Limits...
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.

Show MeSH

Related in: MedlinePlus

Reduction in IRF-3 promoter activity in prion-infected cells.(a) The levels of IRF-3 mRNA in N2a58 and ScN2a58 cells. Results represent the mean ± SD. P value was determined by Student's t test (**: P < 0.01). The quantitative, real time-PCR data were normalized by the β-actin mRNA levels. (b) N2a58 and ScN2a58 cells were transiently transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity in N2a58 cells was set to 100%. Results represent the mean ± SD, n = 4. P value was determined by Student's-t test (***: P < 0.001). (c) The promoter activities of ScN2a58 cells continuously treated with CR or PPS. Cells were transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity of each control cell was set to 1.0. Results represent the mean ± SD, n = 3. P values were determined by Student's-t test (*: P < 0.05, ***: P < 0.001). (d) The levels of PK-resistant PrP [M20, PK (+)] in drug-treated N2a58 and ScN2a58 cells were analyzed by immunoblotting. PPS: pentosan polysulfate, CR: Congo red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126003&req=5

f2: Reduction in IRF-3 promoter activity in prion-infected cells.(a) The levels of IRF-3 mRNA in N2a58 and ScN2a58 cells. Results represent the mean ± SD. P value was determined by Student's t test (**: P < 0.01). The quantitative, real time-PCR data were normalized by the β-actin mRNA levels. (b) N2a58 and ScN2a58 cells were transiently transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity in N2a58 cells was set to 100%. Results represent the mean ± SD, n = 4. P value was determined by Student's-t test (***: P < 0.001). (c) The promoter activities of ScN2a58 cells continuously treated with CR or PPS. Cells were transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity of each control cell was set to 1.0. Results represent the mean ± SD, n = 3. P values were determined by Student's-t test (*: P < 0.05, ***: P < 0.001). (d) The levels of PK-resistant PrP [M20, PK (+)] in drug-treated N2a58 and ScN2a58 cells were analyzed by immunoblotting. PPS: pentosan polysulfate, CR: Congo red.

Mentions: To examine the relationship between the IRF-3 promoter activity and prion infection, we analyzed N2a58 and ScN2a58 (N2a58 cells persistently infected with 22L prion) cells. As shown in Fig. 2a, mRNA of IRF-3 had a significant reduction in ScN2a58 cells. Reduction of the promoter activity was confirmed in ScN2a58 cells which was transiently transfected with pGL3 -119/-1 (Fig. 2b). Furthermore, in the persistently other prion strain-infected cells by the mouse-adapted Gerstmann-Sträussler-Schenker syndrome (GSS) Fukuoka-1 strain (FK-N2a58), IRF-3 mRNA and the promoter activity were also significantly decreased (Supplementary Fig. S6a and S6b).


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)

Reduction in IRF-3 promoter activity in prion-infected cells.(a) The levels of IRF-3 mRNA in N2a58 and ScN2a58 cells. Results represent the mean ± SD. P value was determined by Student's t test (**: P < 0.01). The quantitative, real time-PCR data were normalized by the β-actin mRNA levels. (b) N2a58 and ScN2a58 cells were transiently transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity in N2a58 cells was set to 100%. Results represent the mean ± SD, n = 4. P value was determined by Student's-t test (***: P < 0.001). (c) The promoter activities of ScN2a58 cells continuously treated with CR or PPS. Cells were transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity of each control cell was set to 1.0. Results represent the mean ± SD, n = 3. P values were determined by Student's-t test (*: P < 0.05, ***: P < 0.001). (d) The levels of PK-resistant PrP [M20, PK (+)] in drug-treated N2a58 and ScN2a58 cells were analyzed by immunoblotting. PPS: pentosan polysulfate, CR: Congo red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Reduction in IRF-3 promoter activity in prion-infected cells.(a) The levels of IRF-3 mRNA in N2a58 and ScN2a58 cells. Results represent the mean ± SD. P value was determined by Student's t test (**: P < 0.01). The quantitative, real time-PCR data were normalized by the β-actin mRNA levels. (b) N2a58 and ScN2a58 cells were transiently transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity in N2a58 cells was set to 100%. Results represent the mean ± SD, n = 4. P value was determined by Student's-t test (***: P < 0.001). (c) The promoter activities of ScN2a58 cells continuously treated with CR or PPS. Cells were transfected with pGL3 -119/-1 and pRL-. Results were normalized to the co-expressed renilla luciferase activity, and the activity of each control cell was set to 1.0. Results represent the mean ± SD, n = 3. P values were determined by Student's-t test (*: P < 0.05, ***: P < 0.001). (d) The levels of PK-resistant PrP [M20, PK (+)] in drug-treated N2a58 and ScN2a58 cells were analyzed by immunoblotting. PPS: pentosan polysulfate, CR: Congo red.
Mentions: To examine the relationship between the IRF-3 promoter activity and prion infection, we analyzed N2a58 and ScN2a58 (N2a58 cells persistently infected with 22L prion) cells. As shown in Fig. 2a, mRNA of IRF-3 had a significant reduction in ScN2a58 cells. Reduction of the promoter activity was confirmed in ScN2a58 cells which was transiently transfected with pGL3 -119/-1 (Fig. 2b). Furthermore, in the persistently other prion strain-infected cells by the mouse-adapted Gerstmann-Sträussler-Schenker syndrome (GSS) Fukuoka-1 strain (FK-N2a58), IRF-3 mRNA and the promoter activity were also significantly decreased (Supplementary Fig. S6a and S6b).

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