Interleukin-2 Enhancer Binding Factor 2 Research Articles

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3′—untranslated region (3′—UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3′—UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3′—UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Protein regulation strategies of the mouse spleen in response to Babesia microti infection

BackgroundBabesia is a protozoan parasite that infects red blood cells in some vertebrates. Some species of Babesia can induce zoonoses and cause considerable harm. As the largest immune organ in mammals, the spleen plays an important role in defending against Babesia infection. When infected with Babesia, the spleen is seriously injured but still actively initiates immunomodulatory responses.MethodsTo explore the molecular mechanisms underlying the immune regulation and self-repair of the spleen in response to infection, this study used data-independent acquisition (DIA) quantitative proteomics to analyse changes in expression levels of global proteins and in phosphorylation modification in spleen tissue after Babesia microti infection in mice.ResultsAfter mice were infected with B. microti, their spleens were seriously damaged. Using bioinformatics methods to analyse dynamic changes in a large number of proteins, we found that the spleen still initiated immune responses to combat the infection, with immune-related proteins playing an important role, including cathepsin D (CTSD), interferon-induced protein 44 (IFI44), interleukin-2 enhancer-binding factor 2 (ILF2), interleukin enhancer-binding factor 3 (ILF3) and signal transducer and activator of transcription 5A (STAT5A). In addition, some proteins related to iron metabolism were also involved in the repair of the spleen after B. microti infection, including serotransferrin, lactoferrin, transferrin receptor protein 1 (TfR1) and glutamate-cysteine ligase (GCL). At the same time, the expression and phosphorylation of proteins related to the growth and development of the spleen also changed, including protein kinase C-δ (PKC-δ), mitogen-activated protein kinase (MAPK) 3/1, growth factor receptor-bound protein 2 (Grb2) and P21-activated kinase 2 (PAK2).ConclusionsImmune-related proteins, iron metabolism-related proteins and growth and development-related proteins play an important role in the regulation of spleen injury and maintenance of homeostasis. This study provides an important basis for the diagnosis and treatment of babesiosis.Graphical

Interleukin-2 enhancer binding factor 2 (ILF2) in pacific white shrimp (Litopenaeus vannamei): Alternatively spliced isoforms with different responses in the immune defenses against vibrio infection

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates the functions of interleukin-2 (IL-2) at the levels of transcription, splicing and translation, and plays other critical roles in the immune system. ILF2 is well-documented in vertebrates, while little is currently known in crustacean species such as the Pacific white shrimp (Litopenaeus vannamei). In the present study, five cDNA for spliced isoforms of Lv-ILF2 were identified, in which four of them are the full-length long isoforms (Lv-ILF2-L1, Lv-ILF2-L2, Lv-ILF2-L3 and Lv-ILF2-L4) and one of them is a truncated short isoform (Lv-ILF2-S). The whole sequence of ILF2 gene from L. vannamei was obtained, which is 11,680 bp in length with 9 exons separated by 8 introns. All five isoforms contain a domain associated with zinc fingers (DZF). Two alternative splicing types (alternative 5′ splice site and alternative 3′ splice site) were identified in the five isoforms. The Lv-ILF2 mRNA showed a broad distribution in all detected tissues, and the Lv-ILF2-L transcript levels were higher than those of Lv-ILF2-S in corresponding tissues. The mRNA levels of Lv-ILF2-S in the hepatopancreas, heart, muscle and stomach, but not in the eyestalk, were significantly increased after challenges with Vibrio harveyi or lipopolysaccharide (LPS), while no significant changes were observed for the transcript levels of Lv-ILF2-L in these tissues under the same immune stimulants. On the contrary, the transcript levels of neither Lv-ILF2-S nor Lv-ILF2-L were affected by challenges of polyinosinic: polycytidylic acid [Poly (I:C)]. In addition, after knockdown of the Lv-ILF2 mRNA level by siRNA, the mortality of shrimp and the hepatopancreatic bacterial numbers were significantly increased under V. harveyi challenge, indicating that Lv-ILF2 might participate in the immune defenses against V. harveyi invasion. Collectively, our study here supplied the first evidence for a novel splicing mechanism of ILF2 transcripts, and provided a functional link between the Lv-ILF2 isoforms and the capacity against pathogenic Vibrio in penaeid shrimp.

Effects of dietary fructooligosaccharide on the growth, antioxidants, immunity and disease resistance of Chinese mitten crab

This study aimed to investigate the effects of dietary fructooligosaccharide (FOS) on the growth, antioxidant capability, immune response and disease resistance of Chinese mitten crab Eriocheir sinensis. Groups of crab (17.74±0.39g) were distributed into 18 cement pools (33 crabs per pool) with replicates and fed experimental diets supplemented with FOS at 0, 0.5, 1.0, 1.5, 2.0 and 2.5g/kg (designated as control, TF1, TF2, TF3, TF4 and TF5, respectively). After 60days of feeding trial, crabs were challenged by Aeromonas hydrophila and the survival was recorded for the next 96h. The results indicated that crabs fed TF4 had significantly higher weight gain (WG) than crabs fed control diet, whereas WG decreased with FOS level increasing to 2.5g/kg. There was no significant difference (P>0.05) in SGR among all experimental groups. Catalase (CAT) and superoxide dismutase (SOD) activities in crabs fed TF4 diet were significantly higher than those in control group, while malondialdehyde (MDA) concentrations in crabs fed TF4 diet were significantly lower than that in control group. Crabs fed TF4 diet showed significantly higher alkaline phosphatase (AKP) activity compared to the control group. There was a trend that acid phosphatase (ACP) activity was elevated with FOS levels increasing up to 2.0g/kg and decreased subsequently, and crabs fed TF4 diet showed significantly higher ACP activity compared to the control group. Supplementation of 2.0g/kg FOS up-regulated the mRNA expression of interleukin-2 enhancer binding factor 2 (ILF2) and peroxinectin (PX), and down-regulated the mRNA expression of lipopolysaccharide-induced TNF-α factor (LITAF). Bifidobacteriales abundance in the digestive tract increased significantly with dietary FOS levels increasing from 1.5 to 2.5g/kg. Crabs fed TF4 showed significantly higher Bacteroides abundance compare to the other groups. After Aeromonas hydrophila challenge, the highest relative percent survival (25.00%) was observed in crabs fed TF4 diet, which was significantly higher than that in control group. Taken together, 2.0g/kg FOS supplementation might be effective in improving growth performance, antioxidant capability, immune response and disease resistance of Chinese mitten crab.

Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication.MethodsThe interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively.ResultsThe interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells.ConclusionOur findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.

ILF2-YB1 Protein Interaction Modulates RNA Splicing to Induce Resistance to Chemotherapy in High Risk Multiple Myeloma

The 1q21 amplification, which occurs in approximately 40% of de novo and 70% of relapsed MM, is among the most frequent chromosomal aberrations in multiple myeloma (MM) patients and is considered a very high-risk genetic feature that is especially correlated with disease progression and drug resistance.To uncover novel 1q21 MM-critical genes, we first identified a list of 78 potential 1q21 drivers, which were located in the minimal common region of amplification of 254 MM samples and showed copy number-driven expression. These 78 candidates were then subjected to an shRNA screen to identify those genes involved in selective death and/or growth inhibition of MM cells carrying the 1q21 amplification.Using this approach, we identified and functionally validated the Interleukin-2 enhancer binding factor 2 (ILF2) as one of key 1q21 amplification-specific genes.ILF2 downregulation in 1q21-amplified MM cells resulted in multinucleated phenotypes and abnormal nuclear morphologies, findings that are consistent with the DNA damage-induced genomic instability that is associated with DNA repair defects that occur during cellular replication. Correspondingly, ILF2 downregulation was associated with a significant increase in the activation of the ATM (but not ATR or DNA-PK) pathway and accumulation of gH2AX foci, which are indicative of double-strand DNA breaks, and resulted in caspase 3-mediated apoptosis. Therefore, we sought to determine whether ILF2 is involved in the genome damage repair that occurs during cellular replication. To this end, we evaluated whether ILF2 depletion could affect the efficiency of non-homologous end joining (NHEJ) or homologous recombination (HR), the two major repair pathways in mammalian cells. We observed a profound impairment of HR in ILF2-depleted cells (p=0.038), whereas NHEJ was unaltered after ILF2 downregulation. Conversely, enforced ILF2 expression significantly enhanced HR efficiency in MM cells (p=0.008). To further support the role of ILF2 in the regulation of the DNA repair pathway in MM cells, we evaluated whether ILF2 downregulation increased MM sensitivity to DNA-damaging agents routinely used in the treatment of MM. Employing the interstrand crosslinker melphalan as an instigator of double-strand DNA breaks, we found that ILF2-depleted MM cells subjected to continuous melphalan treatment showed increased accumulation of γH2AX and apoptosis. Consistent with these findings, elevated ILF2 expression significantly correlated with poor survival in MM patients treated with high-dose melphalan followed by tandem autologous transplantation (n=256, p=0.01).Mechanistically, mass spectrometry analysis showed that ILF2 interacted with numerous RNA binding proteins directly involved in the regulation of DNA damage response by modulating alternative splicing of specific pre-mRNAs. RNA-sequencing experiments confirmed that ILF2 depletion resulted in aberrant splicing of genes involved in the DNA repair pathway, including ERCC1, FANCD2, and EXO1. RNA immunoprecipitation sequencing experiments showed that ILF2 directly bound to transcripts involved in the regulation of the HR pathway, including components of BRCA1 protein complex. Furthermore, in an attempt to dissect the ILF2 protein interacting network involved in the DNA repair regulation in response to DNA damage activation, we found that ILF2 mediated drug resistance in a dose-dependent manner by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65 to promote mRNA processing and stabilization of DNA repair genes, including FANCD2 and EXO1, in response to DNA damage.In conclusion, our study reveals an intimate relationship among 1q21 amplification, mRNA splicing, and DNA repair in the control of DNA damage response in MM.Given that 1q21 amplification is one of the most frequent copy number alterations in cancer, synthetic lethality approaches based on targeting gain-of-functions associated with ILF2 may have a broad spectrum of applications to potentiate the sensitivity of cancer cells to chemotherapeutic agents. DisclosuresGiuliani:Janssen: Research Funding; Celgene: Research Funding.

An interleukin-2 enhancer binding factor 2 homolog involved in immune response from Chinese mitten crab Eriocheir sinensis

As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159 bp, containing a 5′ untranslated region (UTR) of 90 bp, a 3′ UTR of 866 bp with a poly (A) tail, and an open reading frame (ORF) of 1203 bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3 kDa, which shared 59.6–64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly ( P < 0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6 h and 12 h after the stimulation of L. anguillarum, while P. pastoris significantly ( P < 0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6 h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris.

Cloning, characterization, and expression analysis of orange-spotted grouper ( Epinephelus coioides) ILF2 gene (EcILF2)

Interleukin-2 enhancer binding factor 2 (ILF2), also named as nuclear factor 45 (NF45), plays important roles in regulating interleukin-2 expression in mammals. In the present study, a novel ILF2 gene (designated EcILF2) was cloned and well characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcILF2 cDNA is composed of 1544 bp and encodes a polypeptide of 387 amino acids with 98% identity to ILF2 of Atlantic salmon. The genomic DNA of EcILF2 consists of 14 exons and 13 introns, with a length of approximately 6.9 kb. EcILF2 contains two conserved domains including an RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. Recombinant EcILF2 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcILF2 serum preparation. Subcellular localization analysis revealed that EcILF2 was distributed predominantly in the nucleus. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcILF2, with a relative strong expression in skin, liver, brain, head kidney and spleen. The expression of EcILF2 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Furthermore, EcILF2 was able to activate human IL-2 promoter in different cell lines and promote the endogenous IL-2 transcription in human H9 T cells. These results suggest that EcILF2 is potentially involved in grouper immune responses to invasion of bacterial and viral pathogens.

Molecular Cloning, Characterization and Expression Analysis of an ILF2 Homologue from Tetraodon nigroviridis

Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2 (IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2 homologue was cloned and well characterized from Tetraodon nigrovirid is for the further investigation of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a molecular weight of 42.9 kDa, a 5' untranslated region (UTR) of 57 bp, and a 3' UTR of 159 bp containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of 58%~93% with other known ILF2 sequences, and contained two Nglycosylation sites, two N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites, one amino-terminal RGG-rich single-stranded RNAbinding domain, and a DZF zinc-finger nucleic acid binding domain, most of which were highly conserved through species compared. Constitutive expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney, spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than other known ILF2 genes in human and mouse. Genes upand down-stream from the Tetraodon ILF2 were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in human and mouse.