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Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3.

Homma T, Ishibashi D, Nakagaki T, Fuse T, Sano K, Satoh K, Atarashi R, Nishida N - Sci Rep (2014)

Bottom Line: We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity.Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups.Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2].

ABSTRACT
As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

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Deletion analysis of murine IRF-3 promoter.N2a (a) and 3T3 (b) cells were transiently transfected with pRL-, and a series of 5′-deletion plasmids of murine IRF-3 promoter (pGL3 -2000/-1, pGL3 -1000/-524, pGL3 -1000/-1, pGL3 -523/-1, pGL3 -340/-1 and pGL3 -119/-1) or the empty plasmid (pGL3-Basic). Schematic structures of the plasmids are shown on the left. Results were normalized to the co-expressed renilla luciferase activity, and the activity of pGL3-Basic was set to 1.0. Results represent the mean ± SD.
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f1: Deletion analysis of murine IRF-3 promoter.N2a (a) and 3T3 (b) cells were transiently transfected with pRL-, and a series of 5′-deletion plasmids of murine IRF-3 promoter (pGL3 -2000/-1, pGL3 -1000/-524, pGL3 -1000/-1, pGL3 -523/-1, pGL3 -340/-1 and pGL3 -119/-1) or the empty plasmid (pGL3-Basic). Schematic structures of the plasmids are shown on the left. Results were normalized to the co-expressed renilla luciferase activity, and the activity of pGL3-Basic was set to 1.0. Results represent the mean ± SD.

Mentions: To determine the specific promoter region responsible for murine IRF-3 induction, we generated a series of plasmids that included various sizes of the 5′-flanking region of the murine IRF-3 gene fused to the luciferase gene and transfected them into N2a and 3T3 cells. As shown in Fig. 1, a plasmid containing nt -1000 to -1 relative to the transcription start site (pGL3 -1000/-1) showed approximately 33-fold (in N2a cells) and 6-fold (in 3T3 cells) activity compared with the control (pGL3 Basic), while plasmids containing nt -2000 to -1001 (pGL3 -2000/-1001) and nt -1000 to -524 (pGL3 -1000/-524) completely lost their responsiveness. Plasmids containing nt -523 to -1 (pGL3 -523/-1), nt -340 to -1 (pGL3 -340/-1) and nt -119 to -1 (pGL3 -119/-1) maintained similar levels of their promoter activity with the full-length promoter (pGL3 -1000/-1), suggesting that nt -119 to -1 was responsible for the promoter activity.


Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3.

Homma T, Ishibashi D, Nakagaki T, Fuse T, Sano K, Satoh K, Atarashi R, Nishida N - Sci Rep (2014)

Deletion analysis of murine IRF-3 promoter.N2a (a) and 3T3 (b) cells were transiently transfected with pRL-, and a series of 5′-deletion plasmids of murine IRF-3 promoter (pGL3 -2000/-1, pGL3 -1000/-524, pGL3 -1000/-1, pGL3 -523/-1, pGL3 -340/-1 and pGL3 -119/-1) or the empty plasmid (pGL3-Basic). Schematic structures of the plasmids are shown on the left. Results were normalized to the co-expressed renilla luciferase activity, and the activity of pGL3-Basic was set to 1.0. Results represent the mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126003&req=5

f1: Deletion analysis of murine IRF-3 promoter.N2a (a) and 3T3 (b) cells were transiently transfected with pRL-, and a series of 5′-deletion plasmids of murine IRF-3 promoter (pGL3 -2000/-1, pGL3 -1000/-524, pGL3 -1000/-1, pGL3 -523/-1, pGL3 -340/-1 and pGL3 -119/-1) or the empty plasmid (pGL3-Basic). Schematic structures of the plasmids are shown on the left. Results were normalized to the co-expressed renilla luciferase activity, and the activity of pGL3-Basic was set to 1.0. Results represent the mean ± SD.
Mentions: To determine the specific promoter region responsible for murine IRF-3 induction, we generated a series of plasmids that included various sizes of the 5′-flanking region of the murine IRF-3 gene fused to the luciferase gene and transfected them into N2a and 3T3 cells. As shown in Fig. 1, a plasmid containing nt -1000 to -1 relative to the transcription start site (pGL3 -1000/-1) showed approximately 33-fold (in N2a cells) and 6-fold (in 3T3 cells) activity compared with the control (pGL3 Basic), while plasmids containing nt -2000 to -1001 (pGL3 -2000/-1001) and nt -1000 to -524 (pGL3 -1000/-524) completely lost their responsiveness. Plasmids containing nt -523 to -1 (pGL3 -523/-1), nt -340 to -1 (pGL3 -340/-1) and nt -119 to -1 (pGL3 -119/-1) maintained similar levels of their promoter activity with the full-length promoter (pGL3 -1000/-1), suggesting that nt -119 to -1 was responsible for the promoter activity.

Bottom Line: We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity.Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups.Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2].

ABSTRACT
As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

Show MeSH
Related in: MedlinePlus