Abstract
Background
IRF9 is a transcription factor that mediates the expression of interferon-stimulated genes (ISGs) through the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway. The JAK-STAT pathway is regulated through phosphorylation reactions, in which all components of the pathway are known to be phosphorylated except IRF9. The enigma surrounding IRF9 regulation by a phosphorylation event is intriguing. As IRF9 plays a major role in establishing an antiviral state in host cells, the topic of IRF9 regulation warrants deeper investigation.
Methods
Initially, total lysates of 2fTGH and U2A cells (transfected with recombinant IRF9) were filter-selected and concentrated using phosphoprotein enrichment assay. The phosphoprotein state of IRF9 was further confirmed using Phos-tag™ assay. All protein expression was determined using Western blotting. Tandem mass spectrometry was conducted on immunoprecipitated IRF9 to identify the phosphorylated amino acids. Finally, site-directed mutagenesis was performed and the effects of mutated IRF9 on relevant ISGs (i.e., USP18 and Mx1) was evaluated using qPCR.
Results
IRF9 is phosphorylated at S252 and S253 under IFNβ-induced condition and R242 under non-induced condition. Site-directed mutagenesis of S252 and S253 to either alanine or aspartic acid has a modest effect on the upregulation of USP18 gene—a negative regulator of type I interferon (IFN) response—but not Mx1 gene.
Conclusion
Our preliminary study shows that IRF9 is phosphorylated and possibly regulates USP18 gene expression. However, further in vivo studies are needed to determine the significance of IRF9 phosphorylation.
Supplementary Information
The online version contains supplementary material available at 10.1007/s11033-023-08253-3.
Keywords: Interferon regulatory factor 9, Phosphorylation, JAK-STAT pathway, Innate immunity, Type I IFN response
Introduction
Interferon regulatory factors (IRFs) are transcription factors that mediate the transcription of pathogen-, and IFN-induced signaling pathways [1]. There are nine IRFs found in humans. The ninth IRF was discovered as a subunit of the interferon-stimulated gene factor 3 (ISGF3) complex; a trimeric unit that includes STAT1 and STAT2 proteins [2]. Following stimulation by type I IFNs, tyrosine kinase 2 and Janus kinase 1 phosphorylates STAT1 and STAT2 on tyrosine [3]. The phosphorylated STATs subsequently bind to IRF9 to form ISGF3, which then translocates to the nucleus and binds to interferon-stimulated response elements (ISRE), where it activates the transcription of hundreds of interferon-stimulated genes (ISGs) providing host protection against multiple types of pathogen infection [4, 5]. While both STAT1 and STAT2 of the ISGF3 are phosphorylated, phosphorylation of IRF9 has been largely unknown. Though, an earlier report had suggested that IRF9 is constitutively phosphorylated within its DNA-binding domain despite the absence of an IFN stimuli [6]. In the 1993 study, dephosphorylation of IRF9 using calf intestinal phosphatase was shown to abrogate ISRE binding in vitro, indicating a role for phosphorylation in DNA association [6], however the exact amino acid(s) was not reported. Acetylation is a possible regulating mechanism for IRF9. Site-directed mutagenesis of IRF9 at lysine 81 to arginine (K81R, which is unable to be acetylated) was shown to abolish its DNA-binding activity under IFNα-inducing condition [7]. However, no follow-up study was conducted since.
Given the role of IRF9 in mediating the innate immune response [8–10] and its pleiotropic function [11], discovering the regulatory mechanism of IRF9 is of biological importance. In our preliminary study, we found that IRF9 is phosphorylated at amino acid residues S252 and S253 under IFNβ-inducing condition and at R242 under non-inducing condition. IRF9 mutants of S252 and S253 were observed to affect the upregulation of USP18, but not Mx1 gene. No significant difference was observed on OAS1 and PKR gene expressions.
Materials and methods
Cell culture and reagents
HEK293T cells were grown in DMEM with HEPES (Gibco). 2fTGH and U2A cells were grown in DMEM without HEPES (Gibco). All culture media were supplemented with 10% fetal bovine serum (Gibco). TrypLE (Gibco) was used for detaching the cells during maintenance of cell culture. Cells were washed using PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Cells were induced with recombinant IFNβ (R&D Systems) at either 100 or 1000 IU/ml. All cells were maintained in an incubator at 37 °C with 5% CO2.
Phosphoprotein enrichment assay
Fractionation and enrichment of phosphoproteins from cell lysate was performed using the Pierce™ Phosphoprotein Enrichment Kit (Thermo Scientific), following manufacturer’s recommendations. Briefly, cells were washed with ice-cold HEPES buffer (115 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 2.4 mM K2HPO4, 20 mM HEPES) pH 7.4, scraped, and pelleted by centrifugation at 10,000×g at 4 °C for 5 min. Pelleted cells were lysed with the supplied Lysis/Binding/Wash buffer (added with 0.25% (v/v) CHAPS, protease inhibitor, and phosphatase inhibitor cocktail) and incubated on ice for 45 min. Four mg of total protein was applied through the phosphoprotein enrichment column and concentrated using the supplied concentrator column to a final volume of ~ 100 µl and kept in −80 °C until further analysis. Following manufacturer’s protocol, cytochrome c (a non-phosphoprotein) was used as a negative control.
Phos-tag™ polyacrylamide gel assay
Phos-tag™ (Wako Pure Chemical Industries) polyacrylamide assay was performed as described by [12], with modifications. Briefly, 25 µM of Phos-tag™ and 50 µM of MnCl2 were supplemented into 12% SDS-PAGE resolving gel solution and gels were allowed to polymerize for 2 h. Thirty-five µg of total protein was loaded per well. SDS-PAGE was carried out for 20 h under constant 20 V. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) followed by Western blotting analysis.
Plasmid constructs and site-directed mutagenesis
The cDNA for human IRF9 was purchased from GE Dharmacon (USA) and amplified by PCR using primers (Table 1) containing EcoRI and XbaI restriction sites and ligated into the vector plasmid. The vector plasmid used was p3×FLAG-CMV-7.1™ (Sigma-Aldrich). For PCR subcloning reaction, 10 ng of plasmid DNA was used as starting template. Site-directed mutagenesis assay was performed as described by [13], with modifications. For site-directed mutagenesis PCR reaction, 500 ng of plasmid DNA was used as a starting template. All constructs were confirmed by Sanger DNA sequencing. All oligonucleotides were manufactured by Integrated DNA Technologies (Singapore). All oligonucleotides used are listed in Table 1. The protein expression of IRF9 mutants is available as Supplemental Fig. S1.
Table 1.
Oligonucleotides used in the design of 3×FlagIRF9 plasmid construct and in site-directed mutagenesis
| Oligonucleotides used for 3×FlagIRF9 plasmid construct | ||
| 1.1 | 3×FlagIRF9 (forward) | 5′-GTCAGAATTCAATGGCATCAGGCAG |
| 1.2 | 3×FlagIRF9 (reverse) | 5′-CGTATCTAGACTACACCAGGGACAGAATG |
| Oligonucleotides used in site-directed mutagenesis assay | ||
| 2.1 | S252A (forward) | 5′-GAGCCCTCAGGCTCTGAGGCCAGCATGGAGCAGGTGCTG |
| 2.2 | S252A (reverse) | 5′-CAGCACCTGCTCCATGCTGGCCTCAGAGCCTGAGGGCTC |
| 2.3 | S252D (forward) | 5′-GAGCCCTCAGGCTCTGAGGACAGCATGGAGCAGGTGCTG |
| 2.4 | S252D (reverse) | 5′-CAGCACCTGCTCCATGCTGTCCTCAGAGCCTGAGGGCTC |
| 2.5 | S253A (forward) | 5′-CCTCAGGCTCTGAGAGCGCCATGGAGCAGGTGCTGTTC |
| 2.6 | S253A (reverse) | 5′-GAACAGCACCTGCTCCATGGCGCTCTCAGAGCCTGAGG |
| 2.7 | S253D (forward) | 5′-CCTCAGGCTCTGAGAGCGACATGGAGCAGGTGCTGTTC |
| 2.8 | S253D (reverse) | 5′-GAACAGCACCTGCTCCATGTCGCTCTCAGAGCCTGAGG |
| 2.9 | S252-S253A (forward) | 5′-GAGCCCTCAGGCTCTGAGGCCGCCATGGAGCAGGTGCTGTTC |
| 2.10 | S252-S253A (reverse) | 5′-GAACAGCACCTGCTCCATGGCGGCCTCAGAGCCTGAGGGCTC |
| 2.11 | S252-S253D (forward) | 5′-GAGCCCTCAGGCTCTGAGGACGACATGGAGCAGGTGCTGTTC |
| 2.12 | S252-S253D (reverse) | 5′-GAACAGCACCTGCTCCATGTCGTCCTCAGAGCCTGAGGGCTC |
RNA isolation and quantitative real-time PCR (qPCR) assay
Total RNA was extracted using the Tri-Reagent (MRC, USA) according to manufacturer’s protocol, and purified using the phenol–chloroform-isoamyl alcohol extraction method. cDNA was synthesized from 2 µg of total RNA using 20-mer anchored oligo-dT mix and AMV-reverse transcriptase (Promega, USA). qPCR was performed using LightCycler® SYBR Green I Master (Roche) in a LightCycler® 480 II (Roche). Ct values were converted into relative gene expression levels compared to that of the internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Samples were run in duplicates and relative gene levels were calculated using the ΔΔCt method [14]. The PCR protocol was as follows: initial activation at 95°C for 5 min, amplification for 45 cycles at 95°C for 10s, 54°C for 10s and 72°C for 10s. All oligonucleotides used are listed in Table 2.
Table 2.
Oligonucleotides used in qPCR assay
| Oligonucleotides used in qPCR assay | ||
| 1.1 | Mx1 (forward) | 5′-CATCCAGTTCTTCATGCTCC |
| 1.2 | Mx1 (reverse) | 5′-GAACTTCCGCTTGTCGCT |
| 1.3 | USP18 (forward) | 5′-GAAGCGAGAGTCTTGTGATG |
| 1.4 | USP18 (reverse) | 5′-GAGTCATTGAAGCAGAACCA |
| 1.5 | OAS1 (forward) | 5′-GAAACTTGGGTGGTGGAG |
| 1.6 | OAS1 (reverse) | 5′-CTCATCGTCTGCACTGTTG |
| 1.7 | PKR (forward) | 5′-CGATACATGAGCCCAGAAC |
| 1.8 | PKR (reverse) | 5′-CCATCCCGTAGGTCTGTG |
| 1.9 | GAPDH (forward) | 5′-GACCACAGTCCATGCCATC |
| 1.10 | GAPDH (reverse) | 5′-GCTCAGGGATGACCTTGC |
Western blot
Cells were washed with ice-cold TBS buffer (25 mM Tris–HCl (pH 7.5), 150 mM NaCl), scraped, and pelleted by centrifugation at 10,000×g at 4°C for 5 min. Pelleted cells were lysed with complete NET buffer (0.15 M NaCl, 0.5% (v/v) NP-40, 1 mM EDTA pH 8.0, 0.05 M Tris–HCl pH 7.5, 0.02% (w/v) sodium azide, PhosSTOP™ phosphatase inhibitor cocktail (Roche), and cOmplete™ EDTA-free protease inhibitor cocktail (Roche)). After incubation on ice for 40 min, cell debris was pelleted by centrifugation at 10,000×g for 10 min at 4°C. Total protein concentration was determined by the Bradford assay [15], separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked using 4% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST). All antibodies were diluted in 4% BSA in TBST. Enhanced chemiluminescence solution WesternBright ECL HRP (Advansta) was used to visualize the target protein and were either developed on autoradiography films (MTC Bio) or detected using Molecular Imager® VersaDoc™ MP 4000 system (Bio-Rad). The primary antibodies used are IRF9 (1:500, Cell Signaling Technology Cat# 76684), pSTAT1 Y701 (1:1000, Cell Signaling Technology Cat# 9167), STAT1 (1:1000, Cell Signaling Technology Cat# 9172), PKR (1:200, Santa Cruz Biotechnology Cat# sc-6282), Cytochrome c (1:200, Cell Signaling Technology Cat# 11940), β-actin (1:1000, Cell Signaling Technology Cat# 4967). The secondary antibodies used are anti-rabbit IgG HRP-linked (1:2500, Cell Signaling Technology Cat# 7074), and anti-mouse IgG HRP-linked (1:2500, Cell Signaling Technology Cat# 7076).
Immunoprecipitation
HEK293T cells were grown in 10 cm dishes and transfected with 3×FlagIRF9 plasmid constructs using Lipofectamine™ 2000 (Thermo Scientific). After 36 h, cells were induced with IFNβ for 24 h, then lysed with complete NET buffer. Twenty-eight milligram of total protein from whole cell lysate was incubated by mixing using an end-over-end rotator with Anti-Flag® M2 magnetic beads (Sigma) at 4°C, overnight. The following day, M2-bound 3×FlagIRF9 proteins were washed 3 times with ice-cold TBS buffer (50 mM Tris–HCl, 150 mM NaCl pH 7.4), and heated to 95°C with 5× SDS-PAGE loading buffer containing 10% (v/v) β-mercaptoethanol to elute 3×FlagIRF9 proteins out. The immunoprecipitated 3×FlagIRF9 was then subjected to SDS-PAGE. A protein band corresponding to the approximate size of 3×FlagIRF9 (51 kDa) was visualized with Coomassie Blue (G250) stain and excised.
Tandem mass spectrometry (LC–MS/MS)
Following SDS-PAGE, the gel bands with immunoprecipitated 3×FlagIRF9 were excised and de-stained until gel appearance was clear. The gel pieces were dehydrated with acetonitrile, dried in a Concentrator Plus (Eppendorf), reduced with 10 mM dithiothreitol and alkylated with 55 mM of iodoacetamide. The gel pieces were then incubated in 20 ng/µl of SOLu-Trypsin (Merck) in 50 mM ammonium bicarbonate, at 37°C for overnight. The following day, an additional 50 µl of 20 mM ammonium bicarbonate was added to the gel pieces and incubated in 37°C for 10 min. Then, the supernatant was transferred to clean tubes. Peptides were further extracted in two portions of 50 µl each in 40% acetonitrile, 2% formic acid. The combined extracts were evaporated to < 10 µl using Concentrator Plus (Eppendorf) and then resuspended in 0.1% formic acid to a final volume of ~ 100 µl. The peptides were then filtered using Minisart® RC4 0.2 µm syringe filter (Sartorius) to remove particulates and kept in − 80 °C until further analysis. LC–MS/MS analyses were performed using LTQ-Orbitrap Velos Pro mass spectrometer coupled to Easy-nLC II nano-LC system (Thermo Scientific) with 0.1% formic acid in deionized water and 0.1% formic acid in 100% acetonitrile as the mobile phase. The flow rate was at 0.3 μl/min. The Easy column C18 (10 cm, 0.75 mm i.d., 3 μm; Thermo Scientific) was used as the analytical column, whereas the Easy column C18 (2 cm, 0.1 mm i.d., 5 μm; Thermo Scientific) was used as the pre-column. Sample eluent was sprayed at 220 °C capillary temperature and a 2.1 kV source. Full-scan mass analysis from m/z 300 to 2000 was used to detect the protein and peptides at a resolving power of 60,000 (at m/z 400, FWHM; 1/s acquisition). Data-dependent MS/MS analyses were triggered by the eight most abundant ions from the MS stage. Fragmentation technique used was the collision-induced dissociation (CID) with a collision energy of 35. In addition, high-energy collision-induced dissociation was also applied with a similar setting as the CID.
Protein sequencing and database searching
PEAKS® Studio Version 7 (Bioinformatics Solution Inc.) was used to perform de novo sequencing and database matching to confirm the identity of IRF9 and identify phosphorylation site(s). The standard identification workflow includes the targeted post-translational modifications of Oxidation (M), Carbamidomethylation (C), and Phosphorylation (STY and HCDR), as well as mutational analysis. Carbamidomethylation of cysteines and oxidation of methionine were set as constant modifications. The peptide sequences obtained were then matched to the human protein database from UniProt. 1% false discovery rate was applied to filter the result. The mass spectrometry data can be publicly accessed in the MassIVE database (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) using the data identifier MSV000089924.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 9.2.0 (GraphPad Software Inc.). Data were analyzed using two-way ANOVA and Tukey’s multiple comparison test when comparing the mean of one group to the mean of any other group. P values of ≤ 0.05 are considered statistically significant.
Results
IRF9 is enriched as part of a phosphoprotein extraction and enrichment assay
First, we performed the phosphoprotein extraction and enrichment assay on 2fTGH lysate, which was previously induced with IFNβ at 1000 IU/ml for 24 h. We found the enrichment of IRF9 in the phosphoprotein fraction (Fig. 1A). We then performed a mobility shift-based SDS-PAGE Phos-tag™ assay. Phos-tag™ is a di-nuclear metal complex that transiently binds to phosphoproteins and, as a result, decreases the migration rate of phosphoproteins relative to non-phosphoproteins in polyacrylamide gel [12]. The Phos-tag™ assay showed two separate IRF9 protein bands (Fig. 1B, right) as opposed to one IRF9 band in the normal gel (Fig. 1B, left). The two separate bands in Fig. 1B indicate to a phosphorylated and a non-phosphorylated form of IRF9. Endogenous IRF9 was not detected in the non-induced cell lysates (Fig. 1A,B), prompting us to construct and use recombinant IRF9 (termed as 3×FlagIRF9). The functionality of 3×FlagIRF9 in IFNβ-induced U2A cells (IRF9-null) was confirmed by determining the protein expression of protein kinase R (PKR). PKR expression was upregulated in 3×FlagIRF9 transfected-U2A cells, similar to its 2fTGH parent cell (Fig. 1C). Relative gene expressions of USP18 and Mx1 were also found to be upregulated (Fig. 1D, refer Supplemental Fig. 5 for gene expressions of USP18 and Mx1 in 2fTGH cells). We detected 3×FlagIRF9 in the phosphoprotein fractions (Fig. 1E), which is similar to the observation in Fig. 1A. Given that the phosphoprotein column only fractionates and concentrates phosphoproteins, any protein bands appearing in the output lanes would then be phosphoprotein. The reduction in band intensity of cytochrome c (a negative control) is apparent in Fig. 1E, indicating to a functional phosphoprotein fractionation assay. Note that IRF9 is phosphorylated both under IFNβ-induced and non-induced conditions (Fig. 1E), which suggests a constitutive phosphorylation.
Fig. 1.
Phosphorylation of IRF9. A Phosphoprotein extraction and enrichment assay of 2fTGH cell lysate. Endogenous IRF9 is undetectable in non-induced cells. Cytochrome c and phospho-STAT1 Y701 were used as negative and positive control, respectively. Thirty-five microgram of proteins were loaded into SDS-PAGE. “Input” lane refers to unprocessed total cell lysate. “Output” lane refers to the cell lysate after filter selection and concentration (i.e., enriched) of phosphoproteins using the phosphoprotein column. B Mobility of IRF9 in normal polyacrylamide gel vs. Phos-tag™ polyacrylamide gel. Two arrows on IRF9 panel indicate two isoforms detected in Phos-tag™ gel (right) compared to one in the normal polyacrylamide gel (left). Distance of two isoforms of phospho-STAT1 (Y701) is more pronounced in Phos-tag™ gel, which is indicative of Phos-tag™ gel’s efficacy. C PKR protein expression in 2fTGH, mock-transfected U2A, empty vector-transfected U2A, and 3×FlagIRF9-transfected U2A cells in varying IFNβ concentration. D mRNA expression of USP18 and Mx1 in vector-transfected U2A cells and normalized to GAPDH and calculated using the ΔΔCt method. Data presented as the mean ± s.e.m. fold change of two independent experiments. P values were calculated using two-way ANOVA with Tukey’s post-test (**P < 0.01, ***P < 0.001). ns not statistically significant. E Phosphoprotein enrichment assay of total lysate from 3×FlagIRF9-transfected U2A cells. 3×FlagIRF9 protein was enriched as shown in the output lane, which indicates to the phosphorylation of IRF9. Twenty-five microgram of proteins were loaded into SDS-PAGE
IRF9 is phosphorylated at S252 and S253 under IFNβ-condition; and R242 under non-induced condition
Our initial finding prompted us to carry out phosphorylation site-mapping of IRF9. 3×FlagIRF9 immunoprecipitated from HEK293T cells—either in IFNβ-induced or non-induced conditions—were subjected to liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis, revealing S252 and S253 as the phosphorylated amino acid residues in IFNβ-induced cells (Fig. 2A, bottom). Interestingly, a non-canonical phosphorylation on arginine reside (R242) was also detected under non-induced conditions (Fig. 2A, top). Residues R242, S252 and S253 are within the IRF-associated domain (IAD) of IRF9. All three residues are conserved across several other mammalian species (Fig. 2B), suggesting an important function. The LC–MS/MS data (Fig. 2A) indicates to a constitutive phosphorylation of IRF9, which corroborates our earlier observation (Fig. 1E). Taken together, these results demonstrated the phosphorylation event of IRF9. A schematic figure of IRF9 (human) is illustrated to show the phosphorylation site at its IAD with and without IFNβ induction (Fig. 2C). The tryptic peptide segment of IRF9 is available as Supplemental Fig. S2.
Fig. 2.
Phosphorylation of IRF9 at R242, S252 and S253. A Detection of phosphorylated peptides of 3×FlagIRF9 from non-induced (top) and IFNβ-induced (bottom) HEK293T cells. Phosphorylation at R242 (non-induced); S252 and S253 (IFNβ-induced). B Conservation of R242, S252 and S253 of IRF9 across mammalian species. C Schematic representation of human IRF9 showing the location of phosphorylated amino acids within its IAD with and without IFNβ induction. DBD DNA-binding domain, IAD IRF-association domain
Mutant S252 and S253 of IRF9 have modest effect on the upregulation of USP18, but not Mx1
Encouraged by the LC–MS/MS data, we mutated the S252 and S253 to alanine (S252A, S253A) as a non-phosphomimic mutation and aspartic acid (S252D, S253D) to mimic phosphorylation. The mutants were then transfected into U2A cells and induced with IFNβ for 24 h followed by RNA extraction and qPCR analysis. We tested the effects of mutant IRF9 on the expression of four important antiviral genes; USP18, Mx1, OAS1 and PKR. In this assay, U2A cells were used because it does not express endogenous IRF9. Consequently, all effects are attributable to the mutated IRF9 in response to IFNβ induction. All mutants of IRF9 have a modest effect on the upregulation of USP18 gene expression compared to wildtype IRF9 (Fig. 3, Supplemental Table 1). However, all mutants of IRF9—except S252-3A mutant—did not affect the expression of Mx1 gene (Fig. 3, Supplemental Table 1). Meanwhile, we found no significant difference between induced wildtype and IRF9 mutants on its effect on the expression of OAS1 and PKR genes (Supplemental Fig. 3, Supplemental Table 2). Similarly, we found no significant difference between each mutant of non-induced and induced conditions for OAS1 and PKR genes (Supplemental Table 1).
Fig. 3.
S252 and S253 IRF9 mutations mildly negates the upregulation of USP18, but not Mx1. qPCR for USP18, and Mx1 mRNA in U2A cells transfected with mock, empty vector, wildtype (WT), and mutants of IRF9; S252A, S252D, S253A, S253D, S252–253A, and S252–253D. Genes were normalized to GAPDH and determined using the ΔΔCt method. Data presented as the mean ± s.e.m. fold change of two independent experiments. P values were calculated using two-way ANOVA with Tukey’s post-test (*P < 0.05, ***P < 0.001, ****P < 0.0001). ns not statistically significant
Discussion
The primary aim of this study was to understand the regulatory control of IRF9 under type I IFN responses. In particular, we sought to identify a potential post-translational modification reaction of IRF9 due to the dominant role of the phosphorylation reaction in mediating the JAK-STAT pathway. Moreover, several of IRF family members undergo phosphorylation as a way of activating/deactivating its functions.
In this study, we have determined the phosphorylation sites in IRF9, which was previously unknown. We identified residues S252 and S253 as phosphorylated under IFNβ-induced condition. A non-canonical phosphorylation amino acid arginine 242 was also identified. However, the effect of R242 phosphorylation was not explored in this study. Residues S252 and S253 may play a role in regulating ISGs, whereby mutational change of serine to either alanine or aspartic acid has a modest effect on the upregulation of gene expression of USP18, but not Mx1. The effect of IRF9 mutants on the expression of OAS1 and PKR genes was also investigated, but no significant differences in expression levels were found. It appears that neither the addition of a negative charge residue nor abolishing phosphorylation function of serine affects Mx1 gene expression—except for USP18—suggesting an unknown mechanism.
There are nine amino acid residues (e.g., L233, R236, S247, M248, L274, A276, N278, F283, and Q285) on mouse IRF9 involved in STAT2 interaction [16]. R236, S246 and S247 residues of mouse IRF9 are homologs to R242, S252 and S253 residues of human IRF9, respectively (Fig. 2B). Based on the Protein Data Bank (PDB) structure information (PDB ID: 5OEN), R236 interacts with D167 of mouse STAT2, S246 interacts with E168 of mouse STAT2, and S247 interacts with E168 and R175 of mouse STAT2 through hydrogen bonding. The downregulation of USP18 by both phosphomimetic and non-phosphomimetic IRF9 is confounding. We speculate that alteration on S252 and S253 disrupted binding affinity with STAT2, and that this disruption interfered with USP18 expression. More intriguing is the detection of phosphorylated R242 of IRF9 under non-induced condition. Rengachari, Groiss [16] also showed that R236 mutation to glutamic acid of mouse IRF9 resulted in reduced interaction with STAT2. By virtue of its homology, R242 phosphorylation may play a role in IRF9-STAT2 interaction. Given that IRF9 and STAT2 is constitutively coupled [17], we posit that phosphorylation of IRF9 at R242 causes destabilization of IRF9-STAT2 dimer (Supplemental Fig. 3). It may be that a phosphorylation site-switch mechanism is involved in maintaining a state of constitutive phosphorylation in IRF9. We surmise that IRF9 is phosphorylated at multiple sites throughout the IFNβ-induction period, and thus investigation into varying time-points of IFNβ-induction in relation to its phosphorylation state would be of immediate interest. In addition to the phosphorylated ISGF3 complex, the role of unphosphorylated ISGF3 (U-ISGF3) cannot be ignored. Previous studies have shown that unphosphorylated STAT1 (U-STAT1) is capable of sustaining the response to IFNβ induction for days as it induces the expression of a subset of ISGs, distinct from the phosphorylated ISGF3-induced genes [18]. Subsequent findings indicate that induction by IFNβ also induces the expression of U-STAT2, which would then dimerize with U-STAT1 and IRF9 to form the unphosphorylated ISGF3 complex (U-ISGF3) [19]. Interestingly, independent of IFN induction, the U-ISGF3 could also form in the nucleus and was found to maintain a basal ISG expression under homeostasis, which provided antiviral protection against hepatitis C and E virus [20]. We foresee that the phosphorylation of IRF9 will add a new dimension to the functional dynamics of ISGF3, U-ISGF3 or STAT2-IRF9 protein complex. The role of arginine phosphorylation remains largely unknown. In bacterial cells, phosphorylation of arginine earmarks protein for degradation by Clp protease, a process similar to ubiquitination [21]. Although the function of arginine phosphorylation in human cells is still unknown [22], it will be interesting to determine whether phosphorylation at R242 actually marks IRF9 for degradation.
Our study has several limitations. First, our results show that phosphorylation occurs in vitro. This requires further work to determine if phosphorylation occurs in vivo. Second, the mechanistic detail of phosphorylation is not reported in this study. Future studies may include identifying the enzymes kinase and phosphatase involved in the phosphorylation reaction. Third, the exact mechanism of how mutant IRF9 affects the expression of USP18 was not investigated in this study, and thus provides an avenue for further research into selective gene regulation by IRF9. A recent study reported that in severe COVID-19 cases, there was an increase in phosphorylated STAT1 relative to total STAT1 expression compared to mild cases [23]. Similarly, the study also found a lower IRF9 expression level in severe COVID-19 cases compared to mild, with the authors noting the IRF9 decrease as “consistent with a reduced STAT1 expression” [23]. It is therefore tempting to hypothesize that, in severe COVID-19 cases, phosphorylated IRF9 may be present in larger amounts relative to an increase in total IRF9 expression. Our finding could thus facilitate verification of this hypothesis.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors would like to thank George R. Stark (Cleveland Clinic, Ohio) for gifting us the 2fTGH and U2A cells. We also thank Timofey Rozhdestvensky (University of Münster, Germany) and our RNA-Bio Group (Universiti Sains Malaysia) members for the critical review of this manuscript.
Author contributions
AP—methodology, investigation, data curation, visualization, writing (original draft, review & editing), analysis. MNI—investigation, data curation, analysis, resources, supervision. THT—resources, funding acquisition, supervision. SKN—conceptualization, resources, funding acquisition, writing (review & editing), analysis, supervision.
Funding
This research was funded by the Fundamental Research Grant Scheme (FRGS/1/2016/STG04/USM/03/1) from the Ministry of Higher Education Malaysia and the Short-Term Research Grant (304/CIPPT/6313229) from Universiti Sains Malaysia.
Declarations
Competing interests
The authors declare no competing interests exist.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Footnotes
Publisher's Note
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Contributor Information
Alvin Paul, Email: alvin.amdi1@gmail.com.
Mohd Nazri Ismail, Email: mdnazri@usm.my.
Thean Hock Tang, Email: tangth@usm.my.
Siew Kit Ng, Email: skng@usm.my.
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