Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

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

The innate immune system initiates innate immune responses by recognizing pathogen-related molecular patterns on the surface of pathogenic microorganisms through pattern recognition receptors. Through cascade signal transduction, it activates downstream transcription factors NF-κB and interferon regulatory factors (IRFs), and then leads to the production of inflammatory cytokines and type Ⅰ interferon, which resists the infection of pathogenic microorganism. TBK1 is a central adapter protein of innate immune signaling pathway and can activate both NF-κB and IRFs. It is a key protein kinase in the process of anti-infection. The finetuning regulation of TBK1 is essential to maintain immune homeostasis and resist pathogen invasion. This paper reviews the biological functions and ubiquitin modification of TBK1 in innate immunity, to provide theoretical basis for clinical treatment of pathogenic infections and autoimmune diseases.
Immunity, Innate ; Interferon Regulatory Factor-3/metabolism* ; Protein-Serine-Threonine Kinases/genetics* ; Signal Transduction ; Ubiquitin

The innate immune response is the first line of host defense to eliminate viral infection. Pattern recognition receptors in the cytosol, such as RIG-I-like receptors (RLR) and Nod-like receptors (NLR), and membrane bound Toll like receptors (TLR) detect viral infection and initiate transcription of a cohort of antiviral genes, including interferon (IFN) and interferon stimulated genes (ISGs), which ultimately block viral replication. Another mechanism to reduce viral spread is through RIPA, the RLR-induced IRF3-mediated pathway of apoptosis, which causes infected cells to undergo premature death. The transcription factor IRF3 can mediate cellular antiviral responses by both inducing antiviral genes and triggering apoptosis through the activation of RIPA. The mechanism of IRF3 activation in RIPA is distinct from that of transcriptional activation; it requires linear polyubiquitination of specific lysine residues of IRF3. Using RIPA-active, but transcriptionally inactive, IRF3 mutants, it was shown that RIPA can prevent viral replication and pathogenesis in mice.
Animals ; Apoptosis ; DEAD Box Protein 58 ; genetics ; immunology ; metabolism ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3 ; genetics ; immunology ; metabolism ; Mice ; Virus Diseases ; genetics ; immunology ; metabolism

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.
DNA, Bacterial ; immunology ; metabolism ; DNA, Viral ; immunology ; metabolism ; Gene Expression Regulation ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3 ; genetics ; immunology ; Interferon Type I ; biosynthesis ; immunology ; Membrane Proteins ; genetics ; immunology ; Models, Molecular ; NF-kappa B ; genetics ; immunology ; Nucleotides, Cyclic ; biosynthesis ; immunology ; Nucleotidyltransferases ; genetics ; immunology ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.
Adaptor Proteins, Signal Transducing ; biosynthesis ; chemistry ; genetics ; metabolism ; Cell Aggregation ; genetics ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; HSC70 Heat-Shock Proteins ; genetics ; metabolism ; Heat-Shock Response ; genetics ; Humans ; Interferon Regulatory Factor-3 ; genetics ; metabolism ; Interferon-beta ; genetics ; NF-kappa B ; genetics ; Prions ; metabolism ; Receptors, Retinoic Acid ; metabolism ; Viruses ; drug effects ; metabolism ; pathogenicity

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

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Leukemic ; drug effects ; Genes, Regulator ; drug effects ; Humans ; Interferon Regulatory Factor-1 ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; STAT2 Transcription Factor ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

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