinct conformational change that results in a 66-kd trypsin - resistant fragment , indicating that pamp - bound rig - i undergoes a distinct conformational change depending on the nature of its ligand .
structural studies validate that rig - i prefers ssrna ligands , whereas dsrna binds only inefficiently to rig - i , possibly due to constrains imposed by the location of basic residues within the rna binding groove of its repressor domain .
the charged residues are proposed to anchor 5ppp ssrna within the pocket , which would be less amenable to binding dsrna ligands ( 15 , 16 ) .
these observations suggest that rig - i may have two ways to distinguish different rna species : efficient recognition of 5ppp ssrna through its repressor domain ( 10 , 15 , 16 ) and comparably inefficient recognition and binding of dsrna ( 10 , 15 ) . by extension , these conformational alterations of rig - i might provide clues about how dsrna ligands interact with mda5 to stimulate its activation .
thus , mda5 might become active upon binding long dsrna through a ligand - induced conformation change that places its card into a signaling - active conformation .
this might involve dsrna interactions with its helicase domain and c - terminal region in a fashion similar to the binding of poly i : c to rig - i .
others have demonstrated that although very short dsrna ( 2030 bp ) fail to trigger rig - i  dependent signaling ( 15 , 17 ) , longer dsrnas ranging from 70 kb to 2 kb can at least weakly engage rig - i .
these data suggest that the length of dsrnas may dictate the interaction with the rig - i helicase domain and/or repressor domain in a manner that supports a rig - i  dsrna complex .
however , the mechanism of length discrimination of dsrna pamps by these rlrs has not yet been revealed .
the third rlr family member , lgp2 , has been defined as a regulator of rig - i signaling based on its suppression of rig - i function when overexpressed in cultured cells ( 10 , 38 , 39 ) .
however , recent studies imply that lgp2 may also function as either a positive or negative regulator of rlr signaling by forming a heterodimer with rig - i or mda5 when bound to rna ligand ( 40 , 41 ) .
such interactions between the rlrs may also broaden their pamp recognition profiles to accommodate other ligands , including ssrna or dsrna of varied length and composition , or even dna  rna duplexes .
structure and function analyses of rna interactions with rlrs or heteromeric rlr complexes will further define the molecular basis of self- versus nonself discrimination .
the rlrs are adenosine triphosphate ( atp ) binding proteins that hydrolyze atp as a result of binding rna ligand ( 15 ) .
however , the role of the atpase activity in the function of rlrs is not fully understood .
rlrs contain multiple domains , including two domains known as the walker a motif and the walker b ( also known as the dexd / h - box ) motif , each located within the helicase domain ( 42 ) .
walker a and b motifs comprise an atp binding region wherein specific amino acid residues contact the -phosphate of the nucleotide and mediate atp hydrolysis upon substrate binding ( 4345 ) .
mutant rig - i with an inactive walker a motif retains ssrna binding function but fails to trigger downstream signaling ( 15 , 46 ) .
thus , atp hydrolysis is not essential for pamp binding but is required for downstream signaling .
recent studies revealed that the rig - i repressor domain , in addition to binding to the rig - i cards , forms a complex with the helicase domain linker region that likely stabilizes rna ligand binding ( 10 , 15 , 16 ) .
moreover , it seems that motifs in the rlr helicase domain could also be involved in rna substrate binding according to structure and function studies of other dexd / h - box rna helicases ( 42 ) .
such studies indicate that the rna binding sites and the walker a and b motifs mediate specific interactions ( 47 ) .
these observations present a model in which a repressor domain  helicase domain internal complex forms an rna binding pocket whose association with pamp rna may serve to initiate the atpase activity of rig - i , which then triggers a conformation change that allows innate immune signaling .
the repressor  helicase domain interaction of rlrs may thus be the key determinant of pamp discrimination by rig - i and mda5 ( 15 , 17 ) .
it also seems that rig - i atpase activity is involved in the unwinding of dsrna ( 15 ) .
the importance of this activity for innate immune function is unclear , but it might support rig - i interaction with dsrna ligands by catalyzing their conversion to ssrnas .
the breadth of prrs expressed in different cells and tissues and their distinct intracellular distribution provide the means for pamp detection and immune activation against a variety of microbial pathogens at the local site of infection or in the  microbial niche . 
these include the membrane - associated cytosolic replication sites of rna viruses , extracellular sites of microbe interaction , and endosomal sites of microbial trafficking and metabolism ( fig .
the recent identification of the dai ( dlm-1/zbp1 ) protein as a cytoplasmic sensor of microbial dna ( 48 ) suggests the existence of other prrs that detect cytosolic pamp dna .
it is reasonable to speculate that , similar to tlrs and rlrs , the cytoplasmic dna sensor molecules also exhibit ligand specificity to cover a variety of dna pamps in association with their specific microbial niche , thus providing a further basis for self- versus nonself discrimination .
understanding the processes of pamp ligand recognition within each microbial niche will provide a foundation for the design of appropriate vaccines , adjuvants , and immunotherapies .
 retinoic acid  inducible gene ( rig)-i  like receptors ( rlrs ) are cytosolic rna helicases that sense viral rna and trigger signaling pathways that induce the production of type i interferons ( ifns ) and proinflammatory cytokines . 
 rlrs recognize distinct and overlapping sets of viruses , but the mechanisms that dictate this specificity were unknown . 
 a new study now provides evidence for size - based discrimination of double - stranded rna ( dsrna ) by rlrs and suggests how host cells recognize a variety of rna viruses . 