key: cord-0782753-z6ajwdya authors: Perčulija, Vanja; Ouyang, Songying title: Chapter 9 Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases date: 2019-12-31 journal: Helicases from All Domains of Life DOI: 10.1016/b978-0-12-814685-9.00009-9 sha: 46b355f72e611c05ba32943b7e90219dea804cca doc_id: 782753 cord_uid: z6ajwdya Abstract DEAD/DEAH-box helicases are enzymes that belong to the DEAD/H-box family of SF2 helicase superfamily. These enzymes are essential in RNA metabolism, where they are involved in a number of processes that require manipulation of RNA structure. Recent studies have found that some DEAD/DEAH-box helicases play important roles in innate immunity, where they act as sensors of cytosolic DNA/RNA, as adaptor proteins, or as regulators of signaling and gene expression. In spite of their function in immunity, DEAD/DEAH-box helicases can also be hijacked and exploited by viruses to circumvent detection and aid in viral replication. These findings not only imply that DEAD/DEAH-box helicases have a broader function than previously thought, but also give us a much better understanding of immune mechanisms and diseases that arise due to the dysregulation or evasion thereof. In this chapter, we demonstrate the known scope of activities of human DEAD/DEAH-box helicases in innate immunity and interaction with viruses or other pathogens. Additionally, we give an outline of diseases in which they are, or may be, involved in the context of immunity. DEAD-box and DEAH-box helicases (DEAD/DEAH-box helicases) are enzymes that bind and hydrolyze NTP to unwind double-stranded RNA molecules or remodel RNA-protein complexes [1] . They constitute the DEAD/H-box family, the largest group of helicases in the SF2 helicase superfamily [2] . Structurally, these enzymes are characterized by two tandem repeats of RecA globular domains, with nine conserved motifs: Q (only in DEAD-box helicases), I, Ia, Ib, II, III in the N-terminal domain, and IV, V, and VI in the C-terminal domain [2] . Among them, motifs Q, I (Walker A Motif), II (Walker B Motif), and IV are required for ATP binding and hydrolysis, motifs Ia, Ib, III, IV, and V participate in intramolecular rearrangement and interaction with RNA, and motifs III and V are implied in coordination between the ATP binding and nucleic binding sites [2] . The Walker B Motif comprises a distinctive amino acid sequence, D-E-A-D (asp-glu-ala-glu) or D-E-A-H (asp-glu-ala-his), hence giving the name to the entire protein family. In total, the human genome encodes for 42 DEAD-box and 16 DEAH-box helicases [3, 4] . The nomenclature of DEAD/DEAH-box helicases uses DDX or DHX and a number for DEAD-box proteins and DEAHbox proteins, respectively. DEAD/DEAH-box helicases are primarily involved in all facets of RNA metabolism, including pre-mRNA splicing, micro RNA processing, RNA export, RNA editing, storage and decay, ribosome biogenesis, transcription, and translation [5] . However, DEAD/DEAH-box helicases can perform important roles unrelated to RNA metabolism, for which they sometimes do not even require interaction with RNA [6] . Some of these include transcriptional coregulation [7] , monitoring of cell cycle progression [8] , regulation of protein localization [9] and protein activity [10, 11] . Furthermore, an accumulating number of studies associate DEAD/DEAH-box helicases with multiple functions in innate immunity. INTRODUCTION [37] . dendritic cells [37] HIV-1 [38, 39] coronavirus [40, 41] JC virus [42] DDX21 [37] À DDX1 interacts with DDX21 in the DDX1-DDX21-DHX36 complex and transduces the signal to DDX21-DHX36 upon sensing viral dsRNA DDX3 (DDX3X) Positively regulates translation of PACT [43] . Binds RLRs or directly recognizes viral RNA and binds MAVS to augment or sensitize downstream signaling. Known to sense poly I:C, viral stem-loop RNA and VSV [44] . Scaffolding adaptor in the MAVS signaling pathway. Interacts with MAVS, TRAF3 and IKKε [45, 46] . Direct transcription factor for IFN-β upon Listeria monocytogenes infection [47] . Regulation of NF-κB pathway [48, 49] . Assembly of processing bodies and stress granules [50, 51] . Participates in DAI-dependent sensing of viral dsDNA [52] . Inhibition of hepatitis B virus polymerase [53] . hepatocyte lineage cells [43À46,48,49,53] A549 cells [45, 46] RAW264.7 macrophages [47] THF cells [52] hepatitis C [54À57] HIV-1 [55, 58, 59] norovirus, West Nile virus, Japanese encephalitis virus [55] MAVS À DDX3 can activate MAVS and act as a scaffolding adaptor in downstream signaling TRAF3 [46] À Interaction between DDX3 and TRAF3 triggers K63-linked polyubiquitination and oligomerization of TRAF3 IKKε [45, 46] À IKKε and DDX3 phosphorylate each other to promote downstream signaling in the MAVS pathway TBK1 [47] À DDX3 undergoes phosphorylation by TBK1 before it can act as a transcription factor for IFN-β PP2A [48] À Positive regulation of NF-κB transcriptional activity by modulation of PP2A activity NF-κB subunit p65 [49] À Suppression of NF-κB transcriptional activity eIF4E and PABP1 [50] À Promotion of stress granule formation and inhibition of translation Component of the cytosolic RNA-sensing DDX1-DDX21-DHX36 complex [37] . Enhances inflammation through expression of S100A9 during infection [60] . Sensor of bacterial and viral RNA, activates the NLRP3 inflammasome in macrophages. [85, 86] . RLR-independent viral dsRNA sensor in the MAVS pathway in myeloid dendritic cells. Known to sense poly I:C and reovirus [87] . Macrophages [85, 86] Myeloid dendritic cells [87] N/A NLRP3 [85, 86] À DHX15 interacts with NLRP3 to activate the NLRP3 inflammasome TRIM33 [86] À Promotes interaction between DHX33 and NLRP3 by K63-linked ubiquitination MAVS [87] À Downstream signal transduction [111] . ZAP-mediated degradation of retroviral RNA [112] Putative role in the RLR-mediated antiviral response [113] . [117] ZAP [112] À DDX17 is a cofactor in the ZAPmediated RNA degradation complex DHX9 [118] interaction between DDX17 and DHX9 (not studied in the context of innate immunity) Negative regulator of the IFN signaling pathway. Inhibits NF-κB and IRF3 activation [119] . Ubiquitously expressed [119] dengue virus, VSV and Zika virus [119] DDX25 inhibits NF-κB and IRF3 [119] DHX29 Upstream viral RNA sensor and activator of RIG-I [120] . Human airway epithelial cells and fibroblasts [120] salivirus [121] RIG-I and MAVS [120] À DHX29 directly interacts with RIG-I and MAVS to transduce signal The role of DDX41 as sensor of cytosolic DNA in STING pathway was first reported by Zhang et al. in 2011 after noticing that knockout of IFI16, another cytosolic DNA sensor, does not significantly decrease IFN-β levels [72] . Constitutively expressed in dendritic cells, macrophages and neutrophils, the cytosolic DDX41 is bound to the E3 ubiquitin ligase TRIM21, which keeps DDX41 in inactivated state [75, 77] . Upon appearance of B-form DNA, DNA viruses or cyclic dinucleotides (CDNs), DDX41 dissociates from TRIM21, and is then phosphorylated by Bruton's tyrosine kinase (BTK), which enables its sensing function [76] . BTK kinase activity also facilitates the interaction between DDX41 and STING, which triggers downstream STING signaling via TBK1 and subsequently activates transcription factors such as IRF3 and NF-κB [72, 73, 76] . These transcription factors induce expression of type I IFNs and proinflammatory cytokines, including TNF and IL-6. Type I IFN signaling also stimulates the expression of interferon-stimulated genes, which further promote or regulate the immune response. Among the interferon-stimulated genes, the expression of cGAS is dependent on the initial production of type I IFN mediated by basal DDX41 [79] . TRIM21 is responsible for negative regulation of DDX41 by inducing its degradation via K48-linked ubiquitination [77] . The expression of APOBEC3, a cytidine deaminase important in antiretroviral innate immunity, can also be induced by IFN signaling through DDX41/STING axis [74] . This suggests that DDX41 has a role in sensing retroviral reverse transcripts. Negative regulation of DDX41 is essential because excessive expression of type I interferons may cause various pathological conditions, such as systemic lupus erythematosus [122] , or lymphocyte apoptosis during Listeria monocytogenes infection. Besides TRIM21, the complement anaphylatoxins C3a and C5a have also been reported to negatively regulate DDX41 by suppressing its expression [78] . Structural analyses of DDX41 have given us a better insight into the mechanisms behind its roles in the innate immunity. DDX41 contains a disordered N-terminal region, a DEAD domain and a helicase domain. The N-terminal region of DDX41 was demonstrated to play a role in translocation of the protein to the nucleus [123] . This may explain the fact that DDX41 was found localized both in cytoplasm and nucleus of MCAT-293T (murine cationic amino acid transporterexpressing 293T) cells upon the infection with murine leukemia virus [74] . Such observation implies that DDX41 may participate in another, unknown pathway associated with innate immunity. The DEAD domain plays the central role in DNA/CDN sensing, a feature specific for DDX41 among DEAD/DEAH-box helicases. Several highly conserved amino acid residues (Arg267, Lys304, Lys331, and Lys381; Lys331 only binds CDNs) are crucial for the ligand binding [124] . In addition, deletion of motif I or motif II impairs both DNA/CDN sensing and binding to STING [36] . Interestingly, the process of DNA/CDN sensing was also shown to be inhibited by the helicase domain [72, 124] . The possible explanation is that the linker region between DEAD and helicase domain may position the helicase domain in a conformation which disturbs the interaction between DEAD domain and its ligands [124] . Moreover, Q motif, motif I, and motif II collectively form an ATP binding and hydrolysis site, as predicted in the overall structures of the DEAD domain solved by Jiang et al. [123] and Omura et al. [124] . Omura et al. reported three different conformations of the N-terminal region, among which only the "closed form," characterized by Q motif adjacent to motif I, is predicted to bind ATP [124] . Contrarily, the two "opened forms" and a similar structure reported by Jiang et al. are all characterized by conformation that sterically clashes with the putative ATP binding site [123, 124] . This is further supported by the fact that Jiang et al. did not detect any significant affinity of the protein for ATP in vitro [123] . Based on their findings, Omura et al. propose that the DEAD domain undergoes a structural transition between an ATP-bound closed conformation and an opened conformation after ATP hydrolysis and ADP release, thus playing a role in DNA/CDN sensing by accelerating the ligand turnover of DDX41 [124] . Lastly, phosphorylation of Tyr364 in the DEAD domain and Tyr414 from the linker region between the DEAD and helicase domain by BTK was demonstrated to be of key importance in DNA/CDN sensing and binding to STING [76] ; however, a pull-down assay of phosphorylated full-length DDX41 from another study failed to validate the effect of BTK on DNA/CDN sensing [124] . Altogether, our understanding of DDX41 is insufficient to clearly explain the exact mechanisms behind DNA/CDN sensing, signal transduction via STING, and the regulation of DDX41 activity. However, the results of the abovementioned studies strongly indicate the existence of additional cofactors or posttranslational modifications involved in these mechanisms. Mutations in DDX41 have been reported to be associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [125, 126] . A number of these mutations, namely p.M155I, p.R164W, p.F183I, p.A225D, p.E247K, p.P321L, and p.I396T, are located in the DEAD domain of DDX41 [123] . The presence of mutations in the DEAD domain suggests the role of deregulated innate immune signaling in MDS and AML. Furthermore, the whole STING signaling pathway is implicated in a number of inflammatory diseases and tumors, where it exhibits both pro-and anticancer functions. The topic has been reviewed in detail by He et al. [127] . Nevertheless, the involvement of DDX41 in the context of STING signaling pathway has not been reported in pathogenesis of these diseases, but it should be considered in future studies. Human DDX3 has two homologs, DDX3X and DDX3Y, which are located on chromosomes X and Y, respectively. Out of the two, DDX3X (here referred to as DDX3) is a nucleocytoplasmic helicase expressed in a wide range of tissues and involved in fundamental biological processes such as cell-cycle progression, lipid metabolism, gene expression, as well as transcription, mRNA splicing, mRNA export and translation [8,54,128À130] . Research on DDX3 in antiviral immunity conducted before 2015 has been reviewed in detail by Ariumi [131] and Valiente-Echeverría et al. [55] . In brief, DDX3 participates in a plethora of processes associated with immune responses, ranging from multiple functions in RLR-MAVS signaling pathway [44À47] and sensing of viral dsDNA via DAI [52] , to constitution of cytoplasmic granules [50, 51] , and direct inhibition of hepatitis B virus polymerase [53] . Upstream of RLRs, DDX3 enhances the sensitivity of RLR-MAVS pathway by positively regulating translation of PACT, a dsRNA-binding protein that facilitates RIG-I sensing and activation [43] . Also, DDX3 can either bind RLRs or directly recognize viral RNA and bind MAVS to sensitize downstream signaling [44] . While RLR binding could be important for intensifying the signal, direct RNA sensing may be essential during the initial antiviral response, when the IFN-inducible RLRs are present in low concentration. With no need for its enzymatic activity, DDX3 also serves as a scaffolding adaptor that interacts with important signaling molecules downstream of MAVS [46] . Upon infection, the interaction between DDX3 and TRAF3 triggers the K63-linked polyubiquitination and oligomerization of TRAF3. This event is important for the stabilization of MAVS-TRAF3 complex and recruitment of additional signaling molecules to the complex [46] . CK1ε, another kinase whose activity is regulated by DDX3, may also be recruited to the MAVS-TRAF3 complex to phosphorylate TRAF3 and thereby promote its autoubiquitination [132] . However, this notion needs to be further confirmed. Concurrently, DDX3 binds the scaffolding/dimerization domain of IKKε to induce the autophosphorylation of IKKε [45] . In turn, IKKε phosphorylates serine 102 in DDX3, leading to recruitment of IRF3 to the signaling complex. IKKε phosphorylates IRF3, which ultimately activates transcription of IFN-β [45] . In addition, DDX3 appears to directly act as a transcription factor for IFN-β after being phosphorylated by TBK1 [47] . Besides the stimulation of interferon production, DDX3 has been implied in the regulation of NF-κB pathway [48, 49] . Interestingly, while one study showed the positive regulatory effect achieved by modulation of PP2A (a phosphatase that inactivates the NF-κB activator IKK-β and NF-κB subunit p65) [48] , another study showed that DDX3 interacts with p65 to suppress its transcriptional activity [49] . The discrepancy between the effects is possibly due to different experimental conditions. The positive regulation was observed when the cells were stimulated by the synthetic dsRNA analog poly I:C [48] , which resembles antiviral immune response, whereas the repression was observed during normal cell culture conditions [49] . This would suggest that DDX3 is an arbitrary regulator of NF-κB pathway whose activity depends on the cellular conditions. DDX3 is the main component promoting the assembly of cytoplasmic granules, such as processing bodies (P-bodies) and stress granules [50, 130] . P-bodies are constitutively present in cells, where they carry out mRNA degradation. On the other hand, stress granules are assembled during cellular stress conditions to store mRNA and ribonucleoproteins, and to stall translation initiation. Both of these structures participate in cellular response against viruses and restriction of viral replication [51, 133] . Importantly, the fact that RLRs can also be recruited to and activated in stress granules further strengthens and interconnects the multiple roles of DDX3 in innate immunity [134] . Considering the importance of DDX3 in a number of innate immune mechanisms, it is not surprising that viruses have evolved strategies to evade host defenses by preventing DDX3 from participating in innate immunity. A number of viruses have been reported to interfere with formation of cytoplasmic granules and exploit DDX3 as a host factor for replication. This is seen in cases of hepatitis C virus, HIV-1, norovirus, West Nile virus, and Japanese encephalitis virus [55] . For example, during HCV infection the binding of HCV core protein to DDX3 disrupts the interaction between DDX3 and MAVS [56] , decreases the expression of DDX3 and redistributes DDX3 to lipid droplets (the cytoplasmic sites of HCV replication) [57] . Since DDX3 is important for the maintenance of lipid homeostasis, such decrease in expression levels and sequestration of DDX3 does not only aid in the viral evasion of the host immunity, but also results in retention of lipids in cells and subsequent steatosis [54] . Similarly, HIV-1 interrupts MAVS signaling as viral feedback to the initial sensing of HIV-1 RNA by DDX3 [58] . At the same time, HIV-1 utilizes the CRM1/ DDX3 complex in nucleus for the Rev-dependent nuclear export of viral RNAs, or DDX3 itself to promote the translation of the HIV-1 unspliced mRNA [59] . DHX 9, also known as RNA helicase A or Nuclear DNA Helicase II, is a DNA/RNA helicase from the DEAH-box helicase family involved in regulation of transcription [135À137] and translation [138, 139] , DNA replication [140] , embryonic stem cell differentiation [141] , as well as genome repair and maintenance [142À144] . In addition, DHX9 is also present in several immune pathways. Along with DHX36, DHX9 has been demonstrated to serve as a microbial DNA sensor in the cytosol of plasmacytoid dendritic cells [88] . DHX9 specifically binds class B CpG motifs via its DUF (domain of unknown function) domain. Its DUF and helicase C domains then interact with the TIR (Toll/interleukin-1 receptor) domain of MyD88 (myeloid differentiation primary response gene 88, the adapter protein commonly associated with the Toll-like receptors) [88] . Upon receiving the signal from DHX9, MyD88 induces downstream signaling cascade to activate NF-κB subunit 50, ultimately resulting in the production of IL-6, TNF and, partly, IFN-α [88] . DHX9 is also an important sensor of viral dsRNA in myeloid dendritic cells, and has been found to sense both long and short poly I:C, as well as reovirus and influenza A virus [89] . DHX9 binds dsRNA via dsRBD (dsRNA-binding domain) domains and transduces the signal to MAVS, thereby triggering IRF3 and NF-κB-dependent expression of response genes [89] . Furthermore, DHX9 has been discovered to sense short dsRNA and activate the formation of NLRP9b (NOD-like receptor family, pyrin domain containing 9B) inflammasome as a response to rotavirus infection in intestinal epithelial cells [90] . Inflammasomes, such as NLRP9b inflammasome, are cytosolic protein complexes in the innate immune cells (such as myeloid cells and epithelial cells) capable of initiating caspase-1mediated IL-1β and IL-18 maturation and/or gasdermin-induced pyroptosis in response to pathogens, cellular stress, and other danger signals [90, 145] . Interestingly, a structural analysis of the DHX9 dsRBD domains in complex with dsRNA elucidated the mechanisms underlying dsRNA binding by DHX9, and provided evidence on how the sensing of the small interfering RNA by DHX9 plays a role in the assembly of RISC (RNAinduced silencing complex) [146] . Given that RNA interference is also an element of the mammalian antiviral immune response [91] , the assembly or RISC may be another way for DHX9 to counter virus infection. DHX9 is a known target of immune evasion and exploitation by several viruses. For example, the ATPase-dependent helicase activity of DHX9 is required for HIV-1 RNA translation [92] . DHX9 can also bind to the primer binding site segment of the 5 0 untranslated region (UTR) in the genomic HIV-1 RNA during the virion assembly, which increases the overall infectivity of HIV-1 [92, 93] . The myxoma virus (rabbit-targeting member of the Poxviridae family) protein M029 recruits DHX9 to promote viral infection in myeloid cells [94] . Additionally, the influenza B virus NS1 protein interacts with DHX9, but the interaction hasn't been functionally characterized yet [95] . Lastly, DHX9 is an autoantigen associated with the early stages of systemic lupus erythematosus and possibly with primary Sjögren's syndrome [147, 148] . In accord with high female-to-male incidence ratios in these two diseases, the expression of DHX9 has also been found to be higher in females than in males [148] . Considering the fact that the autoimmune diseases are thought to be caused by microbial infection, the time context of DHX9 autogenicity and its importance in cytosolic DNA/RNA sensing, it is possible that the function of DHX9 in innate immunity is closely related to the onset of lupus and primary Sjögren's syndrome. Further research would be required to verify this assumption. DHX15 is a DEAH-box helicase ubiquitously expressed in human organs and tissues [149] . Within a cell, DHX15 is distributed across nucleus, nucleolus, cytosol, and mitochondria, with different functions in each location [97, 150] . Nuclear DHX15 is important for early stages pre-mRNA splicing (in nuclear speckles) [151, 152] , during which it is regulated by a number of proteins, such as RBM5 (RNA-binding motif protein 5) [99] and TFIP11 (tuftelin-interacting protein 11) [153] . RBM5 is an alternative splicing regulator of apoptosis-related genes, thus implying that DHX15 regulates apoptosis through splicing of these genes [99] . In nucleolus, DHX15 participates in ribosome biogenesis, forming a complex with NKRF (NF-κB-repressing factor) and exonuclease XRN2 [100] . Apart from these functions, DHX15 located in cytosol and on mitochondrial membrane plays a role in in antiviral innate immune response and signaling [96À98]. Most interestingly, these roles are independent of ATPase activity and are characteristically cell type-specific, with distinct roles reported in HeLa cells, embryonic kidney cell lines (HEK 293 and HEK 293T), myeloid dendritic cells and intestinal epithelial cells. During poly I:C stimulation or encephalomyocarditis virus (EMCV) and Sendai virus infection in HeLa and HEK 293 cells, DHX15 forms complex with MAVS and interacts with TRAF6 to stimulate proinflammatory cytokine response and caspase-3-mediated apoptosis through NF-κB and JNK/p38 MAPK (mitogen-activated protein kinase) pathways [97] . DHX15 was found to weakly bind dsRNA and was not required for IRF3 activation, but was necessary for the optimal cytokine expression (including IFN-β) [97] . Likewise, DHX15 triggers the aggregation of MAVS in myeloid dendritic cells in the presence of viral dsRNA [96] . However, not only was DHX15 observed to be critical for the activation of IRF3, NF-κB, and MAPK, but it also specifically bound poly I:C (size 1.5À8 kb) or Sendai virus and reovirus dsRNA in tandem with DHX9, thus acting as a direct sensor of viral dsRNA [96] . In the intestinal epithelial cells, DHX15 exhibits yet another mode of action: it associates with the NLRP6 (nucleotide-binding oligomerization domain-like receptor 6) to sense long viral dsRNA and transduce the signal to MAVS, eventually inducing the expression of both type I and type III IFNs by activation of the transcription factors IRF3 and IRF7 [98] . In contrast to these cells, DHX15 is dispensable for the expression of IFN-β as an immune response to viral infection in HEK 293T cells [96] . These differences suggest existence of interaction between DHX15 and cell/tissue type-specific proteins which modulate the activity of DHX15 and MAVS signaling pathway. Moreover, the fact that the nucleolar DHX15 interacts with NKRF points out to the possibility that this DEAH-box helicase may participate in NF-κB signaling on two different levels [100, 154, 155] . Further research is needed to gain better understanding of the activities of DHX15. As of May 2017, DHX15 is the only DEAH-box helicase whose crystal structure has been revealed in a nearly-complete form, lacking only the N-terminal extension domain [156] . DHX15 is divided into six structural domains, namely the N-terminal extension, the RecA1 and RecA2 domains, the winged-helix domain, the ratchet domain and the oligonucleotide /oligosaccharidebinding (OB)-fold domain. Among them, the OB-fold domain is important for interaction with the G-patch proteins, such as NKRF [100, 156] . The putative RNA binding pocket is located between the two RecA domains and the C-terminal domain (containing the winged-helix domain, the ratchet domain and the OB-fold domain) [156] . Based on the mechanism by which Prp43 (the fungal orthologue of DHX15) binds ssRNA, the authors suggest that binding of ATP and RNA triggers a structural rearrangement in the RNA binding pocket to form an RNA-accommodating groove [156] . However, DHX15 senses viral RNA in an ATPase-independent manner, indicating existence of other factors that cause the structural rearrangement of the RNA binding pocket. Similarly to DDX41, DHX15 has also been implied in the pathogenesis of acute myeloid leukemia (AML) [157] . The mutation of the highly conserved arginine significant for RNA binding (R222G) is related to AML, and the overexpression of DHX15 in the AML patients indicates poor cytogenic prognosis and overall survival [156, 157] . Of note, overexpressed DHX15 in AML acts through activation of NF-κB pathway just as during the viral infection, but it decreases the levels of caspase-3 and PARP, thereby promoting cell survival [157] . Considering that during the viral response DHX15 also activates apoptotic MAPK signaling, it is possible that DHX15 can activate NF-κB pathway independently of MAVS, or that the transformed myeloid cells in AML use aberrant MAVS signaling. DDX21, also known as nucleolar RNA helicase II, is an ATP-independent helicase associated with many nuclear and nucleolar events, such as ribosomal RNA biogenesis [158] or unwinding of R-loops [159] and RNA guanine quadruplexes [160] . DDX21 also interacts with mitotic regulator PP1 [161] and oncoprotein DEK [162] . Additionally, upregulated DDX21 has been found to promote tumorigenesis in breast cancer by phosphorylating c-Jun, a component of AP-1 transcription factor involved in cell survival pathways [163] . With respect to innate immune pathways, DDX21 occurs as a component of a constitutive, IFN-, TL3-, and RLR-independent viral RNA-sensing cytosolic complex in dendritic cells and macrophages, wherein DDX21 serves as a bridge between DDX1 and DHX36 by binding the two helicases through its PRK domain [37, 60] . Following DDX1-mediated RNA sensing, DDX21 and DHX36 transduce the signal to the adaptor TRIF, which sensitizes NF-κB and IRF3 pathways [37] . Presumably as a part of the same complex, DDX21 also triggers the expression of a DAMP protein S100A9, which induces production of proinflammatory cytokines and apoptosis [60] . DDX21mediated expression of S100A9 is unfavorable during influenza A virus infection, but may be effective against some bacterial infections [60] . DDX21 has been shown to interact with a number of RNA viruses. The DDX1-DDX21-DHX36 complex senses both short and long poly I:C, reovirus and influenza A virus [37] . DDX21 can also act individually to obstruct the replication of influenza A virus through inhibition of the viral RNA polymerase, but this effect is countered in later stages of the infection by the viral NS1 protein, an inhibitor of host antiviral response [61] . Whether DDX21 can similarly impact the replication of other influenza viruses is still unknown. Furthermore, DDX21 translocates from nucleus to inhibit dengue virus replication in the early stages of the infection [64] , participates in cellular defense against Hantaan virus [164] and regulates translation of polycistronic mRNA of Borna disease [62] . However, DDX21 also plays a role in HIV-1 pathogenesis by promoting Rev oligomerization on the Rev-responsive element, thus aiding the export of viral transcripts to cytosol [63] . Collectively, DDX21 appears to uptake a number of distinct roles. Some of these roles influence cells in opposite ways, such as the promotion of cell survival via c-Jun phosphorylation and promotion of apoptosis by triggering expression of S100A9, or ribosomal RNA biogenesis and CHAPTER 9 DIVERSE ROLES OF DEAD/DEAH-BOX HELICASES suppression of ribosomal RNA transcription through formation of rings that encircle RNA polymerase I complexes [165] . We also speculate that, to a certain extent, these activities may be concomitant. For instance, sensing of viral RNA by the DDX1-DDX21-DHX36 complex may stimulate the formation of DDX21 rings containing RNA polymerase I, which would be favorable as a defensive mechanism to attenuate viral replication. Nevertheless, the full scope and determinants of DDX21 activity are still unknown. A notion on the regulation of DDX21 might have been given by a study that identified two micro RNAs (miR-744 and miR-1196) as factors that control DDX21 expression in a NF-κB and Toll-like receptor 4-dependent manner during ischemia-reperfusion injury in skeletal muscles [166] . Further research is required to elucidate the means by which these two mRNAs, as well as other unidentified factors, influence activities of DDX21. DDX24 is an IFN-inducible and STAT1-dependent negative regulator of innate immune signaling in cytosol and nucleus [65] . A study from 2013 by Ma et al. observed that DDX24 exhibits negative regulatory effects on two distinct levels in RLR-MAVS-mediated signaling pathway [65] . In the upstream signaling transduction, DDX24 competes with RIG-I and MDA5 for binding their respective RNA ligands, whereas in the downstream signaling transduction it interferes with formation of complexes comprising FADD/RIP1, the two coregulators significant for the activation of IRF7 [65] . Nonetheless, the regulatory activities of DDX24 are not limited to RLR-MAVS pathway. p300, a histone acetyltransferase that stabilizes the tumor suppressor protein p53, interacts with N-terminal, Q motif and motif I of DDX24. This interaction results in the suppression of p300-mediated p53 acetylation not only during cell homeostasis, but also upon DNA damage stress [66] . Given the well-established roles of p53 in tumor suppression, apoptosis and cellular senescence, and that more and more studies associate p53 with amplified expression of IFN-stimulated genes during antiviral immune response [67, 68] , it is safe to assume that the knowledge on the regulatory activities of DDX24 represents only the very tip of the iceberg. One of the uninvestigated activities of DDX24, among others, is potential restriction of HIV-1 RNA transcription through p300. This is possible due to the fact that HIV-1 transactivator of transcription (Tat) recruits p300 to stimulate the transcription initiation [167] . On the whole, the importance and polyvalence of this helicase is further supported by the fact that polyubiquitinated DDX24 is required for early steps of pre-rRNA processing, and that knockout of DDX24 causes embryonic lethality in mice [65, 168] . DDX60 was demonstrated by the Oshiumi's group to be an interferon-stimulated cytoplasmic helicase with roles both in RLR-dependent and independent antiviral innate immune response [80, 81] . Acting as a cell-type-specific activator of RIG-I and MDA5 in macrophages, splenic CD11c 1 cells and fibroblasts, DDX60 binds viral RNA and possibly DNA via its helicase domain [80] . Afterwards, DDX60 recruits RIG-I or MDA5 with the help of the ATP-binding site located in its DEAD domain [80] . RIG-I and MDA5 further transmit the signal to MAVS adaptor, ultimately leading to the production of type I IFNs and IFN-inducible genes. Alternatively, DDX60 participates in viral RNA degradation pathway, where it functions as a component of RNA exosome complex [81] . Such mode of action is preferred in hepatocytes against hepatitis B and C viruses 155 ROLES OF DEAD/DEAH-BOX HELICASES [81, 169] . The activity of DDX60 in both pathways is negatively regulated by epidermal growth factor (EGF) receptor [81] . EGF receptor phosphorylates Tyr793 and Tyr796 of DDX60 either in response to EGF (which instead activates Toll-like receptor 3), or by virus-mediated activation of EGF receptor for the evasion of the innate immunity [81À83]. DDX60 appears to be ligand-specific with respect to RNA and DNA sensing. Studies led by Oshiumi report that DDX60 is mainly involved in sensing of poly I:C and ssRNA viruses, such as vesicular stomatitis virus, poliovirus, Sendai virus or hepatitis C virus [80, 81] . In addition, DDX60 can sense herpes simplex virus 1 and hepatitis B virus, which respectively belong to dsDNA and dsDNA-RT viruses [80, 81] . On the other hand, DDX60 cannot detect a 63-nucleotide 5 0 triphosphated ssRNA, indicating that this helicase is unable to recognize viral RNA that undergoes 5 0 triphosphate capping [81] . Further studies have also found that Hantaan virus (HNTV) inhibits the transcription of DDX60, but the long noncoding RNA NEAT1 can interfere with these inhibitory effects and thereby deploy DDX60 to counter the HNTV infection [84] . Moreover, the expression of DDX60 is altered during human papillomavirus (HPV) infection in HaCaT cells. HPV-6 infection cause elevated DDX60 levels, whereas HPV-11 and HPV-45 have the opposite effect [170] . Interestingly, upregulated DDX60 has also been discovered in dermatomyositis and advanced oral squamous cell carcinoma, where it may participate in pathogenesis by stimulating secretion of type I IFNs [171, 172] . However, no correlation between DDX60 expression and HPV (a common cause of oral squamous cell carcinoma) was found [172] . Not all studies support the reported roles of DDX60 in antiviral immunity. In a study similar to those from the Oshiumi's group, Goubau et al. failed to identify the function of DDX60 in sensing viruses or other pathogens, could not confirm that DDX60 binds RLRs, and found DDX60 to be dispensable for the production of type I IFNs [173] . Further research is needed to explain the reasons behind these ambiguous results. DDX60L neighbors DDX60 on chromosome IV, and its amino acid sequence is B70% homologous to DDX60 [71] . However, our knowledge on functions of DDX60L in innate immunity is still scarce. A study from 2015 describes DDX60L as an innate immune effector of type I, type II, and type III IFN response that acts independently of DDX60 against hepatitis C virus (HCV) in liver cells [71] . The expression of DDX60L is induced soon after the acute HCV infection, during which it represses the viral RNA replication in a manner independent of IRES-mediated translation or RNA stability, and enhances RIG-I-dependent IRF3 activation [71] . DDX60L is likely to be highly specific in its antiviral functions, as it was unable to inhibit replication of hepatitis A virus, regardless of its similarity to HCV [71] . In addition, it is noteworthy to mention that DDX60L is also upregulated in IFN-β-treated EpsteinBarr-transformed B cells derived from multiple sclerosis patients [174] . DDX46 is the only nuclear DEAD/DEAH-box helicase proven to negatively regulate the antiviral innate immune response [69] . Viral infection induces the acetylation of DDX46 at Lys470, which facilitates the interaction between the DEAD domain of DDX46 and ALKBH5, a demethylase that 156 CHAPTER 9 DIVERSE ROLES OF DEAD/DEAH-BOX HELICASES erases the N 6 -methyladenosine (m 6 A) modification responsible for the export of mRNA transcripts to cytoplasm and their subsequent translation [69] . The negative regulation of antiviral innate immune response performed by DDX46 specifically targets mRNA transcripts containing CCGGUU motif and m 6 A modification, such as Mavs, Traf3 and Traf6 mRNA [69] . As a consequence, these antiviral mRNA transcripts remain in nucleus, which impedes the production of type I IFNs. With regard to its role in pathogenesis of diseases, the upregulated expression of DDX46 found in esophageal squamous cell carcinoma (ESCC) was shown to promote cell growth and survival [175] . Conversely, knockdown of DDX46 substantially reduced phosphorylation of Akt and IκBα, inhibited cell growth and induced apoptosis by suppressing NF-κB signaling [175] . Although the knockdown of DDX46 in macrophages had a different effect on NF-κB signaling during antiviral immune response (i.e. enhanced activation of NF-κB and increased production of proinflammatory cytokines and IFN-β), we speculate that overexpression of DDX46 might benefit ESCC cell growth and survival by increasing the nuclear retention of mRNA transcripts involved in tumor inhibition and apoptosis. DHX33 is a DEAH-box helicase important for 47S rRNA synthesis, mRNA translation initiation and cell cycle control on the transcriptional level [176À178] . Moreover, DHX33 is the first identified sensor of viral dsRNA and bacterial RNA capable of activating the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome [85] . In order to activate NLRP3 inflammasome, DHX33 first binds microbial RNA with its helicase C domain [85] . TRIM33 then induces the K63linked polyubiquitination of DHX33 at lysine 218 [86] . This event is crucial for the interaction between DHX33 and NLRP3, leading to subsequent activation of the inflammasome, caspase-1 cleavage, and processing of pro-IL-1β and pro-IL-18 [85, 86] . In addition to the activation of the NLRP3 inflammasome, DHX33 also acts as a viral dsRNA sensor participating in MAVS-mediated innate immune response in myeloid dendritic cells [87] . DHX33 was shown to sense poly I:C and reovirus in a RLR-independent manner, after which it interacted with the C-terminal domain of MAVS via its helicase C domain, ultimately leading to the activation of MAPK, NF-κB and IRF3 [87] . As demonstrated in this chapter and summarized in Fig. 9 .1, DEAD/DEAH-box helicases have indisputably confirmed their participation in the human innate immunity. What's more, they have shown a remarkable range of functions in the innate immune pathways (Table 9 .1), particularly in the STING-and MAVS-mediated signaling. In innate immunity, DEAD/DEAH-box helicases have been found to act as sensors or cosensors of microbial nucleic acids (e.g., DDX17, DDX41, DDX60, DHX9, DHX15, DHX29, DHX33, and DDX1-DDX21-DHX36), adapter proteins mediating downstream signaling (e.g., DDX3), negative regulators of the signaling pathways or gene expression (e.g., DDX6, DDX24, DDX25, and DDX46), and components of processing bodies and stress granules (e.g., DDX3 and DDX6). Indeed, the fact that viruses either hijack DEAD/DEAHbox proteins or exploit them as host factors of viral replication only underscores the significance of Roles of DEAD/DEAH box helicases in innate immune signaling pathways. Note that the signaling pathways have been simplified to place the focus on DEAD/DEAH-box proteins and their immediate interaction with other proteins. For that reason, many signaling proteins were omitted and some differences between different pathways were not considered. MyD88 signaling utilizes a very distinctive pathway that is not shown in the figure. Instead, MyD88 was connected to other pathways because it also results in activation of IRF7 and NF-κB. these helicases in innate immunity. However, many questions remain to be answered with regard to the roles of DEAD/DEAH-box helicases of helicases in the innate immunity. Some of these questions are addressed below: Originally associated only with RNA metabolism, an increasing number of DEAD/DEAH-box helicases is being found to exhibit multifunctionality in pathways unrelated to RNA metabolism, such as innate immune signaling. Therefore, it is likely that new DEAD/DEAH-box helicases will be identified as effectors of innate immunity. To this end, our knowledge combined with phylogenetic and structural comparison of would be of great use in identification of new DEAD/DEAH-box helicases in innate immunity. However, when comparing, it should be noted that closely related DEAD/DEAH-box helicases might exhibit different functions, such as in cases of DDX60 and DDX60L [71] . Cell type-specific innate immune responses, different stimuli and conditions should also be taken into account. Moreover, future studies should consider that many DEAD/DEAH-box helicases are constitutively expressed in cells, and that their levels may not increase during innate immune response (author's observation based on the literature). This would mean that the functions of some DEAD/DEAH-box helicases in innate immunity may not be easily detected. As demonstrated in this chapter, a large number of DEAD/DEAH-box helicases serve as sensors of microbial DNA and RNA. We propose several explanations for this phenomenon: I. Tissue and cell type specificity. DEAD/DEAH-box helicases serve as sensors in different types of cells. For example, the viral dsRNA cosensors DDX60 and DHX29 have a similar mode of action [80, 120] . Upon sensing of viral RNA, both helicases activate RLRs, thus triggering downstream RLR-MAVS signaling. However, the importance of DDX60 as a cosensor is limited to macrophages, splenic CD11c 1 cells and fibroblasts, whereas DHX29 is known to specifically act in human airway epithelial cells and fibroblasts [80, 120] . The possible reason for this might be that DEAD/DEAH-box helicases are known to have (slightly) different functions in different cells. DHX15 is an interesting example, as it exhibits at least three cell type-dependent roles in antiviral immunity [96À98]. II. Complementarity. DEAD/DEAH-box helicases may act as nucleic acid sensors in the same type of cells, but possess specificity for different ligands or signal through different pathways, thus achieving complementarity through specialization in their sensing of microbial nucleic acids and downstream signaling. This is the case with DDX1-DDX21-DHX36, DHX9, DHX15 and RLRs, which sense distinct (but overlapping) viral dsRNA and utilize different signaling pathways in dendritic cells [20, 37, 89, 96] . Complementarity might be useful due to the fact that the organism counters viral hijacking by the virtue of adding complexity to its PRR arsenal. In other words, while one viral nucleic acid sensor and its signaling pathway can be targeted by all viruses, a set of sensors with different signaling pathways could be more beneficial for the host defenses. III. Redundancy. DEAD/DEAH-box helicases may act as nucleic acid sensors or cosensors in the same type of cells and bind the same type of ligands. For example, both DHX9 and DHX33 sense reovirus in myeloid dendritic cells [87, 89] . Similarly to the complementarity, redundancy could also be useful for countering the viral hijacking, as virus may possess specificity to inhibit only one of the two sensors. Moreover, redundancy can be used to enhance the innate immune response. IV. Prevention of aberrant/exuberant activation of innate immune signaling. The viral RNAsensing RLRs and DNA-sensing cGAS are interferon-stimulated genes [79, 179] , and are expressed in relatively small concentrations in the absence of infection. We hypothesize that these sensors may have a strong affinity for their respective ligands and produce a robust immune response against them. However, if the strong ligand binding is paired with relatively low ligand specificity, such kind of cytosolic nucleic acid sensors may be detrimental for cells in the absence of infection, resulting in a strong, unwanted immune response against self [180] . To prevent that from happening, constitutively expressed DEAD/DEAH-box helicase sensors, such as DDX41 [72] , DHX9 [89] and DHX33 [87] , may mediate the first wave of innate immune signaling and interferon production during infections, only later to be replaced by the interferon-stimulated RLRs and cGAS. Since most of the DEAD/DEAH-box helicases in innate immunity have only been discovered in the latest decade, their regulators and interacting partners are still mostly unknown. Finding those proteins would aid us in gaining a better insight into the pathways in which DEAD/DEAH-box helicases are known to participate. This is particularly intriguing for DDX3 [48, 49] and DDX21 [60, 158, 163, 165] , as both helicases have shown to achieve two opposite effects through their regulatory activities, which possibly depends on the actual cellular conditions. DEAD/DEAH-box helicases are involved in a number of different cellular pathways. Even in the innate immunity, these helicases participate in different pathways, such as STING-, MAVS-, TRIF-, and MyD88-mediated innate immune signaling, inflammasome activation and assembly of cytoplasmic granules. Interestingly, several DEAD/DEAH-box helicases are already known or are thought to play roles in multiple immune pathways. For example, DDX3 is involved in both RLR-MAVS signaling [44À47] and formation of cytoplasmic granules [50, 51] ; DDX24 is a negative regulator of both RLR-MAVS signaling [65] and tumor suppressor p53 signaling [66] , which has recently also been implied in innate immunity [67, 68] ; DDX25 is a suppressor of NF-κB and IRF3/ IRF7 signaling [119] , meaning that it is a common negative regulator of all innate immune signaling pathways that converge on these transcription factors; DDX41 senses cytosolic DNA, CDNs and, possibly, retroviral reverse transcripts [72À74], and thus may contribute to the alreadyobserved crosstalk between STING and RLR-MAVS signaling pathways [181] . DHX9 is an DNA [88] and RNA sensor [89] , as well as a factor in assembly of RISC and RNA interference [90] , another potential aspect of antiviral response. Such pathway interconnectivity would be essential for more efficient and concerted innate immune response. Apart from innate immunity, DEAD/DEAH-box helicases participate in many other cellular events, ranging from RNA metabolism to cell-cycle regulation and transcriptional coregulation. With this in mind, we speculate that the activity of DEAD/DEAH-box helicases in innate immunity might cause concomitant effects on the other side of their functional spectrum, as proposed with DDX21 in this chapter. It would be very interesting to identify the broader connection between these distinct pathways, as well as means of their mutual regulation. Innate immunity has well-established roles in immune surveillance and cancer immunoediting [182, 183] . STING signaling pathway, type I IFNs and proinflammatory cytokines are implicated in a number of tumors, where they exhibit both pro-and anticancer functions [126, 184, 185] . Moreover, cancers are often associated with chronic inflammation and inflammasomes [186, 187] . Although this makes the involvement of DEAD/DEAH-box helicases in cancer highly possible, the innate immunity-related roles of DEAD/DEAH-box helicases in cancer restriction and pathogenesis have not been investigated yet. However, a large number of DEAD/DEAH-box helicases have been implied in cancer [188, 189] . Poor regulation of innate immune signaling, including STING and MAVS pathways, is known to cause inflammatory diseases and autoimmune diseases [119, 190] . Interestingly, indications of involvement of DEAD/DEAH-box helicases in autoimmune diseases have already been reported, albeit scarcely [147, 148] . Nonetheless, none of those studies focused on mechanisms through which DEAD/DEAH-box helicases participate in inflammation and autoimmune diseases in the context of innate immunity. DEAD-box proteins as RNA helicases and chaperones DEAD-box proteins: the driving forces behind RNA metabolism DEAD-box helicases (DDX) Gene Family | HUGO Gene Nomenclature Committee DEAH-box helicases (DHX) Gene Family | HUGO Gene Nomenclature Committee From unwinding to clamping À the DEAD box RNA helicase family DEAD-box helicases as integrators of RNA, nucleotide and protein binding DEAD-box RNA helicases as transcription cofactors Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation DEAD box protein DDX1 regulates cytoplasmic localization of KSRP RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-β-catenin signaling A CK1 FRET biosensor reveals that DDX3X is an essential activator of CK1ε Toll-like receptors Intracellular detection of viral nucleic acids Sweeten PAMPs: role of sugar complexed PAMPs in innate immunity and vaccine biology DAMPs from cell death to new life C-type lectins in immunity: recent developments NOD-like recept.ors: versatile cytosolic sentinels Recognition of cytosolic DNA by cGAS and other STING-dependent sensors Viral RNA detection by RIG-I-like receptors DNA recognition in immunity and disease Innate immune recognition of DNA: a recent history Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response New insights into the structural basis of DNA recognition by HINa and HINb domains of IFI16 AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway STING regulates intracellular DNA-mediated, type I interferondependent innate immunity NEMO-IKKβ Are Essential for IRF3 and NF-κB Activation in the cGAS-STING Pathway How RIG-I like receptors activate MAVS MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif MAVS activates TBK1 and IKKε through TRAFs in NEMO dependent and independent manner Innate immune sensing and signaling of cytosolic nucleic acids DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1 A DEAD-box helicase mediates an RNA structural transition in the HIV-1 rev response element The cellular RNA helicase DDX1 interacts with coronavirus nonstructural protein 14 and enhances viral replication Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription DDX1 promotes proliferation of the JC virus through transactivation of its promoter DDX3 functions in antiviral innate immunity through translational control of PACT DEAD/H BOX 3 (DDX3) helicase binds the RIG-I adaptor IPS-1 to up-regulate IFN-beta-inducing potential Human DEAD box helicase 3 couples IκB kinase ε to interferon regulatory factor 3 activation DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response DEAD)-box RNA helicase 3 modulates NF-κB signal pathway by controlling the phosphorylation of PP2A-C subunit The DEAD-box RNA helicase DDX3 interacts with NF-κB subunit p65 and suppresses p65-mediated transcription Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response Antiviral innate immunity and stress granule responses Human cytomegalovirus induces the interferon response via the DNA sensor ZBP1 DDX3 DEAD-box RNA helicase is a host factor that restricts hepatitis B virus replication at the transcriptional level RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP RNA helicase DDX3: at the crossroad of viral replication and antiviral immunity Hepatitis C virus core protein abrogates the DDX3 function that enhances IPS-1-mediated IFN-beta induction Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3 DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain DAMP molecule S100A9 acts as a molecular pattern to enhance inflammation during influenza A virus infection: role of DDX21-TRIF-TLR4-MyD88 pathway Cellular DDX21 RNA helicase inhibits influenza A virus replication but is counteracted by the viral NS1 protein Autogenous translational regulation of the Borna disease virus negative control factor X from polycistronic mRNA using host RNA helicases A survey of DDX21 activity during Rev/RRE complex formation DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling Negative regulation of the p300-p53 interplay by DDX24 The amazing innate immune response to influenza A virus infection Long story short: p53 mediates innate immunity The RNA helicase DDX46 inhibits innate immunity by entrapping m(6)A-demethylated antiviral transcripts in the nucleus The emerging roles of the DDX41 protein in immunity and diseases DDX60L is an interferon-stimulated gene product restricting hepatitis C virus replication in cell culture The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response Nucleic acid recognition orchestrates the anti-viral response to retroviruses IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling Bruton's tyrosine kinase phosphorylates DDX41 and activates its binding of dsDNA and STING to initiate type 1 interferon response The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA The complement anaphylatoxins C5a and C3a suppress IFN-β production in response to Listeria monocytogenes by inhibition of the cyclic dinucleotide-activated cytosolic surveillance pathway Positive feedback regulation of type I IFN production by the IFN-inducible DNA sensor cGAS DDX60, a DEXD/H box helicase, is a novel antiviral factor promoting RIG-I-like receptor-mediated signaling DDX60 is involved in RIG-I-dependent and independent antiviral responses, and its function is attenuated by virusinduced EGFR activation Respiratory virusinduced EGFR activation suppresses IRF1-dependent interferon λ and antiviral defense in airway epithelium Epidermal growth factor receptor is essential for Toll-like receptor 3 signaling The long noncoding RNA NEAT1 exerts antihantaviral effects by acting as positive feedback for RIG-I signaling The DHX33 RNA helicase senses cytosolic RNA and activates the NLRP3 inflammasome The E3 ubiquitin ligase tripartite motif 33 is essential for cytosolic RNA-induced NLRP3 inflammasome activation The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells Aspartate-glutamatealanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells Antiviral innate immune response of RNA interference RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions DHX9/RHA binding to the PBS-segment of the genomic RNA during HIV-1 assembly bolsters virion infectivity Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication Human interactome of the influenza B virus NS1 protein DHX15 senses double-stranded RNA in myeloid dendritic cells The DEAH-box RNA helicase DHX15 activates NF-κB and MAPK signaling downstream of MAVS during antiviral responses Nlrp6 regulates intestinal antiviral innate immunity Tumor suppressor RBM5 directly interacts with the DExD/H-box protein DHX15 and stimulates its helicase activity The G-patch protein NF-κB-repressing factor mediates the recruitment of the exonuclease XRN2 and activation of the RNA helicase DHX15 in human ribosome biogenesis DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation DDX6 represses aberrant activation of interferon-stimulated genes Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly DDX6 (Rck/p54) is required for efficient hepatitis C virus replication but not for internal ribosome entry site-directed translation Modulation of hepatitis C virus RNA accumulation and translation by DDX6 and miR-122 are mediated by separate mechanisms Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5 0 untranslated region of hepatitis C virus RNA HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3 0 UTR structures Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction Integrative genomics-based discovery of novel regulators of the innate antiviral response Host cell interactome of HIV-1 Rev includes RNA helicases involved in multiple facets of virus production DDX17 promotes the production of infectious HIV-1 particles through modulating viral RNA packaging and translation frameshift Identification of RNA helicases in human immunodeficiency virus 1 (HIV-1) replication -a targeted small interfering RNA library screen using pseudotyped and WT HIV-1 Host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins Identification of novel pathway partners of p68 and p72 RNA helicases through Oncomine meta-analysis DEAD-box helicase DDX25 is a negative regulator of type I interferon pathway and facilitates RNA virus infection Helicase proteins DHX29 and RIG-I cosense cytosolic nucleic acids in the human airway system A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae Antiviral immune responses: triggers of or triggered by autoimmunity? Structural and functional analyses of human DDX41 DEAD domain Structural and Functional Analysis of DDX41: a bispecific immune receptor for DNA and cyclic dinucleotide Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms Myeloid neoplasms with germline DDX41 mutation STING signaling in tumorigenesis and cancer therapy: a friend or foe? The Ded1/DDX3 subfamily of DEAD-box RNA helicases DDX3 localizes to the centrosome and prevents multipolar mitosis by epigenetically and translationally modulating p53 expression The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis, and viral infection The kinase CK1ε controls the antiviral immune response by phosphorylating the signaling adaptor TRAF3 DDX3 interacts with influenza A virus NS1 and NP proteins and exerts antiviral function through regulation of stress granule formation Pivotal role of RNAbinding E3 ubiquitin ligase MEX3C in RIG-I-mediated antiviral innate immunity BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A RNA helicase A mediates association of CBP with RNA polymerase II RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus RNA helicase A is necessary for translation of selected messenger RNAs A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs WRN helicase unwinds Okazaki fragment-like hybrids in a reaction stimulated by the human DHX9 helicase The RNA helicase DHX9 establishes nucleolar heterochromatin, and this activity is required for embryonic stem cell differentiation Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) Autoantibodies to RNA helicase A: a new serologic marker of early lupus Sex influences eQTL effects of SLE and Sjögren's syndrome-associated genetic polymorphisms Cloning and characterization of a putative human RNA helicase gene of the DEAH-box protein family The human La (SS-B) autoantigen interacts with DDX15/hPrp43, a putative DEAH-box RNA helicase Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of pre-rRNA processing factors Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly NF-κB repressing factor downregulates basal expression and mycobacterium tuberculosis induced IP-10 and IL-8 synthesis via interference with NF-κB in monocytes Nucleolar localization and mobility analysis of the NF-kappaB repressing factor NRF The crystal structure of human DEAH-box RNA helicase 15 reveals a domain organization of the mammalian DEAH/RHA family DHX15 is associated with poor prognosis in acute myeloid leukemia (AML) and regulates cell apoptosis via the NF-kB signaling pathway RNA helicase DDX21 coordinates transcription and ribosomal RNA processing SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability Human DDX21 binds and unwinds RNA guanine quadruplexes Isolation of human mitotic protein phosphatase complexes: identification of a complex between protein phosphatase 1 and the RNA helicase Ddx21 The nuclear DEK interactome supports multi-functionality Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers In-cell western assays to evaluate Hantaan virus replication as a novel approach to screen antiviral molecules and detect neutralizing antibody titers SLERT Regulates DDX21 Rings Associated with Pol I Transcription TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury From promoting to inhibiting: diverse roles of helicases in HIV-1 replication MDM2 mediates nonproteolytic polyubiquitylation of the DEAD-Box RNA helicase DDX24 Extracellular vesicles including exosomes regulate innate immune responses to hepatitis B virus infection Integrative analyses reveal novel strategies in HPV11, -16 and -45 early infection Altered RIG-I/DDX58-mediated innate immunity in dermatomyositis Subsite-specific association of DEAD box RNA helicase DDX60 with the development and prognosis of oral squamous cell carcinoma Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses Gene expression profiling of the response to interferon beta in Epstein-Barr-transformed and primary B cells of patients with multiple sclerosis Knockdown of DDX46 inhibits proliferation and induces apoptosis in esophageal squamous cell carcinoma cells The DHX33 RNA helicase promotes mRNA translation initiation DHX33 transcriptionally controls genes involved in the cell cycle Identification of DHX33 as a mediator of rRNA synthesis and cell growth A diverse range of gene products are effectors of the type I interferon antiviral response Discrimination of cytosolic self and non-self RNA by RIG-I-like receptors Crosstalk between cytoplasmic RIG-I and STING sensing pathways Cancer immunoediting by the innate immune system in the absence of adaptive immunity Innate immune recognition of cancer New perspectives on type I IFNs in cancer Chronic inflammation and cytokines in the tumor microenvironment Inflammasomes in cancer: a doubleedged sword DEAD box RNA helicases: crucial regulators of gene expression and oncogenesis Wanted DEAD/H or alive: helicases winding up in cancers Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication This work was supported by the National Nature Science Foundation of China grants 31770948, 31570775, 31560727 and 81501353. We thank Fujian Normal University for the financial support. The authors also wish to express their gratitude to Darija Domijan and Denzel Amado Silva for their assistance and advice on language and readability of this review.