Objective To investigate the role and mechanism of the zinc finger protein Kruppel-like transcription factor 9 (KLF9) in the stimulation of type I interferon expression induced by herpes simplex virus type 1 (HSV-1) in macrophages. Methods Agarose Gel electrophoresis, quantitative real-time PCR (qRT-PCR) and western blot analyses were employed to detect the KLF9 relative expression in bone marrow-derived macrophages (BMDMs) from Klf9^-/- (gKO) mice and wild-type (WT) mice. RNA-seq analysis was utilized to identify the potential targeted genes upon HSV-1 stimulation in BMDMs. ELISA was used to measure the potent of IFN-β in the supernatant of BMDMs derived from gKO and WT mice after HSV-1 stimulation. qRT-PCR analysis was employed to further confirm the changes of Ifnb1 and interferon-stimulated gene (ISG) such as interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), interferon-stimulated exonuclease gene 20 (Isg20), cholesterol 25-hydroxylase (Ch25h) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1). Western blot was used to detect the expression of phosphorylated interferon regulatory factor-3 (p-IRF3), IRF3, phosphorylated interferon regulatory factor-7 (p-IRF7), IRF7, phosphorylated nuclear factor-kappa B p65 (p-NF-κB p65) and NF-κB p65. CUT-Tag and ChIP-qPCR assay were utilized to confirm the binding region of KLF9 in Ifnb1. Results The KLF9 expression was significantly decreased in BMDMs from gKO mice compared with that from WT mice. The RNA-seq analysis showed that Klf9 deletion in BMDMs resulted in an impaired type I interferon signaling pathway. The qRT-PCR analysis revealed that Klf9 deletion in BMDMs led to a significant decrease of Ifnb1 and ISG such as Ifit1, Ch25h and Oasl1 except Isg20. Moreover, ELISA revealed that Klf9 knockout in BMDMs resulted in a significant decrease of IFN-β secreted from BMDMs. Mechanistically, KLF9 directly binds to the promoter of Ifnb1. Conclusion KLF9 is essential for macrophages to resist HSV-1 infection.
Animals ; Kruppel-Like Transcription Factors/physiology* ; Interferon-beta/metabolism* ; Macrophages/virology* ; Mice ; Herpesvirus 1, Human/physiology* ; Mice, Knockout ; Signal Transduction ; Mice, Inbred C57BL ; Interferon Regulatory Factor-3/genetics* ; Interferon Regulatory Factor-7/genetics* ; Gene Expression Regulation

Dendritic Cells ; cytology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; HEK293 Cells ; Humans ; Interferon Regulatory Factor-7 ; metabolism ; Interferon Type I ; metabolism ; Interferon-gamma ; analysis ; Interleukin-6 ; analysis ; Oligonucleotides ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor ; antagonists & inhibitors ; genetics ; metabolism

Interferon (IFN)-mediated pathways are a crucial part of the cellular response against viral infection. Type III IFNs, which include IFN-λ1, 2 and 3, mediate antiviral responses similar to Type I IFNs via a distinct receptor complex. IFN-λ1 is more effective than the other two members. Transcription of IFN-λ1 requires activation of IRF3/7 and nuclear factor-kappa B (NF-κB), similar to the transcriptional mechanism of Type I IFNs. Using reporter assays, we discovered that viral infection induced both IFN-λ1 promoter activity and that of the 3'-untranslated region (UTR), indicating that IFN-λ1 expression is also regulated at the post-transcriptional level. After analysis with microRNA (miRNA) prediction programs and 3'UTR targeting site assays, the miRNA-548 family, including miR-548b-5p, miR-548c-5p, miR-548i, miR-548j, and miR-548n, was identified to target the 3'UTR of IFN-λ1. Further study demonstrated that miRNA-548 mimics down-regulated the expression of IFN-λ1. In contrast, their inhibitors, the complementary RNAs, enhanced the expression of IFN-λ1 and IFN-stimulated genes. Furthermore, miRNA-548 mimics promoted infection by enterovirus-71 (EV71) and vesicular stomatitis virus (VSV), whereas their inhibitors significantly suppressed the replication of EV71 and VSV. Endogenous miRNA-548 levels were suppressed during viral infection. In conclusion, our results suggest that miRNA-548 regulates host antiviral response via direct targeting of IFN-λ1, which may offer a potential candidate for antiviral therapy.
3' Untranslated Regions ; Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; Base Sequence ; Down-Regulation ; drug effects ; Female ; Hep G2 Cells ; Hepatitis B, Chronic ; drug therapy ; metabolism ; pathology ; Humans ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Regulatory Factor-7 ; metabolism ; Interleukins ; antagonists & inhibitors ; genetics ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; MicroRNAs ; metabolism ; Middle Aged ; NF-kappa B ; metabolism ; Poly I-C ; pharmacology ; therapeutic use ; Promoter Regions, Genetic ; RNA Interference ; RNA, Small Interfering ; metabolism

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.
ATP-Binding Cassette Transporters/*genetics ; Blotting, Western ; Cells, Cultured ; Endothelial Cells/metabolism/*virology ; GTP-Binding Proteins/*genetics ; *Gene Expression Regulation ; Hantaan virus/*immunology ; Histocompatibility Antigens Class I/analysis ; Humans ; Interferon Regulatory Factor-3/genetics ; Interferon Regulatory Factor-7/*genetics ; Interferons/*genetics ; RNA, Messenger/analysis

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.
Viral Matrix Proteins/*biosynthesis/metabolism ; Trans-Activation (Genetics) ; Signal Transduction ; RNA, Messenger/metabolism ; Plasmids/metabolism ; Microscopy, Fluorescence ; Interferon Regulatory Factor-7/*biosynthesis ; Immunoprecipitation ; Humans ; Herpesvirus 4, Human/metabolism ; *Gene Expression Regulation ; Cytoplasm/metabolism ; Cell Line, Tumor ; B-Lymphocytes/metabolism/virology

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.
Cell Line* ; Cytoplasm ; Fluorescence ; Herpesvirus 4, Human ; Interferon Regulatory Factor-7 ; Membrane Proteins

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.
Cell Line* ; Cytoplasm ; Fluorescence ; Herpesvirus 4, Human ; Interferon Regulatory Factor-7 ; Membrane Proteins