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2.
Cell Death Dis ; 13(8): 707, 2022 08 15.
Article in English | MEDLINE | ID: mdl-35970851

ABSTRACT

RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5'-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.


Subject(s)
Antiviral Agents , Toll-Like Receptor 3 , Antiviral Agents/pharmacology , Apoptosis , Interferon-beta/genetics , RNA, Double-Stranded/genetics , RNA, Viral/metabolism , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
3.
Mol Cell Biol ; 40(5)2020 02 12.
Article in English | MEDLINE | ID: mdl-31818880

ABSTRACT

TRIF is an essential adaptor for Toll-like receptor 3/4 (TLR3/4) signaling to activate transcription factor interferon regulatory factor 3 (IRF-3). We examined the molecular mechanism of TLR3 signaling and found that TLR3 stimulation by double-stranded RNA (dsRNA) induces phosphorylation of TRIF at Ser210 and is required for IRF-3 recruitment. TANK-binding kinase 1 (TBK1) is known to be responsible for IRF-3 phosphorylation and activation. We found that TBK1 is also responsible for phosphorylation of Ser210 in TRIF. Unexpectedly, we discovered that IκB kinase ß (IKKß) plays an essential role in TLR3/4 signaling using a pharmacological inhibitor and gene deletion. Of note, IKKß is essential in TLR3/4 but not in retinoic acid-inducible gene I (RIG-I) signaling. Mechanistically, IKKß transiently associates with and induces the phosphorylation of TBK1 upon TLR3 stimulation. These results suggest a phosphorylation cascade of IKKß and TBK1, where priming phosphorylation of TBK1 by IKKß is required to surpass the threshold to induce signaling, thereby activating IRF-3.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , I-kappa B Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Adaptor Proteins, Vesicular Transport/genetics , HEK293 Cells , Humans , I-kappa B Kinase/genetics , Interferon Regulatory Factor-3/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Serine/metabolism , Toll-Like Receptor 3/genetics , Toll-Like Receptor 4/genetics
4.
Int Immunol ; 31(12): 811-821, 2019 11 08.
Article in English | MEDLINE | ID: mdl-31367737

ABSTRACT

Double-stranded RNA (dsRNA) is well characterized as an inducer of anti-viral interferon responses. We previously reported that dsRNA extracted from a specific edible plant possesses an immune-modulating capacity to confer, in mice, resistance against respiratory viruses, including the H1N1 strain of the influenza A virus (IAV). We report here that the systemic immune-activating capacity of the plant-derived dsRNA protected mice from infection by a highly virulent H5N1 strain of the IAV. In addition, subcutaneous inoculation of the dsRNA together with the inactivated virion of the H5N1 strain of the IAV suppressed the lethality of the viral infection as compared with individual inoculation of either dsRNA or HA protein, suggesting its potential usage as a vaccination adjuvant. Moreover, intra-peritoneal inoculation of the dsRNA limited the growth of B16-F10 melanoma cells through the activation of NK cells in murine models. Taken together, this study demonstrated the systemic immune-modulating capacity of a plant-derived dsRNA and its potential for nucleic acid-based clinical applications.


Subject(s)
Capsicum/chemistry , RNA, Double-Stranded/immunology , Animals , Capsicum/immunology , Cells, Cultured , Interferon Type I/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , RNA, Double-Stranded/isolation & purification , RNA, Double-Stranded/metabolism , Ribonucleases/metabolism
5.
PLoS One ; 14(2): e0212233, 2019.
Article in English | MEDLINE | ID: mdl-30779774

ABSTRACT

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.


Subject(s)
Antiviral Agents/pharmacology , Cell Nucleus/metabolism , DNA, Viral/metabolism , Dicumarol/pharmacology , Hepatitis B virus/metabolism , Plasmids/metabolism , Cell Nucleus/genetics , Cell Nucleus/virology , DNA, Viral/genetics , Drug Evaluation, Preclinical , Genetic Vectors , HEK293 Cells , Hep G2 Cells , Hepatitis B virus/genetics , Humans , Lentivirus , Plasmids/genetics , RNA, Viral/genetics , RNA, Viral/metabolism
6.
Proc Natl Acad Sci U S A ; 111(24): 8877-82, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24889612

ABSTRACT

Paired Ig-like type 2 receptor α (PILRα) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILRα. Furthermore, we determined the crystal structures of PILRα and its complex with an sTn and its attached peptide region. The structures show that PILRα exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.


Subject(s)
Membrane Glycoproteins/chemistry , Mucins/chemistry , Peptides/chemistry , Polysaccharides/chemistry , Receptors, Immunologic/chemistry , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Glycosylation , HEK293 Cells , Humans , Immune System , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Surface Plasmon Resonance
7.
PLoS One ; 8(10): e75297, 2013.
Article in English | MEDLINE | ID: mdl-24130701

ABSTRACT

To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca²âº release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca²âº/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4⁺ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca²âº signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.


Subject(s)
Calcium/metabolism , HIV-1/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Vesicular Transport Proteins/metabolism , Cell Line , Flow Cytometry , Humans , Immunoblotting , Immunoprecipitation , Jurkat Cells , Microscopy, Confocal , Protein Binding , Ryanodine Receptor Calcium Release Channel/genetics , Two-Hybrid System Techniques , Vesicular Transport Proteins/genetics
8.
Biochem Biophys Res Commun ; 405(3): 428-33, 2011 Feb 18.
Article in English | MEDLINE | ID: mdl-21241660

ABSTRACT

PILRα is an immune inhibitory receptor possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain enabling it to deliver inhibitory signals. Binding of PILRα to its ligand CD99 is involved in immune regulation; however, whether there are other PILRα ligands in addition to CD99 is not known. Here, we report that a novel molecule, PILR-associating neural protein (PANP), acts as an additional ligand for PILRα. Transcription of PANP was mainly observed in neural tissues. PILRα-Ig fusion protein bound cells transfected with PANP and the transfectants stimulated PILRα reporter cells. Specific O-glycan structures on PANP were found to be required for PILR recognition of this ligand. These results suggest that PANP is involved in immune regulation as a ligand of the PILRα.


Subject(s)
Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Humans , Ligands , Melanoma, Experimental , Mice , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Rats , Rats, Wistar
9.
Cell ; 132(6): 935-44, 2008 Mar 21.
Article in English | MEDLINE | ID: mdl-18358807

ABSTRACT

Glycoprotein B (gB) is one of the essential components for infection by herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with glycoprotein D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have been identified, specific molecules that mediate HSV-1 infection by associating with gB have not been elucidated. Here, we found that paired immunoglobulin-like type 2 receptor (PILR) alpha associates with gB, and cells transduced with PILRalpha become susceptible to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing both HVEM and PILRalpha was blocked by either anti-PILRalpha or anti-HVEM antibody. Our results demonstrate that cellular receptors for both gB and gD are required for HSV-1 infection and that PILRalpha plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.


Subject(s)
Herpes Simplex/metabolism , Herpesvirus 1, Human/metabolism , Membrane Glycoproteins/metabolism , Receptors, Immunologic/metabolism , Receptors, Virus/metabolism , Viral Envelope Proteins/metabolism , Animals , Antibodies, Monoclonal/metabolism , CHO Cells , Cell Line , Cells, Cultured , Cricetinae , Cricetulus , Herpes Simplex/virology , Humans , Transfection
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