Background:The 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.

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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.

Dear Editor, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems are RNA-guided adaptive immune systems in prokaryotes. 1,2 Class 2 CRISPR-Cas systems (including type II, V, and VI) involve large single effector proteins in complex with crRNA for interference. 3,4 The type II and V effectors, such as Cas9 and Cas12a, have been engineered into powerful tools for genome editing. The type VI system encompasses RNA-guided RNases. Its effectors Cas13a, Cas13b and Cas13d are capable of both precursor CRISPR RNA (pre-crRNA) processing and target RNA cleavage, which protect the host from phage attacks. 5-7 Once bound to a target RNA, they are activated, switching on a non-specific RNase activity. Moreover, they have been utilized to target and edit RNA as programmable RNAbinding modules. 6,[8][9][10][11][12] Although related to Cas13a and Cas13d, Cas13b possesses many distinctive features. These include the lack of significant sequence similarity with Cas13a and Cas13d, disparate crRNA repeat region, double-sided protospacer flanking sequence (PFS)-dependent target RNA cleavage. [5][6][7][8]13 To investigate how Cas13b processes pre-crRNA, recognizes crRNA and settles the spacer nucleotides for target recognition, we solved the crystal structure of Bergeyella zoohelcum Cas13b (BzCas13b) in complex with its crRNA at 2.79 Å resolution (Supplementary information, Table S1). The binary complex was obtained by the SeMet-derived BzCas13b R1177A mutant co-expressed with CRISPR template in vivo. The architecture of BzCas13b assumes a triangular domain distribution around the central L-shaped crRNA ( Fig. 1a-e; Supplementary information, Movie S1). In the binary complex, Helical-1, HEPN-1 and HEPN-2 domains together form one side of the triangular structure. Helical-1 domain comprises six α-helices connected with random loops (Supplementary information, Fig. S1). The second side of the triangle is formed by RRI-1 (the repeat region interacting domain-1), RRI-2 domains and the linker region. RRI-1 domain can be subdivided into two separate motifs (RRI-1 I and II) that stack onto each other. Both motifs contain a short twostranded, antiparallel β-sheet flanked by five α-helices. RRI-2 domain includes a long central two-stranded, antiparallel β-sheet flanked by two α-helices, and a short central two-stranded, antiparallel β-sheet flanked by three α-helices. The linker region consists of random loops that connect two short α-helices, which shows multiple interactions with RRI-2 domain. Helical-2 domain is composed of nine α-helices and its rather long helix-23 extends in parallel with crRNA, thereby forming the third side of the triangle. Helix-8 of Helical-1 domain and helix-23 of Helical-2 domain protrude out of the complex in a crab claw-like manner to clamp the spacer region of crRNA (Supplementary information, Fig. S1). In addition, HEPN-1 domain bridges Helical-1 and Helical-2 domains.A mature 52-nt crRNA, originated from a co-expressed CRISPR encoding sequence and bein...

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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.