The composition and unrevealed immune role of non-RLR DExD/H box RNA helicases in fish

Highlight

• •Fifty-two homologous genes of non-RLR DExD/H box RNA helicases were identified in fish.
• •DDX3a, DDX3b, DHX9, DDX41, DDX43 regulate positively anti-viral/bacterial immunity in fish.
• •DDX1, DDX3a, DDX5, DDX19, DDX23 regulate negatively anti-infection immunity in fish.

Abstract

The large superfamily of DExD/H box RNA helicases are involved in various life processes, such as RNA metabolism, transcriptional regulation, translation, tumorigenesis, cell cycle, and viral infection, and RIG-like receptors (RLRs) in the superfamily have been confirmed with crucial roles in viral infection by recognizing viral RNA. In this review, non-RLR DExD/H box RNA helicases were possibly summarized in relation with their composition and antiviral immune responses in teleost, with at least 52 genes in the helicase subfamily. Compared with mammals, most DExD/H box RNA helicases in teleost are conservatively present, but some are present in a fish-specific gene duplication manner or have been lost in evolution, which may imply the obvious compositional difference in fish. The comparison in innate immune roles of mammalian and teleost DExD/H box RNA helicases indicated that DDX3a, DDX3b, DHX9, DDX41, DDX43 are functional positively in anti-viral/bacterial immunity in fish, while DDX1, DDX3a, DDX5, DDX19, DDX23 negatively in the immunity. In the future, the composition, possible immune recognition, signalling and interaction with other immune pathways, as well as other biological functions are all of significant importance for research, which may contribute to the development of disease prevention and control strategies in aquaculture.

Introduction

DNA and RNA helicases exist widely in life organisms and are classified into six superfamilies (SF1–6) based on structural and functional features [1]. Most members of DExD/H (Asp-Glu-x-Asp/His) box RNA helicases, which belong to the large SF2 with eight shared conserved motifs (I, Ia, Ib, II, III, IV, V and VI), are involved in ATP-binding, ATP hydrolysis, nucleic acid substrate binding and intermolecular interaction [2,3]. These helicases are grouped into different families according to characteristic sequences in Motif II with DEAD, DEAH, DExH or DExD [2]. DExD/H box RNA helicases are involved in multiple biological processes, including RNA metabolism, transcriptional regulation, translation, tumorigenesis, cell cycle, and viral infection [[4], [5], [6], [7]]. RIG-like receptors (RLRs) as the typical DExD/H box RNA helicases are known to be important cytosolic sensors of viral RNA. Mammal RLR family is consist of three members, retinoic acid-inducible gene I (RIG-I, or DEAD box polypeptide 58, DDX58), melanoma differentiation-associated gene 5 (MDA5, or interferon (IFN) induced with helicase C domain 1, IFIH1), and laboratory of genetics and physiology 2 (LGP2, or DExH box polypeptide 58, DHX58) [8]. All RLR members have DExD/H box RNA helicase domain and C-terminal domain (CTD); however, caspase activation and recruitment domains (CARDs) are only found at N-terminal region of RIG-I and MDA5, which are associated with mitochondria antiviral signalling protein (MAVS) to activate TANK binding kinase 1 (TBK1) - IFN regulatory factor 3/7 (IRF3/7) signalling pathway for the production of type I IFNs [8,9].

In fact, non-RLR DExD/H box RNA helicases have been shown to play critical roles in viral infection [4]. Human non-RLR DExD/H box RNA helicase family contains 56 members, while 52 genes have been identified in teleost fish, except ddx25, ddx50, ddx53, and ddx60 [10,11]. To date, roles of non-RLR DExD/H box RNA helicases have been reported in relation with development, cell-cycle progression, ribosomal RNA synthesis, sex differentiation, haematopoiesis, growth switch, and viral replication in teleost fish [[12], [13], [14], [15], [16]]. In consideration of the rapid development of fish immunology and its importance in aquaculture [17], the current understanding on the function of non-RLR DExD/H box RNA helicases in teleost fish was reviewed in comparison with the knowledge reported in mammals in order to highlight future perspectives.

DDX1

DDX1 is a unique DEAD-box RNA helicase due to its helicase core region with SPRY domain and is involved in RNA processing and viral replication. Mammalian DDX1 is utilized by some viruses, including human immunodeficiency virus 1 (HIV-1), hepatitis C virus (HCV), coronaviruses (CoVs) and John Cunningham virus (JCV) for replication by binding with viral RNA or proteins [18]. However, it has been reported that DDX1 can recognize dsRNA, such as polyinosinic-polycytidylic acid (poly I:C), viral nucleic acid of influenza A virus (IAV) and reoviruses, via helicase domain to interact with DDX21 and DHX36 to trigger type I IFN and NF-κB pathways [19]. Unsurprisingly, teleost DDX1 has been identified to possess functions to promote replication of some aquatic viruses. In mandarin fish (Siniperca chuatsi), DDX1 can bind with viral mRNAs to enhance the replication of infectious spleen and kidney necrosis virus (ISKNV), and is also involved in the promotion of Ca²⁺ influx mediated by transient receptor potential vanilloid 4 (TRPV4) - DDX1 axis [20]. In other species of fish, such as zebrafish (Danio rerio) and common carp (Cyprinus carpio), DDX1 homologue is also present, with conserved locus synteny and sequence features in containing DEAD-like helicase superfamily domain (DEXDc) and helicase superfamily C-terminal domain (HELICc) as reported from fish to human [21] (Fig. 1, Fig. 2). Interestingly, the expression of fish ddx1 gene is upregulated following viral infections [21], but the positive regulatory role of DDX1 in type I IFN signalling is unknown in fish.

Fig. 1.

Schematic diagram of domains of DDX1, DHX9, DDX21, DHX36, DDX41 and DDX60. Domains were predicted using the SMART program and Conserved Domain Database (CDD). Abbreviations for domains used in this figure are GUCT (NUC152) domain, dsRNA binding motifs (DSRMs), DEAD-like helicases superfamily (DEXDc), helicase superfamily C-terminal domain (HELICc), helicase associated domain (HA2) and oligonucleotide/oligosaccharide-binding (OB)-fold (OB_NTP_bind).

Fig. 2.

Gene synteny of ddx1 (A), dhx9 (B), ddx21 (C), dhx36 (D) and ddx41 (E). Arrows indicate gene transcription orientation. The gene information is derived from NCBI database, including Homo sapiens (human, assembly version: GRCh38.p14), Latimeria chalumnae (coelacanth, assembly version: fLatCha1.pri), Lepisosteus oculatus (spotted gar, assembly version: LepOcu1), Danio rerio (zebrafish, assembly version: GRCz11), Esox lucius (northern pike, assembly version: ASM72191v2), Oreochromis niloticus (Nile tilapia, assembly version: O_niloticus_UMD_NMBU), Oryzias latipes (Japanese medaka, assembly version: ASM31367v1), Takifugu rubripes (torafugu, assembly version: FUGU5) and Callorhinchus milii (elephant shark, assembly version: IMCB_Cmil_1.0).

DDX3

DDX3 is a highly conserved DEAD-box protein in all eukaryotes [22]. Human DDX3 contains two copies, DDX3X and DDX3Y, which are located separately on chromosomes X and Y [18]. DDX3X, which is usually called DDX3, has a wider tissue distribution and is involved in RNA metabolism, stress response, apoptosis, cell cycle regulation, tumorigenesis, and viral infection [6,18,[22], [23], [24], [25], [26]]. In antiviral response, DDX3 can bind to viral RNA and promote type I IFN production in association with RIG-I, MDA5, MAVS, TRAF3 and TBK1/IKKε [[27], [28], [29]]. DDX3 is also involved in the regulation of NF-κB signalling and NLRP3 inflammasome formation [18]. On the other hand, as an antiviral regulation molecule, DDX3 is usually hijacked by viruses to escape host antiviral immune defense. For example, hepatitis B virus (HBV) polymerase can block the interaction of DDX3 and IKKε/TBK1 for immune evasion [18]. On the contrary, it is also reported that DDX3 as a host factor is required for the replication of viruses, such as HCV and HIV-1 [6,30,31].

Like in mammals, two ddx3 genes (ddx3a and ddx3b) are reported in teleost, which may be originated from teleost-specific genome duplication [32]. In orange-spotted grouper (Epinephelus coioides), DDX3 should be the homologue of DDX3X or DDX3a, which is responsive to viral infection and can inhibit the replication of red-spotted grouper nervous necrosis virus (RGNNV) by inducing the expression of antiviral-related genes in an IRF-dependent manner [33]. Similarly, DDX3 (DDX3a) in rainbow trout (Oncorhynchus mykiss) was found to show antiviral activity against the infection of infectious hematopoietic necrosis virus (IHNV) [34]. In comparison with mammals, fish DDX3 is also reported to have a dual function in inhibiting or facilitating virus replication. For example, channel catfish (Ictalurus punctatus) DDX3X (DDX3a) may contribute to the stability of snakehead vesiculovirus (SHVV) phosphoprotein, thus facilitating the virus replication in CCO cells [35]. In addition, fish DDX3b with ubiquitous constitutive expression shows dsRNA binding activity and also antiviral effect against salmonid alphavirus (SAV) as reported in salmonid fishes [32,36,37]. Thus, the different roles of DDX3 during viral infection in mammals and teleost may indicate the possible different roles of DDX3 in host-virus interactions, which should be of interest for further investigations.

DDX5

Biological functions of DDX5 (also known as p68) have been reported in respects to cell cycle regulation, tumorigenesis, apoptosis, cell proliferation and differentiation, RNA metabolism, and viral infection [[38], [39], [40]]. During the infection of different viruses, DDX5 may be functional as an inhibitor or a helper [40]. For instance, DDX5 shows antiviral activity by decreasing RNA transcription from HBV minichromosome, and may also have a negative effect on antiviral immunity through the promotion of methylation of antiviral genes [40]. However, whether DDX5 is involved in the regulation of IFN signalling is largely unknown in vertebrates. Interestingly, the fish DDX5 was recently reported to be able to suppress the RLR-mediated type I IFN production by degrading TBK1, and also by disrupting the interaction between TRAF3 and TBK1 to prevent the nuclear translocation of IRF3 [41].

DHX9

DHX9 with the DEIH motif belongs to DExH-box helicases and has roles in many cellular processes, including DNA replication, transcription, translation, RNA processing, and genome stability [42]. It is also reported that DHX9 may be essential for host defense against viral infection [43], as it can bind to dsRNA, such as poly I:C, and interact with MAVS to activate NF-κB and IRF3 in myeloid dendritic cells (mDCs) [44]. However, in plasmacytoid dendritic cells (pDCs), DHX9 may sense C-phosphate-G (CpG)-B DNA and recruit MyD88 to promote NF-κB signalling and the production of TNF-α and IL-6 in response to DNA viruses [45]. Interestingly, DHX9 as a transcriptional regulator is directly associated with STAT1 to enhance type I IFN/STAT1-mediated expression of IFN-stimulated genes (ISGs) through recruiting Pol II to the ISG promoter region [43]. To date, the homologous gene of dhx9 has been identified in some fish species, such as zebrafish, common carp, rainbow trout and medaka (Oryzias latipes) [21,36,46], and has conserved features in gene locus and sequence (Fig. 1, Fig. 2). It has been confirmed that the dhx9 gene can be markedly induced by dsRNA (poly I:C) and RNA viruses, including spring viraemia of carp virus (SVCV) and Chum salmon reovirus (CSV) [21,36]. In fact, rainbow trout DHX9 was proved to possess dsRNA binding ability based on pull-down assay [36]. Interestingly, the transcription levels of type I IFNs and ISGs were attenuated by the knockdown of DHX9 in response to the DNA virus, herpes simplex virus 1 (HSV-1), although it is not a piscine virus [47]. It is thus likely that fish DHX9 may have conserved functions in antiviral immunity.

DDX19

DDX19 has been confirmed to be involved in translation, transcription, genome stability and virus infection [[48], [49], [50], [51]]. Mammalian DDX19 was reported as a negative regulator of RLR-mediated type I IFN production by disrupting the interaction between TBK1 / IKKε / IRF3 and promoting the degradation of TBK1 and IKKε [51]. Expectedly, fish DDX19 can negatively regulate RLR-mediated type I IFN signalling and promote SVCV replication [52], and it is reported that fish DDX19 can target IRF3-induced antiviral response and IFN / ISG expression, but not IRF7, providing new understanding for the negative regulation mechanism of DDX19 in vertebrate [52].

DDX23

DDX23 is known to be associated with cancer progression, genome stability and innate antiviral immunity [[53], [54], [55], [56], [57]]. Human DDX23 can bind to LMW poly(I:C) and may act as a dsRNA sensor to inhibit virus replication and activate NF-κB / IRF3 by forming a complex with TRIF or MAVS [55]. However, DDX23 was reported to negatively regulate antiviral response in black carp (Mylopharyngodon piceus) [58], and to interact with MAVS to suppress MAVS-mediated expression of type I IFN and antiviral activity against SVCV [58].

DDX41

DDX41 has been reported as a sensor to recognize intracellular DNA and cyclic di-GMP (c-di-GMP) / cyclic di-AMP (c-di-AMP) in response to viral and bacterial infections [59,60]. The binding with DNA may facilitate the interaction between DDX41 and STING to induce the phosphorylation of TBK1, and the activation of NF-κB / IRF3 and the production of type I IFN [61]. Today, the conserved ddx41 gene has been identified in several species of fish species, including grass carp (Ctenopharyngodon idellus), oliver flounder (Paralichthys olivaceus), Nile tilapia (Oreochromis niloticus), mandarin fish, yellowtail clownfish (Amphiprion clarkia), zebrafish, and orange-spotted grouper [[62], [63], [64], [65], [66], [67], [68], [69]]. Fish ddx41 gene expression can be induced by stimulation / infection of pathogenic analogues or viruses / bacteria, such as poly I:C, poly (dA:dT), grass carp reovirus (GCRV), mandarin fish ranavirus (MRV), Singapore grouper iridovirus (SGIV), red-spotted grouper nervous necrosis virus (RGNNV), lipopolysaccharide (LPS), and Vibrio harveyi [62,64,66,68,69]. Like in mammals, DDX41 in fish as a conserved cytosolic DNA sensor may interact with STING / MITA to induce type I IFN signalling and inhibit virus replication [47,62,64,66,67]. On the other hand, the role of DDX41 in orange-spotted grouper DDX41 was reported in positive regulation of MAVS- and TBK1-mediated IFN response, but not STING / MITA-induced IFN signalling in unstimulated cells [69]. In zebrafish, DDX41 contributes to STING / MITA-STAT6-mediated chemokine (CCL20) production against the invasion of pathogenic bacteria, Aeromonas hydrophilia or Edwardsiella tarda [65].

Other members

DDX6, DHX15, DHX16, DHX29 and DDX60 act as the cytosolic nucleic acids co-sensor of RIG-I to enhance RLR-mediated type I IFN signalling [[70], [71], [72], [73]]. However, the dsRNA sensor DHX33 can interact with MAVS to promote type I IFN production in a RIG-I / MDA5-independent manner against viral infection [74]. DDX21 can regulate ribosomal RNA biogenesis, cancer progression, host innate immunity, tissue differentiation and viral replication [[75], [76], [77], [78], [79]]. In mDCs, DDX21 as a linker protein can interact with DDX1 and DHX36 to promote type I IFN response via TRIF pathway [19]. DHX36 was demonstrated to resolve G-quadruplex structures (G4s) on DNA and RNA in several studies [[80], [81], [82]]. It was reported that DHX36 can interact with RIG-I to enhance its signalling by inducing the formation of PKR-mediated antiviral stress granule [83]. DHX36 can also recognize dsDNA and CpG-DNA to activate downstream IRF7 signalling [61]. Interestingly, DDX4 enhances antiviral function of type I IFN by disrupting the interaction of USP7 / SOCS1 and facilitating the degradation of SOCS1 [84]. Other viral inhibitors include DDX20, DHX30, DDX42 and DDX49 [[85], [86], [87], [88]].

In fish, some DExD/H-box RNA helicase genes, such as ddx4 (vasa), ddx6, dhx15 and dhx30, are reported to be involved in sex differentiation, haematopoiesis or organ development [[13], [14], [15],89,90], but their possible roles in immune functions are seldomly reported. Surprisingly, homologous gene of mammalian DDX50, as an antiviral factor [91,92], is missing in the conserved fish ddx21 loci (Fig. 2). Although the expression of fish ddx21 and dhx36 were upregulated following viral infection [21], the function of these helicases in antiviral immune response needs further investigation. In addition, mammalian DDX43 is known to be involved in nucleic acid unwinding, tumorigenesis, and chromatin remodelling [[93], [94], [95]]. It is reported that in tilapia, DDX43 is associated with MAVS and TRIF to induce IFN-β in HEK-293T cells [96].

Unexpectedly, the ddx60 gene was found absent from the genomes of all analyzed fish species [10]. In fact, it is rather impossible to identify ddx60 homologous gene in teleost, despite of the existence of genes around DDX60 locus in shark or human (Fig. 3). In shark, coelacanth and human, DDX60 locus is conserved at upstream of SPARC / osteonectin, cwcv and kazal-like domains proteoglycan (testican) 3 (SPOCK3), and the syntenic regions, including cluster anxa10-tll1 and gpr78-hmx1 were also found in spotted gar genome (Fig. 3). However, the two genomic clusters are not arranged adjacent to each other, when compared with the cluster in shark (Fig. 3). It is assumed that chromosomal rearrangement event might have occurred, leading to the loss of ddx60 during the evolution of teleost.

Fig. 3.

Comparative analysis of genes around DDX60 loci in elephant shark, spotted gar, coelacanth and human. Arrows indicate gene transcription orientation. The homologous genes are linked in color region.

Moreover, DDX25 can regulate negatively type I IFN production by targeting RLR pathway [97]. DDX46 inhibits antiviral innate immunity by entrapping the transcripts of antivirus-related genes in nucleus [98]. On the other hand, other DExD/H-box RNA helicases, including DDX10, DDX11, DDX18, DDX28, DDX52 and DDX56 in mammals, have been reported to promote viral replication [[99], [100], [101]]. Contradictorily, during virus infection, DDX17, DDX24 and DDX39A may be functional in either promoting or inhibiting viral replications [86,[102], [103], [104], [105], [106]]. In fish, it is reported that homologous genes, such as DDX11, DDX18, DDX46 and DDX52, are required for organ development, cell-cycle progression, ribosomal RNA synthesis, or growth switch [16,[107], [108], [109]], but their possible immune functions are largely unexplored.

Conclusion

It is now confirmed that teleost possesses at least 52 homologous genes of non-RLR DExD/H box RNA helicases, most of which have conserved protein sequences, although some members, such as ddx3, ddx50, ddx60, may have undergone fish-specific gene duplication or loss. Available information has shown that fish non-RLR DExD/H box RNA helicases may have conserved immunological as well as biological functions in virus replication as observed in mammals (Fig. 4). However, the understanding on non-RLR DExD/H box RNA helicases in fish is rather limited, and mostly in relation to their identification and possible interaction with RLR-signalling. In the future, the composition, possible immune recognition, signalling and interaction with other immune pathways, as well as other biological functions should all be of significant importance for research, which may contribute to the development of disease prevention and control strategies in aquaculture.

Fig. 4.

The functions of fish non-RLR DExD/H box RNA helicases in virus replication and bacterial proliferation.

CRediT authorship contribution statement

Shan Nan Chen: Writing – review & editing, Writing – original draft, Validation, Investigation, Formal analysis, Data curation, Conceptualization. Xue Yun Peng: Writing – original draft, Validation, Resources, Formal analysis, Data curation. Pin Nie: Project administration, Resources, Conceptualization, Writing – review & editing, Funding acquisition, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author, Pin Nie, is an Editorial Board Member for [Developmental and Comparative Immunology] and was not involved in the editorial review or the decision to publish this article.

Data availability

• Data will be made available on request.