Integrative Genomics-Based Discovery of Novel Regulators of the Innate Antiviral Response
[['Viruses are a major cause of human disease , as highlighted by the pandemics of influenza viruses , HIV–1 , and the current outbreak of the Ebola virus .', 'Pattern recognition receptors ( PRR ) are among the first molecules that detect viruses during infection .', 'The RIG-I-like receptors ( RLRs , one class of PRRs ) are part of the RLR pathway , which forms a crucial innate antiviral defense system [1 , 2] .', 'Two RLRs , RIG-I and MDA5 , reside in the cytosol where they recognize non-self 5’-triphosphate RNA molecules with short double-stranded regions and long double-stranded RNAs ( dsRNA ) , respectively [3] .', 'Activation of the receptors triggers a complex signaling network , key steps of which are the activation of the mitochondrial adapter MAVS , subsequent recruitment of the TBK1 and IKK complexes , phosphorylation/activation of IRF3 and NFκB , and translocation of these transcription factors to the nucleus .', 'These steps ultimately lead to the production of type I interferons ( IFNα/β ) and proinflammatory cytokines , which are crucial for establishing an antiviral state in infected as well as neighboring cells , and also modulate the adaptive immune response [4] The importance of the RLR system is further demonstrated by the observation that viruses of all types employ strategies to interfere with its activation , often at multiple steps [5 , 6] .', 'Better understanding of viral interaction with the pathway has resulted in novel targets for the development of antiviral therapeutics and attenuated live vaccines , for example viruses lacking functional RLR antagonists [7] .', 'Furthermore , mutations in RIG-I , MDA5 , MAVS and other RLR pathway components are associated not only with strong susceptibility to infections , but also IFN-associated autoimmune disorders [8–10] .', 'Previous studies into virus-host interactions and the innate antiviral pathways have used genomics approaches , often generating large data sets describing physical or genetic interactions [11–14] .', 'Other publications have taken a comparative approach based on model organisms [15] or used over-expression screening systems [16 , 17] .', 'Together , these studies have identified numerous genes with antiviral activity , including members of the RLR pathway .', 'However , it remains important to systematically assess the quality of individual data sets as such screens report distinct sets of genes , often with limited overlap between them .', 'Combining the many available genomics data sets in a statistical framework potentially allows for a more systematic discovery and categorization of genes involved in the RLR pathway .', 'Indeed , Bayesian integration of large-scale data that includes weighing individual datasets for their predictive potential has been successful in other cellular systems , for example identifying novel protein interactions [18] , mitochondrial disease genes [19] , and small RNA pathway genes [20] .', 'In this work we systematically exploit the wealth of available ( gen ) omics data , including transcriptomics and proteomics data , genome sequences , protein domain information , and functional genomics , to discover descriptive molecular signatures of the RLR pathway system .', 'Bayesian integration of these data , together with comprehensive computational and experimental validation , confidently identifies novel genes involved in antiviral RIG-I signaling .'], ['To discover molecular signatures that distinguish RLR pathway components from other genes , we explored a wide variety of genome-scale data describing different aspects of the virology and biology of the pathway .', 'Some of these data we used directly , while other data were used as the basis for further calculations ( Table 1 ) .', 'We quantitatively assessed the predictive power of each data set using a literature-curated standard of 49 known RLR pathway components from InnateDB [21] ( ‘RLR genes’ , S1 Fig ) and a set of 5 , 818 ‘non-RLR genes’ that are unlikely to be part of the pathway ( i . e . genes with known functions not directly related to the innate antiviral response , such as development , housekeeping and neurological processes , see Methods ) .', 'Below we describe 10 signatures for predicting novel RLR pathway components .', 'The first five signatures are based on the relationship of RLR genes with viruses , whereas the second set of five signatures are based on properties of the RLR pathway itself .', 'The RLR pathway components published thus far probably constitute only part of the total proteins with a function in this pathway .', 'To prioritize novel high-confidence genes for a role in the RLR pathway , we integrated the 10 identified molecular signatures of RLR genes in a naive Bayesian classifier [18 , 19] ( see Methods ) .', 'This approach weighs data sets based on their predictive value ( i . e . their ability to separate known positives and negatives; Fig 1A , Tables 1 and S1 ) so that ‘better’ data contribute more to the predictions .', 'Each human gene received a posterior probability score ( ‘RLR score’ ) reflecting the likelihood that the gene is part of the RLR pathway based on its behavior in the collected genomics data .', 'A score of zero indicates equal probabilities of a gene being an RLR versus a non-RLR gene .', 'S6 Table presents the genome-wide ranking of RLR scores ( also available at http://rlr . cmbi . umcn . nl/ ) .', 'As expected , known RLR pathway components have the highest RLR scores ( Figs 1B and S5 ) .', 'Two-thirds ( 32/49 ) of these rank within the first 150 genes .', 'The top ranking genes are IRF7 , RIG-I , IKKε , subunits of NFκB , TRADD , TRAF2 , MDA5 , and IKKγ ( NEMO ) ( Fig 1C ) .', 'Other examples of well-described RLR pathway components include IRF3 ( rank 51 ) , ISG15 ( 52 ) , MAVS ( 102 ) , and LGP2 ( 114 ) .', 'Genes that are unlikely to play a role in the pathway ( the set of non-RLR genes ) generally have very low RLR scores , although some of these received high scores as well ( Fig 1B ) .', 'This is not unexpected , as even though this large set of genes was selected from function annotations generally unrelated to the innate antiviral response , this does not preclude that individual genes ( also ) function in the RLR pathway .', 'To gain insight into what kind of genes are present among the RLR predictions , we examined their functions by pathway and gene ontology enrichment analyses .', 'The top 354 genes with the highest RLR scores ( corresponding to high-confidence predictions , see below ) have strong links with other pathways of the innate immune response , such as TLR , NLR , interferon , and cytokine signaling ( S6 and S7 Figs ) .', 'Antiviral defense functions are also among the most frequent and significant terms associated with the high-scoring genes ( S8 Fig and S7 Table ) .', 'Other important biological processes include various apoptosis-related functions , cancer and cell cycle pathways , and regulation of metabolic processes and protein localization .', 'Furthermore , the top predictions include a wide range of protein families , notably proteasome subunits , ubiquitin ( -like ) conjugating enzymes , and genes involved in phosphatidylinositol signaling ( which was recently shown to affect the type I IFN response [14] ) .', 'Finally , 22% of the top predictions are induced in cells treated with interferons ( i . e . they are interferon-stimulated genes , ISGs ) and ~18% are part of the common host transcription response to pathogens ( Table 2 ) .', 'Together , these observations indicate that our framework successfully predicts genes with a likely role in the innate antiviral response and suggests other cellular systems and functions required for this response .', 'We further computationally assessed the reliability of the integrated RLR score by estimating the sensitivity , specificity and false discovery rate ( FDR ) of the predictions using the positive ( RLR genes ) and negative ( non-RLR genes ) standards .', 'Integration of the data sets achieved better sensitivity and specificity than any of the individual data sets ( Fig 1D ) , thereby enriching for RLR genes and depleting false positives ( S5 , S9 and S10 Figs ) .', 'At an RLR rank threshold of 354 ( RLR score -1 . 10 ) , the framework correctly predicts 78% of the known RLR genes with a specificity of 98 . 4% ( Fig 1D ) .', 'At this threshold , only ~57% of the novel predictions are estimated to be false ( S11 Fig , adjusted FDR to match the expected total number of genes involved in the RLR pathway , see Methods ) .', 'This compares to a genome-wide false discovery rate ( i . e . when predicting genes randomly ) of ~99% .', 'Thus , the integrated RLR score increases the probability of correctly identifying novel RLR genes by a factor of 43 compared to random classification .', 'Because we used the same gene sets for calculating the RLR scores and estimating the performance of the resulting predictions ( i . e . without systematic cross-validation ) , there exists a danger of circular reasoning .', 'Therefore , we also carefully validated the quality of the results using various independent and external data sets .', 'First , we examined the high RLR scores for genes that have a known function in innate immunity , but not in the RLR pathway , and therefore were not part of our training set .', 'Components of other PRR signaling pathways ( TLR , CLR , NLR , cytDNA ) have lower scores than RLR genes , but much higher scores than the rest of the genome ( Fig 1B ) .', 'The same is true for genes functioning in other aspects of the innate immune response ( Fig 1B ) .', 'Of the 225 novel predictions ( i . e . those genes that are not part of the training sets ) in the top 354 ( FDR of 57% , see above ) , 142 ( ~63% ) are part of these innate immunity gene lists ( Fig 1C ) .', 'Thus , the majority of high-scoring genes with no known link to the RLR pathway in fact have a function in other PRR pathways or other parts of innate immunity , supporting the relevance of our predictions .', 'Second , we compared our predictions to six recent data sets that are relevant to the innate ( antiviral ) response but that were in no way part of the RLR score calculations .', 'The overlap with the 354 top genes , excluding known RLR genes , is significantly larger than expected by chance for all these data sets ( Table 2 ) .', 'For example , the top predictions include:', '( i ) 19 of 45 ( 42% ) interferon-stimulated genes with validated antiviral activity against e . g . HIV–1 , HCV , yellow fever , West Nile or chikungunya virus [16] ,', '( ii ) 27 proteins from a set of 241 ( 11% ) that interact with the type I IFN protein network during pattern recognition , among which are five confirmed modulators of IFNβ expression and antiviral activity [12] ,', '( iii ) nine tripartite motif ( TRIM ) family genes , five of which enhance RIG-I-induced activation of IFNβ , NFκB and ISRE ( IFN-stimulated response element ) promoters [17] , and', '( iv ) 38 human proteins interacting with innate immune-modulating viral open reading frames ( viORFs ) from 30 viruses [37] .', '( v ) Furthermore , the type I IFN response has recently been proposed to play a role in antifungal immunity [38 , 39] and the top RLR predictions are strongly enriched for genes expressed in PBMCs stimulated with the fungal pathogen Candida albicans: almost half ( 39/89 = 44% ) of these occur in our top predictions ( P = 4 . 7 × 10−47 , one-tailed Fisher’s exact test , Table 2 ) .', '( vi ) Finally , the overlap between our predictions and a genome-wide screen for regulators of RIG-I-mediated IFNβ production is , at only nine , marginal but significant ( 9/226 genes = 4% , P = 9 . 5 × 10−3 , one-tailed Fisher’s exact test , Table 2 ) [14] .', 'In summary , these diverse and independent experimental data support the validity of our integrated RLR score for predicting genes with a role in the innate antiviral response .', 'To further determine the predictive power of our in silico predictions , we selected 187 candidate RLR genes for experimental validation ( S6 and S8 Tables ) .', 'These include 127 high-confidence candidates from the top 354 , which have not been previously linked to the RLR pathway , supplemented with 60 candidates we selected from the top 1000 predictions , mainly on the basis of limited functional characterization in general ( Fig 2A ) .', 'Importantly , candidates with a known role in RLR signaling , other branches of PRR pathways , or apoptosis were excluded as we were most interested in finding novel components of the RLR pathway .', 'For the selected candidates we performed a medium-throughput RNAi screen ( RNAi screen 1 ) using HeLa cells stably expressing an IFNβ promoter-controlled firefly luciferase reporter ( HeLa-IFNβ-Fluc ) .', 'To activate the RLR pathway and induce Fluc reporter expression we used a known small 5’-ppp-containing RIG-I ligand [40] .', 'This setup led to specific activation of RIG-I , as RIG-I or MAVS siRNA transfection , but not MDA5 or scrambled siRNAs , resulted in loss of reporter activity ( Figs 2B , S12 and S13 ) .', 'All negative controls ( non-transfected , scrambled and MDA5 siRNAs ) scored within 1 . 25 median absolute deviations of the plate normalized IFNβ induction levels ( Z-score cutoff <-1 . 25 or >1 . 25 , Fig 2B ) .', 'At this cutoff , siRNA knockdown of 94 candidates ( 50% of all candidates tested ) affected RIG-I-mediated IFNβ induction ( Figs 2A and S13A–S13D and S8 Table ) .', 'Among these , knockdown of 59 genes decreased RIG-I-mediated IFNβ induction ( down-hits ) and 35 genes increased IFNβ induction ( up-hits ) .', 'It is important to note that the experimental approach only activates the RIG-I branch of the RLR pathway and will not confirm predicted RLR candidates that regulate MDA5 activation and downstream signaling to MAVS .', 'Thus , among the 93 non-confirmed candidates , there might still be novel regulators of the MDA5-mediated IFNβ induction pathway , which should be further investigated .', 'Altogether , the integrated RLR score is clearly a strong and reliable predictor for novel regulators of the RIG-I pathway .', 'From the 94 confirmed hits , we picked the 57 top hits with the largest effect ( stringent Z-score <-2 or >2 ) for a second RNAi screen using a different set of siRNAs ( RNAi screen 2 , Fig 2A ) .', 'In this second RNAi screen , only a single up-hit ( 7% of 15 up-hits tested ) showed a Z-score >1 . 25 .', 'Besides this hit , two negative control wells also had a Z-score >1 . 25 ( Figs 2B , S13E–S13H ) , which suggests that the single confirmed up-hit might be unreliable .', 'The poor reproducibility of the up-hits might be attributed to the screening approach .', 'For instance , we used a large amount of 5’-ppp-containing RIG-I ligand ( see Methods ) , leaving limited room for increased pathway activation .', 'In contrast , the second RNAi screen confirmed 26 down-hits at Z-score <-1 . 25 ( 62% of the 42 down-hits tested ) .', 'Of these , 19 genes ( 45% of tested down-hits ) could be confirmed at a conservative Z-score <-2 ( Fig 2A , 2B and 2C , S8 Table ) .', 'Taken together , the two RNAi screens , guided by the predicted RLR candidates , have substantiated the validity of our approach and have revealed potential novel regulators of the RIG-I receptor pathway .', 'To gain further understanding of how the 19 top hits affect RIG-I-mediated IFNβ promoter activation , another RNAi screen was performed ( RNAi screen 3 ) .', 'In contrast to the first two screens , here we did not use the IFNβ promoter-controlled Fluc reporter translation as readout , but we measured IFNβ mRNA levels using quantitative real-time ( qRT ) -PCR .', 'As expected , knockdown of RIG-I and MAVS abrogated 5’-pppRNA-induced IFNβ mRNA transcription , while MDA5 knockdown [40] and LGP2 knockdown , which regulates only the MDA5-mediated IFNβ mRNA transcription , had no effect ( Fig 2D ) .', 'Of the 19 top hits from the first two RNAi screens , 13 genes ( 68% ) in this third screen again showed a reduction in RIG-I pathway activation .', 'Nine of these showed a significant reduction ( NUP98 , TRIM14 , C16orf57 , PSMA3 , G3BP1 , DDX17 , MAP3K11 , SNW1 , CDK11B; P < 0 . 01 , one-way ANOVA with Dunnett’s post hoc test; Fig 2A and 2D ) , suggesting that these gene products play a so far uncharacterized role in the RIG-I signaling pathway upstream of IFNβ mRNA transcription .', 'In summary , using RNAi-based screening methods we validated more than 50% of the tested candidates .', 'To further assess the predictive power of the in silico integrated RLR score , we ranked the experimentally tested genes based on their RLR score and sequentially calculated the fraction of hits ( either considering all 94 hits from RNAi screen 1 , or only the 57 top hits ) among all tested genes having a certain RLR score or higher ( Fig 2E ) .', 'Higher RLR scores were experimentally confirmed more often , indicating that these indeed correspond to more confident predictions .', 'Further analysis revealed that there is no molecular signature that solely explains the predictions of the validated hits; rather the integrated score of the 10 molecular signatures is important ( S14 Fig ) .'], ['To identify defining signatures of RLR genes in genomics data , we largely depended on current knowledge of the biology of the RLR system and its relationship with viruses .', 'For example , since previous studies had shown that viral antagonism of specific RLR pathway components is prevalent [5 , 6] , one of the first features we investigated , and indeed established , was that human-virus PPIs are a general theme for the RLR pathway as a whole .', 'Similarly , guided by previous observations , we demonstrated that RLR genes conform to the tendency of immunity genes to evolve rapidly and commonly contain innate antiviral TF binding motifs , such as IRF and NFκB , in their promoters .', 'We also included several criteria that are effective for many different biological systems , but were specifically aimed at predicting novel RLR pathway genes in our case , such as the RLR co-expression calculations and RLR protein domain occurrences .', 'We decided not to include associations based on text mining of published literature ( e . g . co-mentioning of gene names in abstracts ) , because such approaches in our hands only enriched for genes already known to be involved in the RLR pathway and therefore compromised our ability to identify novel candidates .', 'Finally , we settled on using a total of 10 molecular signatures that are relevant and predictive for the RLR system .', 'Inclusion of additional data sets , generated for example by future experimental techniques , and substitution of existing data with novel and improved versions , will likely refine this data-driven definition of RLR genes over time and lead to updated Bayesian RLR probabilities that could further improve prediction accuracy .', 'A major challenge in our study arises from the fact that the RLR pathway is highly interconnected with other intracellular pathways , such as other innate PRR pathways ( e . g . TLR and cytosolic DNA sensing ) , the stress response pathway , mitogen-activated protein kinase ( MAPK ) signaling cascades ( e . g . TRAF2 and 6 lead to the p38 MAP kinases ) , and apoptosis ( e . g . via CASP8 and 10 ) ( S1 Fig ) [2 , 12 , 41 , 42] .', 'Although our approach for predicting novel RLR components relied on a well-defined set of genes known to make up the core of RLR signaling , the overlap with other systems was a potential confounding factor .', 'For example , most molecular signatures of RLR genes identified here , especially the virus-based properties such as PPIs with viruses , rapid evolution , and differential expression during infection , could also apply to genes involved in other aspects of antiviral immunity .', 'Nevertheless , combination of the right signatures achieved reasonable specificity for RLR genes ( Fig 1 ) .', 'Thus , we have extended an approach previously used for identifying components of membrane-enclosed organelles such as the mitochondrion [19] and showed that it is also possible to capture the complexity of a diverse and interconnected intracellular signaling pathway .', 'The presented approach for identifying predictive signatures , followed by Bayesian integration , could potentially be applied to any cellular system .', 'Using the sets of known RLR and non-RLR genes , we could systematically assess the relative quality of the individual data sets for predicting novel RLR genes .', 'Indeed the 10 molecular signatures have different predictive values as shown by the likelihood ratio scores ( Fig 1A , Tables 1 and S1 ) , and thus contribute with different weights to the integrated Bayesian RLR score .', 'The data types with the strongest predictive value include NFκB activation mediators , RLR pathway protein domains , and both PPI signatures ( PPIs within the RLR pathway and PPIs between human and viral proteins ) .', 'In contrast to our expectations , antiviral host factors identified in high-throughput RNAi experiments had a relatively small contribution .', 'Besides raw predictive ability , we also considered the coverage of the data sets .', 'For example , there are only few ( <200 ) NFκB activation mediators and antiviral host factors , while the data sets on RLR co-expression , viral miRNA targets , and innate antiviral TF binding motifs identified many more genes ( >4 , 000 ) .', 'Integration of all data sets with their varying coverage and predictive value into a single RLR score resulted in a classifier that is superior to the individual data sets ( Fig 1D ) .', 'This is underscored by the observation that the individual signatures by themselves are unable to explain the predictions for the experimentally validated RLR candidates , and only the integrated RLR score explains all validated genes ( Figs 2E and S14 ) .', 'Aside from our own experimental validation strategies , recent independent studies have confirmed a role for 15 of our predicted RLR candidates in the RLR pathway during viral infection ( Table 3 ) .', 'Most of these publications appeared during the course of our study , and thus were not part of the knowledge or data used for predicting novel RLR genes .', 'For example , TRIM14 ( RLR rank 491 ) has been demonstrated to interact with MAVS leading to activation of IRF3 and NFκB via IKKγ ( NEMO ) [43] .', 'Indeed , our predictions marked TRIM14 as a strong candidate RLR gene and all our RNAi screens confirmed it as a component required for optimal RIG-I signaling ( Fig 2 ) .', 'Two additional high-confidence RLR predictions for which we validated an effect in all three RNAi screens have recently been validated externally as well: G3BP1 [44] and CDC37 [13] .', 'Of the 15 genes recently described in the literature , 11 were part of the candidate RLR genes tested in our RNAi screens ( Table 3 ) .', 'Of these , seven genes affected RIG-I-mediated IFNβ induction in RNAi screen 1 ( Z-score <-1 . 25 or >1 . 25 ) and showed a consistent effect in RNAi screen 2 .', 'Therefore , our experimental screening condition appears to detect these described RIG-I pathway regulators with a sensitivity of ~64% ( 7/11 ) .', 'Furthermore , four out of four down-hits from our experiments ( i . e . genes that decreased IFNβ induction when knocked down , hence positive regulators ) that have been described in the literature were indeed described as positive regulators of RIG-I signaling ( Table 3 ) .', 'Given that our experimental approach detected most , but not all , of the published RIG-I regulators , a substantial number of our predicted RLR candidates not validated by our RNAi screens might still play a role in for example a different cell type , downstream of type I IFN production , or regulate the pathway via MDA5/LGP2 activation .', 'For example , RNF114 ( RLR rank 181 , Z-score RNAi screen 1 = 1 . 68 , Table 3 ) is an ISG and therefore needs to be up-regulated via a positive feedback loop to fully contribute to RLR pathway stimulation [45] .', 'This gene was not confirmed in all RNAi screens , perhaps because the time of RIG-I stimulation in our screens ( 6 hours ) was simply too short .', 'Similar biological reasons could limit the detection of an effect for other genes as well .', 'Therefore we conclude that the hits identified in our RNAi validation experiments may be a conservative estimate of the number of correct RLR predictions .', 'We identified 13 novel RIG-I pathway regulators that reduced IFNβ induction in all three RNAi screens ( Fig 2 ) .', 'These include cell cycle gene CDK11B , heat shock protein HSPA9B , MAP kinase MAP3K11 , proteasome subunit PSMA3 , nucleoporin NUP98 [46] , and the recently identified RLR regulators CDC37 [13] , G3BP1 [44] and TRIM14 [43] ( Table 3 ) .', 'The remaining five genes , DDX17 ( DEAD box helicase 17 ) , C6orf58 , C16orf57 ( USB1 , U6 snRNA biogenesis 1 ) , PKN2 ( serine/threonine protein kinase N2 ) , and SNW1 ( SNW domain containing 1 ) , are overall least characterized .', 'To obtain a first suggestion about how these genes might regulate RLR signaling , we searched for connections with the known human and viral protein interaction networks .', 'Next , we discuss the reported interactions of DDX17 and SNW1 with the RLR pathway .', 'DEAD box RNA helicase DDX17 was recently found to bind Rift Valley fever virus RNA and restrict viral replication in an interferon-independent manner [47] .', 'Our data now suggest a role for DDX17 in RIG-I-mediated IFNβ production as well .', 'DDX17 has reported protein interactions with two other RIG-I regulators identified in our study: CDC37 and CSNK2A1 .', 'Interestingly , DDX17 also interacts with the peptidylprolyl cis/trans isomerase PIN1 [48] , which inhibits RIG-I-mediated IFNβ production by inducing degradation of IRF3 [49] .', 'Furthermore , DDX17 was present among a set of ISG15-modified ( ISGylated ) proteins in HeLa cells treated with IFNβ [50] .', 'Thus , DDX17 could function in IRF3 activation by acting as a negative regulator of PIN1 and might be regulated by ISGylation ( Fig 3 ) .', 'Lastly , DDX17 seems to be a preferred target of viral interference , having reported interactions with six different viruses ( e . g . HIV–1 Rev and influenza virus A NS1 , Fig 3 ) .', 'SNW1 is an intrinsically disordered protein [51 , 52] that interacts with two other newly identified RLR regulators from our study , namely PKN2 [53] and C16orf57 [54] .', 'SNW1 also interacts with the IKBKG ( NEMO ) protein [55] , which is required for NFκB and IRF3 activation [56] .', 'Given that our data shows that knockdown of SNW1 reduces IFNβ induction , SNW1 could be involved in NEMO regulation and thereby contribute to activation of the RLR pathway TFs , NFκB and IRF3 ( Fig 3 ) .', 'The fact that SNW1 was also identified in a siRNA screen for mediators of virus-induced NFκB activation [34] strengthens this hypothesis .', 'Further studies should be conducted to resolve the precise mode-of-action .', 'We have validated the integrated RLR score with various experimental , literature and computational approaches .', 'Our confirmations of a substantial fraction of the predicted RLR genes suggest the value of the prioritized list as a whole .', 'The genome-wide prioritization of RLR pathway components is available in S6 Table and at http://rlr . cmbi . umcn . nl/ , and can be used as a resource in several ways .', 'For example , it can serve in the evaluation of data sets relevant to the innate antiviral and antifungal responses ( Table 2 ) .', 'Many labs routinely consult internal data sets to decide which genes to study further .', 'Comparison of such lists with for example high-scoring RLR candidates could provide insights into the quality of individual data sets for identifying antiviral genes and provide complementary hints about which genes could be important .', 'Finally , the RLR resource could be used for prioritizing genetic variants in patients suffering from severe susceptibility to viral infections or inflammatory disorders caused by inappropriate production of type I interferons .', 'In this work , we have combined integrative genomics with experiments to discover 10 molecular signatures of a cellular signaling system that is central to human infectious disease: the innate antiviral RIG-I-like receptor ( RLR ) pathway .', 'The described signatures span multiple layers of genomics data and provide new insights into the regulation of virus detection and immune signaling .', 'Probabilistic integration of the data resulted in a confident genome-wide ranking of candidate RLR pathway genes .', 'RNAi validation experiments confirmed 94 of 187 novel RLR candidates tested , including 13 novel factors with strong effects on antiviral signaling .', 'These results , together with independent computational and literature-based confirmation , demonstrated the validity and high accuracy of our approach .', 'Our study expands the collection of known antiviral genes , opening up new avenues for research into innate antiviral immunity .'], ['All data sets were calculated for and mapped to a reviewed reference set of 20 , 245 human proteins from UniProtKB/Swiss-Prot , release 2011_11 [57] .', 'This set consists of one manually annotated record for each validated protein-coding gene .', 'Gene/protein identifier mapping was performed using a mapping table from the same UniProt release .', 'Ambiguously mapped identifiers were curated manually .', 'To systematically define RLR pathway components , we mined genome-scale data from a wide variety of sources .', 'The data describe different aspects of the biology of the pathway; from the DNA to the protein level , highlighting evolutionary processes , virus-host interactions , sequence families , etc .', 'We finally settled on 10 data sets that collectively distinguish RLR pathway components from other genes ( see Table 1 for an overview and brief descriptions ) : We assessed the capability of individual data sets to predict novel RLR genes using two ‘gold standard’ training sets: Two additional curated sets of genes were used in our study ( S6 Table ) .', 'The first consists of 153 genes with a known function ( i . e . receptors , signaling components , etc . ) in four PRR signaling pathways; the Toll-like receptor ( TLR ) , C-type lectin receptor ( CLR ) , NOD-like receptor ( NLR ) , and cytosolic DNA sensing ( cytDNA ) pathways , but not in the RLR pathway .', 'TLR , NLR and cytDNA components were obtained from InnateDB ( 27 Mar . 2012 ) .', 'We curated a list of 34 CLR pathway components , based mainly on [78] .', 'The combined PRR pathway gene set was supplemented with several key proteins involved in virus-host interactions .', 'As with the set of RLR genes , cytokines and other secreted proteins were excluded .', 'The second list ( ‘other innate immunity genes’ ) consists of 803 genes with curated annotations from InnateDB ( 12 Jan . 2012 ) for a function in other aspects of the innate immune response , excluding RLR and other PRR signaling pathway genes .', 'Individual ( genomics ) data sets contain important information about the make-up of cellular systems and pathways , but often have limited coverage and introduce data type-specific noise .', 'Combination of multiple heterogeneous types of data , each approaching the characterization of a molecular system from a different angle , therefore has the potential to provide a more complete definition of the system and could have high power for predicting novel components involved .', 'We employed a naive Bayesian framework to facilitate direct comparison and weighing of many data sets describing properties of RIG-I-like receptor pathway components and integrate those data sets that were suitable into a single probabilistic score for each gene .', 'Bayesian integration is well suited to combining evidence from dissimilar types of information and readily accommodates missing data [18–20] .', 'Furthermore , this approach inherently weighs data sets based on their predictive value ( i . e . their ability to separate known positives and negatives , Fig 1A and S1 Table ) so that better data contribute more to the predictions .', 'Indeed , integration enriches for RLR genes and depletes false positive , non-RLR genes ( S5 and S9 Figs ) .', 'Plots , statistics and other calculations were done using custom Perl and SQL scripts , and the R statistical package [83] with additional packages gplots [84] , ROCR [85] and RNAither [86] .', "One-way ANOVA with Dunnett's post hoc test was performed using G
['Introduction', 'Results', 'Discussion', 'Methods']
['The RIG-I-like receptor ( RLR ) pathway is essential for detecting cytosolic viral RNA to trigger the production of type I interferons ( IFNα/β ) that initiate an innate antiviral response .', 'Through systematic assessment of a wide variety of genomics data , we discovered 10 molecular signatures of known RLR pathway components that collectively predict novel members .', 'We demonstrate that RLR pathway genes , among others , tend to evolve rapidly , interact with viral proteins , contain a limited set of protein domains , are regulated by specific transcription factors , and form a tightly connected interaction network .', 'Using a Bayesian approach to integrate these signatures , we propose likely novel RLR regulators .', 'RNAi knockdown experiments revealed a high prediction accuracy , identifying 94 genes among 187 candidates tested ( ~50% ) that affected viral RNA-induced production of IFNβ .', 'The discovered antiviral regulators may participate in a wide range of processes that highlight the complexity of antiviral defense ( e . g . MAP3K11 , CDK11B , PSMA3 , TRIM14 , HSPA9B , CDC37 , NUP98 , G3BP1 ) , and include uncharacterized factors ( DDX17 , C6orf58 , C16orf57 , PKN2 , SNW1 ) .', 'Our validated RLR pathway list ( http://rlr . cmbi . umcn . nl/ ) , obtained using a combination of integrative genomics and experiments , is a new resource for innate antiviral immunity research .']
['Viruses pose a continuous threat to human health , even though our immune systems have evolved to neutralize invading viruses .', 'As part of the innate immune system , the RIG-I-like receptors ( RLRs ) are essential for detecting viruses during infection .', 'Recognition of viral RNA by the RLRs triggers an antiviral response that inhibits viral replication , protects uninfected cells , and attracts specialized immune cells .', 'Better understanding of the innate antiviral response may reveal novel targets for antiviral therapeutics and vaccine development .', 'However , that requires knowledge about which genes and proteins are involved .', 'In the present study , we systematically investigated the wealth of available genomics data ( including gene expression , protein interactions , transcription regulation and genome sequences ) and discovered no less than 10 distinctive properties of genes known to be part of the antiviral RLR pathway .', 'By combining these properties in a statistical framework , we predicted 187 novel RLR pathway components .', 'Our validation experiments showed that ~50% of the predicted candidate genes have a significant effect on antiviral signaling .', 'These results , together with independent computational and literature-based confirmation , demonstrated the validity of our combined bioinformatics and experimental approach .', 'Our study expands the collection of known antiviral genes , opening up new avenues for research into innate antiviral immunity .']
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