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Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2 , serpinc1 , serpinh1a , and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish. • Grass carp KLF2A targets the activation of serpinc1. • KLF2A and serpinc1 are involved in regulating virus-induced interferon responses. • KLF2A upregulates serpinc1 , and suppresses GCRV replication. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5ʹ-untranslated region (UTR) of 52 bp, a 3′-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses. • The molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp. • The CiPrx2 mRNA was ubiquitously expressed in the various tissues. • CiPrx2 was likely to be involved in the immune response against viral and bacterial infection. • Subcellular localization of CiPrx2-GFP fusion protein were only distributed in the cytoplasm. • CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity and protect DNA against oxidative damage. [ABSTRACT FROM AUTHOR]

Abstract Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A , RNF19B , RNF1 44AA, RNF1 44AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, in-between-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))-induced inflammatory cytokines production, including IFN-I , TNF-α , IL-6 , and transcription factor IRF3 , which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes. Highlights • Six RING-between-RING (RBR) genes from Ctenopharyngodon idellus were cloned for the first time. • RNF144AA , RNF144AB , and RNF144B responded substantially after GCRV challenge. • Poly (I: C)-induced proinflammatory cytokines were up-regulated by RNF144B silencing. • The expression of RNF144B in liver, especially in intestine was intensely up-regulated at 1 day post–infection. [ABSTRACT FROM AUTHOR]

The mammalian sterile 20-like (MST) family, which belongs to the serine/threonine protein kinase superfamily, has five members that can be found in mammals: STK3 (also called MST2), STK4 (MST1), STK24 (MST3), STK25 (YSK1 or SOK1), and STK26 (MST4). The MST kinases have key roles in apoptosis, immune regulation, inflammatory responses, cancer, and cell proliferation in mammals, whereas the roles and transcriptional regulatory mechanism of these kinases in teleost fish are still unclear. In this study, four STK genes ( CiSTK3 , CiSTK24 , CiSTK25 , and CiSTK26 ) were cloned and analyzed in grass carp ( Ctenopharyngodon idella ). All four STK genes were broadly expressed in the examined tissues, while their relative expression levels differed. In addition, after exposure to the grass carp reovirus, mRNA expression levels of the four STK genes were altered to different levels in the immune organs, and the levels were dramatically altered in the blood. Subcellular localization indicated that all four STK proteins were localized in the cytoplasm of transfected cells. Moreover, bimolecular fluorescence complementation analysis revealed that mouse protein-25 could interact with CiSTK3, CiSTK24, CiSTK25, and CiSTK26 independently in grass carp. Thus, our findings provide new insights for understanding the functions of the MST family in teleosts. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. [Copyright &y& Elsevier]