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STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Blaszczyk K, Olejnik A, Nowicka H, Ozgyin L, Chen YL, Chmielewski S, Kostyrko K, Wesoly J, Balint BL, Lee CK, Bluyssen HA - Biochem. J. (2015)

Bottom Line: However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3).The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3.Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

View Article: PubMed Central - PubMed

Affiliation: *Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.

ABSTRACT
Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

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2fTGH, U3C, hST2-U3C and Migr1-U3C cell lines, pre-treated for 24 h with 2-fold serial dilutions of IFNα from 250 U/ml, were infected with (A) EMCV or (B) VSV at a MOI of 0.3 for 20 h, or at a MOI of 3 for 20 h (C and D, respectively) followed by visualizing live cells by crystal violet stainingSTAT2/IRF9 mediates a similar antiviral response against EMCV and VSV as ISGF3.
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Figure 8: 2fTGH, U3C, hST2-U3C and Migr1-U3C cell lines, pre-treated for 24 h with 2-fold serial dilutions of IFNα from 250 U/ml, were infected with (A) EMCV or (B) VSV at a MOI of 0.3 for 20 h, or at a MOI of 3 for 20 h (C and D, respectively) followed by visualizing live cells by crystal violet stainingSTAT2/IRF9 mediates a similar antiviral response against EMCV and VSV as ISGF3.

Mentions: To provide further evidence for the functional overlap between STAT2/IRF9 and ISGF3 in the antiviral response, we performed a series of antiviral assays on 2fTGH, U3C, hST2-U3C and Migr1-U3C cells (Figure 8). The cells were first pretreated with 2-fold serial dilutions of IFNα for 24 h and subsequently infected with either EMCV or VSV with MOI=0.3 (Figures 8A and 8B) or 3 (Figures 8C and 8D) for each virus. Indeed, we could observe a restored antiviral response in hST2-U3C cells, as compared with 2fTGH, due to the overexpression of STAT2. U3C cells showed no antiviral protection as well as the Migr1-U3C control cells even when treated with the lower virus concentration of MOI=0.3. In conclusion, STAT2/IRF9 mediates a similar antiviral response against EMCV and VSV virus as ISGF3.


STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Blaszczyk K, Olejnik A, Nowicka H, Ozgyin L, Chen YL, Chmielewski S, Kostyrko K, Wesoly J, Balint BL, Lee CK, Bluyssen HA - Biochem. J. (2015)

2fTGH, U3C, hST2-U3C and Migr1-U3C cell lines, pre-treated for 24 h with 2-fold serial dilutions of IFNα from 250 U/ml, were infected with (A) EMCV or (B) VSV at a MOI of 0.3 for 20 h, or at a MOI of 3 for 20 h (C and D, respectively) followed by visualizing live cells by crystal violet stainingSTAT2/IRF9 mediates a similar antiviral response against EMCV and VSV as ISGF3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403947&req=5

Figure 8: 2fTGH, U3C, hST2-U3C and Migr1-U3C cell lines, pre-treated for 24 h with 2-fold serial dilutions of IFNα from 250 U/ml, were infected with (A) EMCV or (B) VSV at a MOI of 0.3 for 20 h, or at a MOI of 3 for 20 h (C and D, respectively) followed by visualizing live cells by crystal violet stainingSTAT2/IRF9 mediates a similar antiviral response against EMCV and VSV as ISGF3.
Mentions: To provide further evidence for the functional overlap between STAT2/IRF9 and ISGF3 in the antiviral response, we performed a series of antiviral assays on 2fTGH, U3C, hST2-U3C and Migr1-U3C cells (Figure 8). The cells were first pretreated with 2-fold serial dilutions of IFNα for 24 h and subsequently infected with either EMCV or VSV with MOI=0.3 (Figures 8A and 8B) or 3 (Figures 8C and 8D) for each virus. Indeed, we could observe a restored antiviral response in hST2-U3C cells, as compared with 2fTGH, due to the overexpression of STAT2. U3C cells showed no antiviral protection as well as the Migr1-U3C control cells even when treated with the lower virus concentration of MOI=0.3. In conclusion, STAT2/IRF9 mediates a similar antiviral response against EMCV and VSV virus as ISGF3.

Bottom Line: However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3).The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3.Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

View Article: PubMed Central - PubMed

Affiliation: *Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.

ABSTRACT
Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

Show MeSH
Related in: MedlinePlus