Duck IFIT5 differentially regulates Tembusu virus replication and inhibits virus-triggered innate immune response.

Mammalian interferon-induced protein with tetratricopeptide repeats family proteins (IFITs) play important roles in host innate immune response to viruses. Recently, studies have shown that IFIT from poultry also plays a crucial part in antiviral function. This study first reports the regulation of duck Tembusu virus (DTMUV) replication by IFIT5 and the effect of duck IFIT5 (duIFIT5) on the innate immune response after DTMUV infection. Firstly, duIFIT5 was obviously increased in duck embryo fibroblast cells (DEFs) infected with DTMUV. Compared to the negative control, we found that in the duIFIT5-overexpressing group, the DTMUV titer at 24 h post infection (hpi) was significantly reduced, but the viral titer was strikingly increased at 48 hpi. Moreover, overexpression of duIFIT5 could significantly inhibit IFN-ß transcription and IFN-ß promoter activation at indicated time points after DTMUV infection. Further, in DTMUV-infected or poly(I:C)-stimulated DEFs, overexpression of duIFIT5 also significantly inhibited the activation of NF-κB and IRF7 promoters, as well as the activation of downstream IFN induced the interferon-stimulated response element (ISRE) promoter. Meanwhile, the transcription level of antiviral protein Mx, but not OASL, was obviously decreased at various time points. The opposite results were obtained by knockdown of duIFIT5 in DTMUV-infected or poly(I:C)-stimulated DEFs. Compared to the negative control, knockdown of duIFIT5 promoted DTMUV titer and DTMUV envelope (E) protein expression at 24 hpi, but DTMUV titer and E protein expression was markedly decreased at 48 hpi. Additionally, the promoters of IFN-ß, NF-κB, IRF7 and ISRE were significantly activated in the duIFIT5 knockdown group. Collectively, duIFIT5 differentially regulates DTMUV replication and inhibits virus-triggered innate immune response.

Interferon-induced protein with tetratricopeptide repeats 5 of black fruit bat (Pteropus alecto) displays a broad inhibition of RNA viruses.

Bats are natural host reservoirs and have adapted a unique innate immune system that permits them to host many viruses without exhibiting symptoms. Notably, bat interferon stimulated genes (ISGs) have been shown to play antiviral roles. Interferon induced protein with tetratricopeptide repeats 5 (IFIT5) is a well-characterised ISG in humans with antiviral activities against negative-sense RNA viruses via inhibiting viral transcription. Here, we aim to investigate if Pteropus alecto (pa) IFIT5 (paIFIT5) possess the ability to inhibit negative-sense RNA viruses. Initially, gene syntenic and comparative structural analyses of multiple animals highlighted a high level of similarity between Pteropus alecto and human IFIT5 proteins. Our results showed that paIFIT5 was significantly inducible by viral and dsRNA stimulation. Transient overexpression of paIFIT5 inhibited the replication of vesicular stomatitis virus (VSV). Using minireplicon and transcription reporter assays, we demonstrated the ability of paIFIT5 specifically to inhibit H17N10 polymerase activity. Mechanistically, we noticed that the antiviral potential of paIFIT5 against negative sense RNA viruses was retributed to its interaction with 5'ppp containing RNA. Taken together, these findings highlight the genetic and functional conservation of IFIT5 among mammals.

Goose IFIT5 positively regulates goose astrovirus replication in GEF cells.

Interferon-induced protein with tetratricopeptide repeats (IFITs), a family of proteins strongly induced by type I interferon (IFN-I), are deeply involved in many cellular and viral processes. IFIT5, the sole protein in this family found in birds, also plays a crucial role in regulating virus infection. In this study, goose IFIT5 (gIFIT5) was first cloned from peripheral blood lymphocyte (PBL) and phylogenetic analysis showed that it was highly homologous with duck IFIT5 (dIFIT5), sharing 94.6% identity in amino acid sequence. Subsequently, the expression kinetics of gIFIT5 during goose astrovirus (GAstV) infection and the regulatory effect of gIFIT5 on GAstV proliferation were evaluated. Results showed that the mRNA and protein expression level of gIFIT5 was greatly induced by GAstV infection, especially at 12 hpi. Importantly, gIFIT5 could conversely promote GAstV replication in GEF cells. Virus titers in gIFIT5 overexpression group were significantly higher than those in control group at 12 and 24 hpi. Western blot and quantitative real-time PCR (qRT-PCR) further demonstrated that the production of viral cap protein was significantly facilitated in gIFIT5-transfected group. Collectively, GAstV facilitates self-replication via promoting gIFIT5 expression.

IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways.

Humans have a distinct combination of IFIT (IFN-induced protein with tetratricopeptide repeats) family orthologs, including IFIT1 (ISG56), IFIT2 (ISG54), IFIT3 (ISG60), and IFIT5 (ISG58). The function of IFIT1/IFIT2/IFIT3 has been intensively investigated. However, little is known about the role of IFIT5 in any cellular processes. In this study, we reported that both the mRNA and protein levels of IFIT5 are up-regulated in response to RNA virus infection or polyinosinic-cytidylic acid stimulation. Ectopic expression of IFIT5 could synergize IRF3- and NF-κB-mediated gene expression, whereas knockdown of IFIT5 impairs the transcription of these genes. Consistently, anti-viral responses of host cells are significantly increased or decreased in the presence or absence of IFIT5. Mechanistically, IFIT5 co-localizes partly with mitochondria and interacts with RIG-I and MAVS. Our study identified that IFIT5 is an important enhancer in innate immune response.

Screening of interferon-stimulated genes against avian reovirus infection and mechanistic exploration of the antiviral activity of IFIT5.

Avian reovirus (ARV) infection can lead to severe immunosuppression, complications, and secondary diseases, causing immense economic losses to the poultry industry. In-depth study of the mechanism by which the innate immune system combats ARV infection, especially the antiviral effect mediated by interferon, is needed to prevent and contain ARV infection. In this study, ARV strain S1133 was used to artificially infect 7-day-old specific pathogen-free chickens. The results indicated that ARV rapidly proliferated in the immune organs, including the spleen, bursa of Fabricius, and thymus. The viral load peaked early in the infection and led to varying degrees of pathological damage to tissues and organs. Real-time quantitative PCR revealed that the mRNA levels of interferon and multiple interferon-stimulated genes (ISGs) in the spleen, bursa of Fabricius, and thymus were upregulated to varying degrees in the early stage of infection. Among the ISGs, IFIT5, and Mx were the most upregulated in various tissues and organs, suggesting that they are important ISGs for host resistance to ARV infection. Further investigation of the role of IFIT5 in ARV infection showed that overexpression of the IFIT5 gene inhibited ARV replication, whereas inhibition of the endogenously expressed IFIT5 gene by siRNA promoted ARV replication. IFIT5 may be a positive feedback regulator of the innate immune signaling pathways during ARV infection and may induce IFN-α production by promoting the expression of MAD5 and MAVS to exert its antiviral effect. The results of this study help explain the innate immune regulatory mechanism of ARV infection and reveal the important role of IFIT5 in inhibiting ARV replication, which has important theoretical significance and practical application value for the prevention and control of ARV infection.

The driver role of JAK-STAT signalling in cancer stemness capabilities leading to new therapeutic strategies for therapy- and castration-resistant prostate cancer.

BACKGROUND: Lineage plasticity in prostate cancer (PCa) has emerged as an important mechanism leading to the onset of therapy- and castration-resistant PCa (t-CRPC), which is closely associated with cancer stem cell (CSC) activity. This study is to identify critical driver(s) with mechanism of action and explore new targeting strategy. METHODS: Various PCa cell lines with different genetic manipulations were subjected to in vitro prostasphere assay, cell viability assay and in vivo stemness potential. In addition, bioinformatic analyses such as Ingenuity pathway and Gene Set Enrichment Analysis were carried out to determine clinical relevance. The in vivo anti-tumour activity of JAK or STAT1 inhibitors was examined in clinically relevant t-CRPC model. RESULTS: We demonstrated the role of interferon-related signalling pathway in promoting PCa stemness, which correlated with significant elevation of interferon related DNA damage resistance signature genes in metastatic PCa. Inhibition of JAK-STAT1 signalling suppresses the in vitro and in vivo CSC capabilities. Mechanistically, IFIT5, a unique downstream effector of JAK-STAT1 pathway, can facilitate the acquisition of stemness properties in PCa by accelerating the turnover of specific microRNAs (such as miR-128 and -101) that can target several CSC genes (such as BMI1, NANOG, and SOX2). Consistently, knocking down IFIT5 in t-CRPC cell can significantly reduce in vitro prostasphere formation as well as decrease in vivo tumour initiating capability. CONCLUSIONS: This study provides a critical role of STAT1-IFIT5 in the acquisition of PCSC and highlights clinical translation of JAK or STAT1 inhibitors to prevent the outgrowth of t-CRPC.

mRNA expression and functional analysis of chicken IFIT5 after infected with Newcastle disease virus.

Innate immunity is the first line against the invasion of pathogenic microorganisms. Over the past several years, the antiviral activity and mechanisms of the IFIT5 gene have been confirmed in mammals. However, more information is needed on the role of IFIT5 in response to viral infection in chickens. In this study, we examined the mRNA expression profile of chicken IFIT5 (chIFIT5) in different tissues and explored how chIFIT5 transduces upstream signaling to the downstream adaptor. Relative mRNA expression level of chIFIT5 was the highest in spleen and expression level of chIFIT5 was significantly up-regulated following Newcastle disease virus (NDV) infection, and polyinosinic:polycytidylic acid [poly (I:C)]- and poly(deoxyadenylic-thymidylic) [poly (dA:dT)]-triggered antiviral immune responses. Chicken MDA5, MAVS, and IRF7 positively regulated the mRNA expression of chIFIT5. Overexpression of chIFIT5 could promote IRF7- and NF-κB-mediated gene expression following NDV infection or transfection with poly (I:C). These results suggested that chIFIT5 is an important enhancer of the innate immunity response.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

Molecular cloning and mRNA expression of IFIT5 in tissues of ducklings infected with virulent duck hepatitis A virus type 3.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings.

IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells.

Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6 h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, these results suggest a possible role of IFIT5 in the antiviral response of RBCs against VHSV.

Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets.

Teleost red blood cells (RBCs) are nucleated and therefore can propagate cellular responses to exogenous stimuli. RBCs can mount an immune response against a variety of fish viruses, including the viral septicemia hemorrhagic virus (VHSV), which is one of the most prevalent fish viruses resulting in aquaculture losses. In this work, RBCs from blood and head kidney samples of rainbow trout challenged with VHSV were analyzed via transcriptomic and proteomic analyses. We detected an overrepresentation of differentially expressed genes (DEGs) related to the type I interferon response and signaling in RBCs from the head kidney and related to complement activation in RBCs from blood. Antigen processing and presentation of peptide antigen was overrepresented in RBCs from both tissues. DEGs shared by both tissues showed an opposite expression profile. In summary, this work has demonstrated that teleost RBCs can modulate the immune response during an in vivo viral infection, thus implicating RBCs as cell targets for the development of novel immunomodulants.

Interferon-induced IFIT5 promotes epithelial-to-mesenchymal transition leading to renal cancer invasion.

Interferon is known as a pleiotropic factor in innate immunity, cancer immunity and therapy. Despite an objective short-term response of interferon (IFN) therapy in renal cell carcinoma (RCC) patients, the potential adverse effect of IFN on RCC cells is not fully understood. In this study, we demonstrate that IFNs can enhance RCC invasion via a new mechanism of IFIT5-mediated tumor suppressor microRNA (miRNA) degradation resulted in the elevation of Slug and ZEB1 and epithelial-to-mesenchymal transition (EMT). Clinically, a significant upregulation of IFNγ signaling pathway (such as IFNGR1, IFNGR2, STAT1 and STAT2) is observed in RCC patients with metastatic disease. Overall, this study provides a new mechanism of action of IFN-elicited canonical pathway in regulating suppressor miRNAs. Most importantly, it highlights the potential pro-metastatic effect of IFNs, which could undermine the clinical applicability of IFNs for treating RCC patients.

Genotypes of IFIH1 and IFIT5 in seven chicken breeds indicated artificial selection for commercial traits influenced antiviral genes.

Innate immunity is the first line against the invasion of pathogenic microorganisms. Previous reports only demonstrated production traits of commercial importance were often negatively correlated with innate disease resistance. However, whether different purpose of artificial selection influences innate immunity have not been understood. In this study, we cloned exon1, exon6 of IFIH1 and exon2 of IFIT5 by molecular biology techniques in seven different chicken breeds to detect the potential effect of artificial selection for commercial traits on disease resistance for the first time. In total, 8 single nucleotide polymorphisms(SNPs) of IFIH1 gene exon1 and exon6, 19 SNPs of IFIT5 gene exon2 were detected. We found all native chicken breeds had a relatively close relationship to broiler breeds but a remote relationship to layer breed. A great difference between CB and LLH with different selected purpose were observed. The allele frequencies of these two positive antiviral genes were associated with different purpose of artificial selection. Our experiment constituted the foundation for the interaction between commercial traits and immune trait.

IFIT5 positively regulates NF-κB signaling through synergizing the recruitment of IκB kinase (IKK) to TGF-ß-activated kinase 1 (TAK1).

Precise regulation of NF-κB signaling pathways is essential to effective host immune response. However, the specific molecular mechanism underlying NF-κB activation by different stimuli is not fully understood. Here we demonstrate that IFIT5, one of the interferon induced tetratricopeptide repeat family members, enhances IKK phosphorylation and NF-κB activation through interacting with TAK1 and IKK. Following TNF-α treatment, IFIT5 interacted with TAK1 or IKK complex and synergized the recruitment of IKK to TAK1 in a dose dependent manner. Consistent with these observations, knockdown of IFIT5 decreased the recruitment of IKK to TAK1 and markedly weakened IKK phosphorylation, further reducing the production of NF-κB target genes IL-8 and ICAM-1. Moreover, we found that IFIT5 also promoted SeV-induced IKK phosphorylation and NF-κB activation by regulating the recruitment of IKK to TAK1. Our findings identify a previously unrecognized role of IFIT5 as a positive regulator in IKK phosphorylation and NF-κB activation, highlighting that IFIT5 serves as an important mediator in innate immunity.