A murrel interferon regulatory factor-1: molecular characterization,gene expression and cell protection activity

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27 % α-helices (85 aa residues), 5.7 % β-sheets (19 aa residues) and 67.19 % random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (?284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.     

Role of interferon regulatory factor-1 in double-stranded RNA-induced iNOS expression by mouse islets.

Environmental factors, such as viral infection, have been implicated as potential triggering events leading to the initial destruction of pancreatic beta cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, has been shown in combination with interferon-gamma (IFN-gamma) to stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide production and to inhibit beta cell function. Interferon regulatory factor-1 (IRF-1), the activation of which is induced by dsRNA, viral infection, and IFN-gamma, regulates the expression of many antiviral proteins, including PKR, type I IFN, and iNOS. In this study, we show that IRF-1 is not required for dsRNA + IFN-gamma-stimulated iNOS expression and nitric oxide production by mouse islets. In contrast to islets, dsRNA + IFN-gamma fails to induce iNOS expression or nitric oxide production by macrophages isolated from IRF-1(-/-) mice; however, dsRNA + IFN-gamma induces similar levels of IL-1 release by macrophages isolated from both IRF-1(-/-) and IRF-1(+/+) mice. Importantly, we show that dsRNA- or dsRNA + IFN-gamma-stimulated IRF-1 expression by mouse islets and peritoneal macrophages is independent of PKR. These results indicate that IRF-1 is required for dsRNA + IFN-gamma-induced iNOS expression and nitric oxide production by mouse peritoneal macrophages but not by mouse islets. These findings suggest that dsRNA + IFN-gamma stimulates iNOS expression by two distinct PKR-independent mechanisms; one that is IRF-1-dependent in macrophages and another that is IRF-1-independent in islets.     

Induction of antiviral state in fish cells by Japanese flounder, Paralichthys olivaceus, interferon regulatory factor-1

Interferon regulatory factor-1 (IRF-1) mediates an antiviral state in cells by regulating the expression of the interferon (IFN-alpha/beta) system. To elucidate the role of IRF-1 in fish during virus infections, we constructed a recombinant plasmid of the Japanese flounder, Paralichthys olivaceus IRF-1 (JF IRF-1) under the control of the cytomegalovirus (CMV) immediate/early enhancer promoter. The antiviral mechanism of JF IRF-1 was studied using transfection experiments in a homologous cell line. Here, we show that cell supernatants obtained from transiently transfected cells enhanced cell viability of a heterologous cell line upon incubation, reduced the titers of hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), and possessed cytokine-like activity, as shown by their ability to protect cells against virus infections. The supernatants also inhibited the replication of the rhabdoviruses during the early stages of infection as indicated by the reduction of viral titers in the presence of the supernatants obtained from the transfected cells. Further analysis showed that the cell culture supernatants contain cytokine-like substances that possess acid-labile and temperature-resistant properties. These results indicate that JF IRF-1 induces an antiviral state in cells by mediating the production of cytokine-like substances. Thus, JF IRF-1 might be useful as an adjuvant in the development of DNA vaccines against commercially important viral pathogens in Japanese flounder aquaculture.     

Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20-40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106-140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or "docking" of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase.