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3.
Cell Biosci ; 12(1): 36, 2022 Mar 22.
Article in English | MEDLINE | ID: mdl-35317858

ABSTRACT

BACKGROUND: SARS-CoV-2 is the causative agent of COVID-19. Overproduction and release of proinflammatory cytokines are the underlying cause of severe COVID-19. Treatment of this condition with JAK inhibitors is a double-edged sword, which might result in the suppression of proinflammatory cytokine storm and the concurrent enhancement of viral infection, since JAK signaling is essential for host antiviral response. Improving the current JAK inhibitor therapy requires a detailed molecular analysis on how SARS-CoV-2 modulates interferon (IFN)-induced activation of JAK-STAT signaling. RESULTS: In this study, we focused on the molecular mechanism by which SARS-CoV-2 NSP13 helicase suppresses IFN signaling. Expression of SARS-CoV-2 NSP13 alleviated transcriptional activity driven by type I and type II IFN-responsive enhancer elements. It also prevented nuclear translocation of STAT1 and STAT2. The suppression of NSP13 on IFN signaling occurred at the step of STAT1 phosphorylation. Nucleic acid binding-defective mutant K345A K347A and NTPase-deficient mutant E375A of NSP13 were found to have largely lost the ability to suppress IFN-ß-induced STAT1 phosphorylation and transcriptional activation, indicating the requirement of the helicase activity for NSP13-mediated inhibition of STAT1 phosphorylation. NSP13 did not interact with JAK1 nor prevent STAT1-JAK1 complex formation. Mechanistically, NSP13 interacted with STAT1 to prevent JAK1 kinase from phosphorylating STAT1. CONCLUSION: SARS-CoV-2 NSP13 helicase broadly suppresses IFN signaling by targeting JAK1 phosphorylation of STAT1.

4.
Int J Biol Sci ; 17(6): 1547-1554, 2021.
Article in English | MEDLINE | ID: mdl-33907518

ABSTRACT

Suppression of type I interferon (IFN) response is one pathological outcome of the infection of highly pathogenic human coronaviruses. To effect this, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 encode multiple IFN antagonists. In this study, we reported on the IFN antagonism of SARS-CoV-2 main protease NSP5. NSP5 proteins of both SARS-CoV and SARS-CoV-2 counteracted Sendai virus-induced IFN production. NSP5 variants G15S and K90R commonly seen in circulating strains of SARS-CoV-2 retained the IFN-antagonizing property. The suppressive effect of NSP5 on IFN-ß gene transcription induced by RIG-I, MAVS, TBK1 and IKKϵ suggested that NSP5 likely acts at a step downstream of IRF3 phosphorylation in the cytoplasm. NSP5 did not influence steady-state expression or phosphorylation of IRF3, suggesting that IRF3, regardless of its phosphorylation state, might not be the substrate of NSP5 protease. However, nuclear translocation of phosphorylated IRF3 was severely compromised in NSP5-expressing cells. Taken together, our work revealed a new mechanism by which NSP5 proteins encoded by SARS-CoV and SARS-CoV-2 antagonize IFN production by retaining phosphorylated IRF3 in the cytoplasm. Our findings have implications in rational design and development of antiviral agents against SARS-CoV-2.


Subject(s)
Cell Nucleus/metabolism , Coronavirus 3C Proteases/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , SARS-CoV-2/enzymology , Animals , COVID-19/virology , Chlorocebus aethiops , Humans , Phosphorylation , Protein Transport , Vero Cells
5.
Cell ; 184(8): 2212-2228.e12, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33713620

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause acute respiratory disease and multiorgan failure. Finding human host factors that are essential for SARS-CoV-2 infection could facilitate the formulation of treatment strategies. Using a human kidney cell line-HK-2-that is highly susceptible to SARS-CoV-2, we performed a genome-wide RNAi screen and identified virus dependency factors (VDFs), which play regulatory roles in biological pathways linked to clinical manifestations of SARS-CoV-2 infection. We found a role for a secretory form of SARS-CoV-2 receptor, soluble angiotensin converting enzyme 2 (sACE2), in SARS-CoV-2 infection. Further investigation revealed that SARS-CoV-2 exploits receptor-mediated endocytosis through interaction between its spike with sACE2 or sACE2-vasopressin via AT1 or AVPR1B, respectively. Our identification of VDFs and the regulatory effect of sACE2 on SARS-CoV-2 infection shed insight into pathogenesis and cell entry mechanisms of SARS-CoV-2 as well as potential treatment strategies for COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Host Microbial Interactions/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vasopressins/immunology , Virus Internalization , COVID-19/immunology , COVID-19/virology , Cell Line , Humans , Protein Binding
6.
Cancer Cell Int ; 20: 403, 2020.
Article in English | MEDLINE | ID: mdl-32855620

ABSTRACT

BACKGROUND: Long non-coding RNA (lncRNA) BM742401 is a tumor suppressor in gastric cancer and chronic lymphocytic leukemia. As the promoter and coding region of BM742401 are fully embedded in a CpG island, we hypothesized that BM742401 is a tumor suppressor lncRNA epigenetically silenced by promoter DNA methylation in multiple myeloma. METHODS: Methylation-specific PCR and quantitative bisulfite pyrosequencing were performed to detect the methylation of BM742401 in normal plasma cells, myeloma cell lines and primary myeloma samples. The expression of BM742401 was measured by qRT-PCR. The function of BM742401 in multiple myeloma cells was analyzed by lentivirus transduction followed by migration assay. RESULTS: BM742401 methylation was detected in 10 (66.7%) myeloma cell lines but not normal plasma cells, and inversely correlated with expression of BM742401. In primary samples, BM742401 methylation was detected in 3 (12.5%) monoclonal gammopathy of undetermined significance, 9 (15.8%) myeloma at diagnosis and 8 (17.0%) myeloma at relapse/progression. Moreover, BM742401 methylation at diagnosis was associated with inferior overall survival (median OS: 25 vs. 39 months; P = 0.0496). In myeloma cell line JJN-3, stable overexpression of BM742401 by lentivirus transduction resulted in reduced cell migration (P = 0.0001) but not impacting cell death or proliferation. CONCLUSIONS: This is the first report of tumor-specific methylation-mediated silencing of BM742401 in myeloma, which is likely an early event in myelomagenesis with adverse impact on overall survival. Moreover, BM742401 is a tumor suppressor lncRNA by inhibiting myeloma cell migration, hence implicated in myeloma plasma cell homing, metastasis and disease progression.

7.
J Immunol ; 205(6): 1564-1579, 2020 09 15.
Article in English | MEDLINE | ID: mdl-32747502

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus causing severe disease and mortality. MERS-CoV infection failed to elicit robust IFN response, suggesting that the virus might have evolved strategies to evade host innate immune surveillance. In this study, we identified and characterized type I IFN antagonism of MERS-CoV open reading frame (ORF) 8b accessory protein. ORF8b was abundantly expressed in MERS-CoV-infected Huh-7 cells. When ectopically expressed, ORF8b inhibited IRF3-mediated IFN-ß expression induced by Sendai virus and poly(I:C). ORF8b was found to act at a step upstream of IRF3 to impede the interaction between IRF3 kinase IKKε and chaperone protein HSP70, which is required for the activation of IKKε and IRF3. An infection study using recombinant wild-type and ORF8b-deficient MERS-CoV further confirmed the suppressive role of ORF8b in type I IFN induction and its disruption of the colocalization of HSP70 with IKKε. Ectopic expression of HSP70 relieved suppression of IFN-ß expression by ORF8b in an IKKε-dependent manner. Enhancement of IFN-ß induction in cells infected with ORF8b-deficient virus was erased when HSP70 was depleted. Taken together, HSP70 chaperone is important for IKKε activation, and MERS-CoV ORF8b suppresses type I IFN expression by competing with IKKε for interaction with HSP70.


Subject(s)
Enzyme Activation/immunology , I-kappa B Kinase/immunology , Interferon Type I/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Viral Proteins/immunology , Betacoronavirus , COVID-19 , Cell Line , Coronavirus Infections , HSP70 Heat-Shock Proteins/immunology , HSP70 Heat-Shock Proteins/metabolism , Humans , I-kappa B Kinase/metabolism , Interferon Type I/metabolism , Middle East Respiratory Syndrome Coronavirus/metabolism , Pandemics , Pneumonia, Viral , SARS-CoV-2 , Viral Proteins/metabolism
8.
FASEB J ; 33(8): 8865-8877, 2019 08.
Article in English | MEDLINE | ID: mdl-31034780

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1ß gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro-IL-1ß transcription through activation of NF-κB, which was mediated by TRAF3-dependent ubiquitination and processing of p105. ORF3a-induced elevation of IL-1ß secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3-dependent K63-linked ubiquitination of ASC was more pronounced in SARS-CoV-infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS-CoV ORF3a protein activates NF-κB and the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of p105 and ASC.-Siu, K.-L., Yuen, K.-S., Castaño-Rodriguez, C., Ye, Z.-W., Yeung, M.-L., Fung, S.-Y., Yuan, S., Chan, C.-P., Yuen, K.-Y., Enjuanes, L., Jin, D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.


Subject(s)
CARD Signaling Adaptor Proteins/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Ubiquitination , Viral Structural Proteins/metabolism , A549 Cells , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Inflammasomes/metabolism , Severe acute respiratory syndrome-related coronavirus/metabolism , TNF Receptor-Associated Factor 3/metabolism , Vero Cells
9.
EMBO Rep ; 19(10)2018 10.
Article in English | MEDLINE | ID: mdl-30104205

ABSTRACT

Mouse p202 is a disease locus for lupus and a dominant-negative inhibitor of AIM2 inflammasome activation. A human homolog of p202 has not been identified so far. Here, we report a novel transcript isoform of human IFI16-designated IFI16-ß, which has a domain architecture similar to that of mouse p202. Like p202, IFI16-ß contains two HIN domains, but lacks the pyrin domain. IFI16-ß is ubiquitously expressed in various human tissues and cells. Its mRNA levels are also elevated in leukocytes of patients with lupus, virus-infected cells, and cells treated with interferon-ß or phorbol ester. IFI16-ß co-localizes with AIM2 in the cytoplasm, whereas IFI16-α is predominantly found in the nucleus. IFI16-ß interacts with AIM2 to impede the formation of a functional AIM2-ASC complex. In addition, IFI16-ß sequesters cytoplasmic dsDNA and renders it unavailable for AIM2 sensing. Enforced expression of IFI16-ß inhibits the activation of AIM2 inflammasome, whereas knockdown of IFI16-ß augments interleukin-1ß secretion triggered by dsDNA but not dsRNA Thus, cytoplasm-localized IFI16-ß is functionally equivalent to mouse p202 that exerts an inhibitory effect on AIM2 inflammasome.


Subject(s)
DNA-Binding Proteins/genetics , Inflammasomes/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , Animals , Cell Nucleus/genetics , DNA/genetics , DNA-Binding Proteins/antagonists & inhibitors , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Interleukin-1beta/genetics , Mice , Protein Isoforms/genetics , RNA, Double-Stranded/genetics , RNA, Messenger/genetics
10.
J Med Chem ; 61(2): 583-598, 2018 01 25.
Article in English | MEDLINE | ID: mdl-28692295

ABSTRACT

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Proteolysis/drug effects , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Cell Line , Cell Proliferation/drug effects , Chemistry Techniques, Synthetic , Fluorescence Polarization , Genes, ras , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Molecular Structure , Mutation , Protein Serine-Threonine Kinases/genetics , RNA Interference , Structure-Activity Relationship , Von Hippel-Lindau Tumor Suppressor Protein/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/genetics
11.
Proc Natl Acad Sci U S A ; 113(26): 7124-9, 2016 06 28.
Article in English | MEDLINE | ID: mdl-27274052

ABSTRACT

Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.


Subject(s)
Antineoplastic Agents/administration & dosage , Nuclear Proteins/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins , Cell Line, Tumor , Humans , Male , Mice , Nuclear Proteins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Protein Serine-Threonine Kinases/genetics , Proteolysis , RNA-Binding Proteins/genetics , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcription Factors/genetics
12.
Emerg Microbes Infect ; 5: e39, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-27094905

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) infection has claimed hundreds of lives and has become a global threat since its emergence in Saudi Arabia in 2012. The ability of MERS-CoV to evade the host innate antiviral response may contribute to its severe pathogenesis. Many MERS-CoV-encoded proteins were identified to have interferon (IFN)-antagonizing properties, which correlates well with the reduced IFN levels observed in infected patients and ex vivo models. In this study, we fully characterized the IFN-antagonizing property of the MERS-CoV M protein. Expression of MERS-CoV M protein suppressed type I IFN expression in response to Sendai virus infection or poly(I:C) induction. This suppressive effect was found to be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-κB. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3-TBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response.


Subject(s)
Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Phosphotransferases , Protein Serine-Threonine Kinases/metabolism , Severe acute respiratory syndrome-related coronavirus/pathogenicity , Viral Matrix Proteins/physiology , Coronavirus M Proteins , DEAD Box Protein 58/genetics , Gene Expression Regulation , HEK293 Cells , Humans , Immune Evasion , Immunity, Innate , Interferon Regulatory Factor-3/immunology , Interferon Type I/antagonists & inhibitors , Interferon Type I/genetics , Interferon Type I/immunology , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/physiology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/immunology , Saudi Arabia , Sendai virus/genetics , Sendai virus/immunology , Sequence Alignment , TNF Receptor-Associated Factor 3/genetics , TNF Receptor-Associated Factor 3/immunology , Viral Matrix Proteins/genetics
13.
Int J Biochem Cell Biol ; 61: 53-62, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25681686

ABSTRACT

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Neurites/metabolism , Neurons/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Receptor, ErbB-4/metabolism , Animals , Cell Survival/physiology , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Neuregulins/biosynthesis , Neuregulins/genetics , Neuregulins/metabolism , Neurons/cytology , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Quantitative Trait Loci , Receptor, ErbB-4/genetics , Signal Transduction , Transfection
14.
Sci Rep ; 5: 7897, 2015 Jan 20.
Article in English | MEDLINE | ID: mdl-25600293

ABSTRACT

Ferredoxins are iron-sulfur proteins that play important roles in electron transport and redox homeostasis. Yeast Apd1p is a novel member of the family of thioredoxin-like ferredoxins. In this study, we characterized the hydroxyurea (HU)-hypersensitive phenotype of apd1Δ cells. HU is an inhibitor of DNA synthesis, a cellular stressor and an anticancer agent. Although the loss of APD1 did not influence cell proliferation or cell cycle progression, it resulted in HU sensitivity. This sensitivity was reverted in the presence of antioxidant N-acetyl-cysteine, implicating a role for intracellular redox. Mutation of the iron-binding motifs in Apd1p abrogated its ability to rescue HU sensitivity in apd1Δ cells. The iron-binding activity of Apd1p was verified by a color assay. By mass spectrometry two irons were found to be incorporated into one Apd1p protein molecule. Surprisingly, ribonucleotide reductase genes were not induced in apd1Δ cells and the HU sensitivity was unaffected when dNTP production was boosted. A suppressor screen was performed and the expression of stress-regulated transcription factor Yap1p was found to effectively rescue the HU sensitivity in apd1Δ cells. Taken together, our work identified Apd1p as a new ferredoxin which serves critical roles in cellular defense against HU.


Subject(s)
DNA Replication/genetics , Ferredoxins/genetics , Hydroxyurea/pharmacology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Acetylcysteine/chemistry , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , DNA Replication/drug effects , Ferredoxins/chemistry , Iron/chemistry , Oxidation-Reduction , Phenotype , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism
15.
J Virol ; 88(9): 4866-76, 2014 May.
Article in English | MEDLINE | ID: mdl-24522921

ABSTRACT

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity. IMPORTANCE: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing but also might prove useful in revealing new strategies for the development of vaccines and antivirals.


Subject(s)
Coronavirus/immunology , DEAD-box RNA Helicases/antagonists & inhibitors , Host-Pathogen Interactions , Interferons/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Viral Proteins/metabolism , Cell Line , DEAD Box Protein 58 , Humans , Immune Evasion , Interferon-Induced Helicase, IFIH1 , Protein Binding , Protein Interaction Mapping , Receptors, Immunologic
16.
Cell Mol Immunol ; 11(2): 141-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24509444

ABSTRACT

Coronaviruses have developed various measures to evade innate immunity. We have previously shown that severe acute respiratory syndrome (SARS) coronavirus M protein suppresses type I interferon (IFN) production by impeding the formation of functional TRAF3-containing complex. In this study, we demonstrate that the IFN-antagonizing activity is specific to SARS coronavirus M protein and is mediated through its first transmembrane domain (TM1) located at the N terminus. M protein from human coronavirus HKU1 does not inhibit IFN production. Whereas N-linked glycosylation of SARS coronavirus M protein has no influence on IFN antagonism, TM1 is indispensable for the suppression of IFN production. TM1 targets SARS coronavirus M protein and heterologous proteins to the Golgi apparatus, yet Golgi localization is required but not sufficient for IFN antagonism. Mechanistically, TM1 is capable of binding with RIG-I, TRAF3, TBK1 and IKKε, and preventing the interaction of TRAF3 with its downstream effectors. Our work defines the molecular architecture of SARS coronavirus M protein required for suppression of innate antiviral response.


Subject(s)
Coronavirus/immunology , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Severe Acute Respiratory Syndrome/immunology , Viral Matrix Proteins/metabolism , Coronavirus M Proteins , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/metabolism , Immune Evasion , Immunity, Innate , Immunosuppression Therapy , Interferon Type I/metabolism , Membrane Proteins/genetics , Mutation/genetics , Protein Binding/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Sorting Signals/genetics , Protein Structure, Tertiary/genetics , Severe Acute Respiratory Syndrome/virology , TNF Receptor-Associated Factor 3/metabolism , Viral Matrix Proteins/genetics
17.
Cell Biosci ; 4(1): 3, 2014 Jan 13.
Article in English | MEDLINE | ID: mdl-24410900

ABSTRACT

BACKGROUND: Whereas severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is associated with severe disease, human coronavirus HKU1 (HCoV-HKU1) commonly circulates in the human populations causing generally milder illness. Spike (S) protein of SARS-CoV activates the unfolded protein response (UPR). It is not understood whether HCoV-HKU1 S protein has similar activity. In addition, the UPR-activating domain in SARS-CoV S protein remains to be identified. RESULTS: In this study we compared S proteins of SARS-CoV and HCoV-HKU1 for their ability to activate the UPR. Both S proteins were found in the endoplasmic reticulum. Transmembrane serine protease TMPRSS2 catalyzed the cleavage of SARS-CoV S protein, but not the counterpart in HCoV-HKU1. Both S proteins showed a similar pattern of UPR-activating activity. Through PERK kinase they activated the transcription of UPR effector genes such as Grp78, Grp94 and CHOP. N-linked glycosylation was not required for the activation of the UPR by S proteins. S1 subunit of SARS-CoV but not its counterpart in HCoV-HKU1 was capable of activating the UPR. A central region (amino acids 201-400) of SARS-CoV S1 was required for this activity. CONCLUSIONS: SARS-CoV and HCoV-HKU1 S proteins use distinct UPR-activating domains to exert the same modulatory effects on UPR signaling.

18.
Bioorg Med Chem Lett ; 23(16): 4517-22, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23850198

ABSTRACT

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.


Subject(s)
MAP Kinase Kinase Kinases/antagonists & inhibitors , Pyridines , Amines/chemical synthesis , Amines/chemistry , Amines/pharmacology , Animals , Crystallography, X-Ray , Drug Design , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Furans/chemical synthesis , Furans/chemistry , Furans/pharmacology , Humans , Inhibitory Concentration 50 , Mice , Molecular Structure , Neoplasms/drug therapy , Pyridines/chemical synthesis , Pyridines/pharmacokinetics , Pyridines/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor Assays
19.
Bioorg Med Chem Lett ; 23(4): 979-84, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23317569

ABSTRACT

This Letter describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.


Subject(s)
Imidazoles/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazines/chemistry , Administration, Oral , Animals , Humans , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , Mice , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Structure-Activity Relationship
20.
ACS Med Chem Lett ; 4(7): 627-31, 2013 Jul 11.
Article in English | MEDLINE | ID: mdl-24900721

ABSTRACT

This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure-activity and structure-kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t 1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties.

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