Ming Xian Chang

Origin and evolution of the RIG-I like RNA helicase gene family

BackgroundThe DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria.ResultsThe DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis.ConclusionThe RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Expression and functional characterization of the RIG-I like receptors MDA5 and LGP2 in rainbow trout Oncorhynchus mykiss

ABSTRACT The retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) comprise three homologues: RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). They activate the host interferon (IFN) system upon recognition of viral RNA pathogen-associated molecular patterns (PAMPs) in the cytoplasm. Bioinformatic analysis of the sequenced vertebrate genomes suggests that the cytosolic surveillance system is conserved in lower vertebrates, and recent functional studies have confirmed that RIG-I is important to fish antiviral immunity. In this study, we have identified MDA5 and LGP2 homologues from rainbow trout Oncorhynchus mykiss and an additional LGP2 variant with an incomplete C-terminal domain of RIG-I. Trout MDA5 and LGP2 were constitutively produced in fibroblast and macrophage cell lines and upregulated by poly(I:C), recombinant IFN, or infection by RNA viruses (viral hemorrhagic septicemia virus and salmon alphavirus) with a single-stranded positive or negative genome. Overexpression of MDA5 and LGP2 but not of the LGP2 variant resulted in significant accumulation of Mx transcripts in cultured cells, which correlated with a marked enhancement of protection against viral infection. These results demonstrate that both MDA5 and LGP2 are important RLRs in host surveillance against infection of both negative and positive viruses and that the LGP2 variant with a deletion of 54 amino acids at the C terminus acts as a negative regulator for LGP2-elicited antiviral signaling by competing for the viral RNA PAMPs. Interestingly, MDA5 expression was not affected by overexpressed LGP2 in transfected cells and vice versa, suggesting that they likely act in parallel as positive regulators for IFN production.

Identification of an additional two-cysteine containing type I interferon in rainbow trout Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts

Multiple type I interferons (IFNs) have recently been identified in salmonids, containing two or four conserved cysteines. In this work, a novel two-cysteine containing (2C) IFN gene was identified in rainbow trout. This novel trout IFN gene (termed IFN5) formed a phylogenetic group that is distinct from the other three salmonid IFN groups sequenced to date and had a close evolutionary relationship with IFNs from advanced fish species. Our data demonstrate that two subgroups are apparent within each of the 2C and 4C type I IFNs, an evolutionary outcome possibly due to two rounds of genome duplication events that have occurred within teleosts. We have examined gene expression of the trout 2C type I IFN in cultured cells following stimulation with lipopolysaccharide, phytohaemagglutinin, polyI:C or recombinant IFN, or after transfection with polyI:C. The kinetics of gene expression was also studied after viral infection. Analysis of the regulatory elements in the IFN promoter region predicted several binding sites for key transcription factors that potentially play an important role in mediating IFN5 gene expression.

Cloning and expression of Toll-like receptors 1 and 2 from a teleost fish, the orange-spotted grouper Epinephelus coioides

The two Toll-like receptors, TLR1 and TLR2 were cloned from orange-spotted grouper, Epinephelus coioides, an important teleost fish in mariculture of Asia. The cDNA sequences of TLR1 and TLR2 are 3195 and 3439 bp long, with an open reading frame (ORF) of 2406 and 2466 bp, encoding 801 and 821 amino acids, respectively. The TLR family motifs, i.e. leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains are conserved in the TLR1 and TLR2, with nine and ten leucine-rich repeat (LRR) domains, and with one TIR domain, respectively. The TLR1 and TLR2 had a constitutive expression in examined organs/tissues of naïve orange-spotted grouper, and an increased expression of TLR1 and TLR2 at mRNA level was observed in immune organs such as in spleen of LPS and Poly(I:C) stimulated fish. An increased expression of TLR1 and TLR2 was also recorded in immune organs of the fish injected with the bacterial pathogen, Vibrio alginolyticus. Similarly, a significant rise in the expression of MyD88, an adaptor molecule which forms signalling complex with intracellular TIR domain, thus leading to the production of pro-inflammatory cytokines, such as IL-1β, was also observed in the LPS- and Poly(I:C)-stimulated, and V. alginolyticus-infected fish, indicating the possible role of TLR1 and TLR2 in the MyD88 signalling pathway. However, the mechanism involved in the increased expression of TLR1 and TLR2 following LPS and Poly(I:C) stimulation is at present unknown in fish, and further research should be carried out to identify ligands of fish TLR1 and TLR2 in order to understand the function of these receptors.

Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants, trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61% similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The expression of trNOD2a/b was modulated by the inflammatory stimulant interferon-γ (IFN-γ) or interleukin-1β (IL-1β). Overexpression of trout NOD2 effector domains resulted in induced expression of proinflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and IL-8, the antibacterial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II IFN. These results suggest that fish NOD2 functions in inflammatory events, possibly via NF-κB activation, regulation of apoptosis, and triggering of antibacterial and antiviral defences.

Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant

Melanoma differentiation‐associated gene 5 (MDA5) is one of the three members in the retinoic acid‐inducible gene I‐like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up‐regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram‐negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over‐expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co‐transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

Intracellular Interferons in Fish: A Unique Means to Combat Viral Infection

We demonstrate for the first time in vertebrates, that alternative splicing of interferon (IFN) genes can lead to a functional intracellular IFN (iIFN). Fish IFN genes possess introns and in rainbow trout three alternatively spliced transcripts of the IFN1 gene exist. Two of the encoded IFNs are predicted to lack a signal peptide. When overexpressed these iIFNs induce antiviral responses. Variants of the two IFNR receptor chains (IFNAR1 and IFNAR2) lacking a signal peptide are also present in trout. Transfection of HEK 293T cells with the iIFN and iIFNR molecules results in STAT phosphorylation and induction of antiviral genes. These results show that fish possess a functioning iIFN system that may act as a novel defence to combat viral infection.

Sequence and Expression Analysis of Interferon Regulatory Factor 10 (IRF10) in Three Diverse Teleost Fish Reveals Its Role in Antiviral Defense

Background Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. Molecular Characterization of IRF10 in Three Fish Species In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5’-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. Expression Analysis of IRF10 In Vivo and In Vitro Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5′ untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites’ polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine–cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

MAVS splicing variants contribute to the induction of interferon and interferon-stimulated genes mediated by RIG-I-like receptors

The mitochondrial antiviral signaling protein (MAVS) plays a key role in the signal transduction of RIG-I-like receptors (RLRs)-mediated antiviral response. In the present study, zebrafish MAVS transcript variants, namely MAVS_tv1 and MAVS_tv2, were cloned from zebrafish embryos. The putative MAVS_tv1 protein (full length form) contains an N-terminal CARD domain, a central proline region, and a C-terminal transmembrane domain (TM). MAVS_tv2 is generated by a 190 bp intron fragment insertion. The putative MAVS_tv2 protein lacked TM domain due to a frame shift, with the N-terminal 303 aa residues identical to MAVS_tv1, and no sequence homology for the C-terminal 41 aa residues. Real-time PCR showed that the expression of MAVS_tv1 in ZF4 cells was higher than that of MAVS_tv2, and MAVS variants were induced by Edwardsiella tarda and SVCV infection during the early time points of infection, whereas MAVS_tv1 unchanged or MAVS_tv2 decreased at a later time point after the infection, respectively. Overexpression of MAVS_tv1 and MAVS_tv2 in fish cells conferred antiviral resistance, and activated zebrafish IFN1 and IFN3 promoters. MAVS_tv1 overexpression induced a slow (48 hpf) increased expression of IFN1, mxa, mxb, mxe and RSAD2. In contrast, MAVS_tv2 overexpression increased rapidly and transiently the expression of IFN1, IFN2, IFN3, mxc and rsad2 at 6 or 24 hpf. The simultaneous overexpression of MAVS variants and RIG-I in zebrafish embryos led to an accumulative induction of IFNs and IFN-stimulated genes including IFN1, IFN4, mxc, mxe and rsad. Furthermore, MAVS_tv1 cooperated with RIG-I in the accumulation of RIG-I transcript in a positive feedback loop; MAVS_tv2 synergized with MDA5 in the accumulation of MAVS_tv2 transcript. Collectively, these data suggest the molecular mechanisms of fish MAVS variants in antiviral immunity.