Structural and expression analysis of golden pompano Trachinotus ovatus IRF5 and its role in regulation of type I IFN.

The interferon regulatory factor 5 (IRF5) is a mediator of the type I IFN signalling pathways, thereby playing a key role in innate immunity. However, the detailed mechanism through which IRF5 regulates type I IFN in fish remains unclearly. In the present study, we first describe the identification of IRF5 (ToIRF5) from golden pompano (Trachinotus ovatus) and its features at the genomic sequence and expression level. The genomic DNA sequence consists of eight exons and seven introns. The full-length ToIRF5 cDNA is composed of 2, 059 bp and encodes for 499 amino acid polypeptides. The putative protein sequence shares 66.3%-82.9% identity to fish IRF5 and possesses three representative conserved domains (a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus) and one highly variable domain (middle region (MR)). Furthermore, the ToIRF5 transcript is constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. The mRNA levels of ToIRF5 are increased by polyinosinic: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin stimulation in the immune- and nonimmune-related tissues. The subcellular localization indicates that ToIRF5 is mainly localized in the cytoplasm with or without poly (I: C) induction. In addition, to explore whether ToIRF5 is a modulator of ToIFNa3, promoter analysis is performed. The region from -200 bp to +1 bp is identified as the core promoter by different truncated mutants of ToIFNa3. Mutation analyse declares that the activity of the ToIFNa3-5 promoter significantly decreases after targeted mutation of M2 binding sites. Moreover, overexpression of ToIRF5 in vitro memorably aggrandizes the expression of some IFN/IRF-based signalling pathway genes. These results provide new insights into the roles of teleost IRF5 in transcriptional mechanisms of type I IFN in the immunity process.

Functional characterization of interferon regulatory factor 2 and its role in the transcription of interferon a3 in golden pompano Trachinotus ovatus (Linnaeus 1758).

Similar to mammals, fish possess interferon (IFN) regulatory factor 2 (IRF2)-dependent type I IFN responses. Nevertheless, the detailed mechanism through which IRF2 regulates type I IFNa3 remains largely unknown. In the present study, we first identified two genes from golden pompano (Trachinotus ovatus), IRF2 (ToIRF2) and IFNa3 (ToIFNa3), in the IFN/IRF-based signalling pathway. The open reading frame (ORF) sequence of ToIRF2 encoded 335 amino acids possessing four typical characteristic domains, including a conserved DNA-binding domain (DBD), an interferon association domain 2 (IAD2), a transcriptional activation domain (TAD), and a transcriptional repression domain (TRD). Furthermore, transcripts of ToIRF2 were significantly upregulated after stimulation by polyinosinic: polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS) and flagellin in immune-related tissues (blood, liver, and head-kidney). Moreover, to investigate whether ToIRF2 was a regulator of ToIFNa3, promoter analysis was performed. The results showed that the region from -896 bp to -200 bp is defined as the core promoter using progressive deletion mutations of IFNa3. Additionally, ToIRF2 overexpression led to a clear time-dependent enhancement of ToIFNa3 promoter expression in HEK293T cells. Mutation analyses indicated that the activity of the ToIFNa3 promoter significantly decreased after targeted mutation of M4/5 binding sites. Electrophoretic mobile shift assays (EMSAs) verified that IRF2 interacted with the binding site of the ToIFNa3 promoter region to regulate ToIFNa3 transcription. Last, the promoter activity of ToIFNa3-2 was more responsive to treatment with poly (I:C) than LPS and flagellin. Furthermore, overexpression of ToIRF2 in vitro obviously increased the expression of several IFN/IRF-based signalling pathway genes after poly (I:C) abduction. In conclusion, the present study provides the first evidence of the positive regulation of ToIFNa3 transcription by ToIRF2 and contributes to a better understanding of the transcriptional mechanisms of ToIRF2 in fish.

IFN regulatory factor 3 of golden pompano and its NLS domain are involved in antibacterial innate immunity and regulate the expression of type I interferon (IFNa3).

Introduction: The transcription factor interferon regulatory factor 3 (IRF3) plays an important role in host defence against viral infections. However, its role during bacterial infection in teleosts remains unclear. In the present study, we evaluated the antibacterial effects of Trachinotus ovatus IRF3 (TroIRF3) and how it regulates type I interferon (IFN). Methods: Subcellular localisation experiments, overexpression, and quantitative real-time PCR (qRT-PCR) were performed to examine the nuclear localisation signal (NLS) of TroIRF3 and its role in the antibacterial regulatory function of TroIRF3. We assessed the binding activity of TroIRF3 to the IFNa3 promoter by luciferase reporter assay. Results and Discussion: The results showed that TroIRF3 was constitutively expressed at high levels in the gill and liver. TroIRF3 was significantly upregulated and transferred from the cytoplasm to the nucleus after Vibrio harveyi infection. By overexpressing TroIRF3, the fish were able to inhibit the replication of V. harveyi, whereas knocking it down increased bacterial replication. Moreover, the overexpression of TroIRF3 increased type I interferon (IFNa3) production and the IFN signalling molecules. The NLS, which is from the 64-127 amino acids of TroIRF3, contains the basic amino acids KR74/75 and RK82/84. The results proved that NLS is required for the efficient nuclear import of TroIRF3 and that the NLS domain of TroIRF3 consists of the key amino acids KR74/75 and RK82/84. The findings also showed that NLS plays a key role in the antibacterial immunity and upregulation of TroIFNa3 induced by TroIRF3. Moreover, TroIRF3 induces TroIFNa3 promoter activity, whereas these effects are inhibited when the NLS domain is deficient. Overall, our results suggested that TroIRF3 is involved in the antibacterial immunity and regulation of type I IFN in T. ovatus and that the NLS of TroIRF3 is vital for IRF3-mediated antibacterial responses, which will aid in understanding the immune role of fish IRF3.