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Cheng H, Zhang H, Cai H, Liu M, Wen S, Ren J. Molecular biology of canine parainfluenza virus V protein and its potential applications in tumor immunotherapy. Front Microbiol 2023; 14:1282112. [PMID: 38173672 PMCID: PMC10761501 DOI: 10.3389/fmicb.2023.1282112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Accepted: 11/29/2023] [Indexed: 01/05/2024] Open
Abstract
Canine parainfluenza virus (CPIV) is a zoonotic virus that is widely distributed and is the main pathogen causing canine infectious respiratory disease (CIRD), also known as "kennel cough," in dogs. The CPIV-V protein is the only nonstructural protein of the virus and plays an important role in multiple stages of the virus life cycle by inhibiting apoptosis, altering the host cell cycle and interfering with the interferon response. In addition, studies have shown that the V protein has potential applications in the field of immunotherapy in oncolytic virus therapy or self-amplifying RNA vaccines. In this review, the biosynthesis, structural characteristics and functions of the CPIV-V protein are reviewed with an emphasis on how it facilitates viral immune escape and its potential applications in the field of immunotherapy. Therefore, this review provides a scientific basis for research into the CPIV-V protein and its potential applications.
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Affiliation(s)
- Huai Cheng
- Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou, China
| | - Hewei Zhang
- College of Food and Drugs, Luoyang Polytechnic, Luoyang, China
- Animal Diseases and Public Health Engineering Research Center of Henan Province, Luoyang, China
| | - Huanchang Cai
- Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou, China
| | - Min Liu
- Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou, China
| | - Shubo Wen
- Preventive Veterinary Laboratory, College of Animal Science and Technology, Inner Mongolia Minzu University, Tongliao, China
| | - Jingqiang Ren
- Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou, China
- Animal Diseases and Public Health Engineering Research Center of Henan Province, Luoyang, China
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Le‐Trilling VTK, Banchenko S, Paydar D, Leipe PM, Binting L, Lauer S, Graziadei A, Klingen R, Gotthold C, Bürger J, Bracht T, Sitek B, Jan Lebbink R, Malyshkina A, Mielke T, Rappsilber J, Spahn CMT, Voigt S, Trilling M, Schwefel D. Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor. EMBO J 2023; 42:e112351. [PMID: 36762436 PMCID: PMC9975947 DOI: 10.15252/embj.2022112351] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2022] [Revised: 12/15/2022] [Accepted: 12/21/2022] [Indexed: 02/11/2023] Open
Abstract
Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.
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Affiliation(s)
| | - Sofia Banchenko
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
| | - Darius Paydar
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
- Zentrum für KinderpsychiatrieUniversitätsklinik ZürichZürichSwitzerland
| | - Pia Madeleine Leipe
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
| | - Lukas Binting
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
| | - Simon Lauer
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
| | - Andrea Graziadei
- Bioanalytics Unit, Institute of BiotechnologyTechnische Universität BerlinBerlinGermany
| | - Robin Klingen
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
| | - Christine Gotthold
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
| | - Jörg Bürger
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
- Microscopy and Cryo‐Electron Microscopy Service GroupMax‐Planck‐Institute for Molecular GeneticsBerlinGermany
| | - Thilo Bracht
- Medizinisches Proteom‐CenterRuhr‐University BochumBochumGermany
- Department of Anesthesia, Intensive Care Medicine and Pain TherapyUniversity Hospital Knappschaftskrankenhaus BochumBochumGermany
| | - Barbara Sitek
- Medizinisches Proteom‐CenterRuhr‐University BochumBochumGermany
- Department of Anesthesia, Intensive Care Medicine and Pain TherapyUniversity Hospital Knappschaftskrankenhaus BochumBochumGermany
| | - Robert Jan Lebbink
- Department of Medical MicrobiologyUniversity Medical Center UtrechtUtrechtthe Netherlands
| | - Anna Malyshkina
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
| | - Thorsten Mielke
- Microscopy and Cryo‐Electron Microscopy Service GroupMax‐Planck‐Institute for Molecular GeneticsBerlinGermany
| | - Juri Rappsilber
- Bioanalytics Unit, Institute of BiotechnologyTechnische Universität BerlinBerlinGermany
- Wellcome Centre for Cell BiologyUniversity of EdinburghEdinburghUK
| | - Christian MT Spahn
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
| | - Sebastian Voigt
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
| | - Mirko Trilling
- Institute for VirologyUniversity Hospital Essen, University of Duisburg‐EssenEssenGermany
| | - David Schwefel
- Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt‐Universität zu BerlinBerlinGermany
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3
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Wick ET, Treadway CJ, Li Z, Nicely NI, Ren Z, Baldwin AS, Xiong Y, Harrison JS, Brown NG. Insight into Viral Hijacking of CRL4 Ubiquitin Ligase through Structural Analysis of the pUL145-DDB1 Complex. J Virol 2022; 96:e0082622. [PMID: 35938868 PMCID: PMC9472758 DOI: 10.1128/jvi.00826-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 07/21/2022] [Indexed: 12/21/2022] Open
Abstract
Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.
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Affiliation(s)
- Elizaveta T. Wick
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Colton J. Treadway
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Zhijun Li
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Nathan I. Nicely
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Zhizhong Ren
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Albert S. Baldwin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Yue Xiong
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Joseph S. Harrison
- Department of Chemistry, University of the Pacific, Stockton, California, USA
| | - Nicholas G. Brown
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
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Barik S. Mechanisms of Viral Degradation of Cellular Signal Transducer and Activator of Transcription 2. Int J Mol Sci 2022; 23:ijms23010489. [PMID: 35008916 PMCID: PMC8745392 DOI: 10.3390/ijms23010489] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Revised: 12/28/2021] [Accepted: 12/31/2021] [Indexed: 12/31/2022] Open
Abstract
Virus infection of eukaryotes triggers cellular innate immune response, a major arm of which is the type I interferon (IFN) family of cytokines. Binding of IFN to cell surface receptors triggers a signaling cascade in which the signal transducer and activator of transcription 2 (STAT2) plays a key role, ultimately leading to an antiviral state of the cell. In retaliation, many viruses counteract the immune response, often by the destruction and/or inactivation of STAT2, promoted by specific viral proteins that do not possess protease activities of their own. This review offers a summary of viral mechanisms of STAT2 subversion with emphasis on degradation. Some viruses also destroy STAT1, another major member of the STAT family, but most viruses are selective in targeting either STAT2 or STAT1. Interestingly, degradation of STAT2 by a few viruses requires the presence of both STAT proteins. Available evidence suggests a mechanism in which multiple sites and domains of STAT2 are required for engagement and degradation by a multi-subunit degradative complex, comprising viral and cellular proteins, including the ubiquitin–proteasomal system. However, the exact molecular nature of this complex and the alternative degradation mechanisms remain largely unknown, as critically presented here with prospective directions of future study.
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Affiliation(s)
- Sailen Barik
- EonBio, 3780 Pelham Drive, Mobile, AL 36619, USA
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Douglas J, Drummond AJ, Kingston RL. Evolutionary history of cotranscriptional editing in the paramyxoviral phosphoprotein gene. Virus Evol 2021; 7:veab028. [PMID: 34141448 PMCID: PMC8204654 DOI: 10.1093/ve/veab028] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The phosphoprotein gene of the paramyxoviruses encodes multiple protein products. The P, V, and W proteins are generated by transcriptional slippage. This process results in the insertion of non-templated guanosine nucleosides into the mRNA at a conserved edit site. The P protein is an essential component of the viral RNA polymerase and is encoded by a faithful copy of the gene in the majority of paramyxoviruses. However, in some cases, the non-essential V protein is encoded by default and guanosines must be inserted into the mRNA in order to encode P. The number of guanosines inserted into the P gene can be described by a probability distribution, which varies between viruses. In this article, we review the nature of these distributions, which can be inferred from mRNA sequencing data, and reconstruct the evolutionary history of cotranscriptional editing in the paramyxovirus family. Our model suggests that, throughout known history of the family, the system has switched from a P default to a V default mode four times; complete loss of the editing system has occurred twice, the canonical zinc finger domain of the V protein has been deleted or heavily mutated a further two times, and the W protein has independently evolved a novel function three times. Finally, we review the physical mechanisms of cotranscriptional editing via slippage of the viral RNA polymerase.
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Affiliation(s)
- Jordan Douglas
- Centre for Computational Evolution, University of Auckland, Auckland 1010, New Zealand
- School of Computer Science, University of Auckland, Auckland 1010, New Zealand
| | - Alexei J Drummond
- Centre for Computational Evolution, University of Auckland, Auckland 1010, New Zealand
- School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
| | - Richard L Kingston
- School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
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6
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Mayor-Ruiz C, Bauer S, Brand M, Kozicka Z, Siklos M, Imrichova H, Kaltheuner IH, Hahn E, Seiler K, Koren A, Petzold G, Fellner M, Bock C, Müller AC, Zuber J, Geyer M, Thomä NH, Kubicek S, Winter GE. Rational discovery of molecular glue degraders via scalable chemical profiling. Nat Chem Biol 2020; 16:1199-1207. [PMID: 32747809 PMCID: PMC7116640 DOI: 10.1038/s41589-020-0594-x] [Citation(s) in RCA: 233] [Impact Index Per Article: 46.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 06/15/2020] [Indexed: 11/09/2022]
Abstract
Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
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Affiliation(s)
- Cristina Mayor-Ruiz
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Sophie Bauer
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Matthias Brand
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Zuzanna Kozicka
- FMI Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
- Faculty of Science, University of Basel, Basel, Switzerland
| | - Marton Siklos
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Hana Imrichova
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | | | - Elisa Hahn
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Kristina Seiler
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Anna Koren
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Georg Petzold
- FMI Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Michaela Fellner
- Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria
| | - Christoph Bock
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
| | - André C Müller
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Johannes Zuber
- Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria
| | - Matthias Geyer
- Institute of Structural Biology, University of Bonn, Bonn, Germany
| | - Nicolas H Thomä
- FMI Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Stefan Kubicek
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Georg E Winter
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
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Syed Lal Badshah, Ullah A, Syed S. The Role of Zinc-Finger Antiviral Proteins in Immunity against Viruses. MOLECULAR GENETICS MICROBIOLOGY AND VIROLOGY 2020. [DOI: 10.3103/s0891416820020020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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Malik T, Ngo L, Bosma T, Rubin S. A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation. Viruses 2019; 11:v11111016. [PMID: 31683999 PMCID: PMC6893744 DOI: 10.3390/v11111016] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Revised: 10/11/2019] [Accepted: 10/30/2019] [Indexed: 01/02/2023] Open
Abstract
Mumps virus (MuV) is a neurotropic non-segmented, negative-stranded, enveloped RNA virus in the Paramyxovirus family. The 15.4 kb genome encodes seven genes, including the V/P, which encodes, among other proteins, the V protein. The MuV V protein has been shown to target the cellular signal transducer and activator of transcription proteins STAT1 and STAT3 for proteasome-mediated degradation. While MuV V protein targeting of STAT1 is generally accepted as a means of limiting innate antiviral responses, the consequence of V protein targeting of STAT3 is less clear. Further, since the MuV V protein targets both STAT1 and STAT3, specifically investigating viral antagonism of STAT3 targeting is challenging. However, a previous study reported that a single amino acid substitution in the MuV V protein (E95D) inhibits targeting of STAT3, but not STAT1. This provided us with a unique opportunity to examine the specific role of STAT 3 in MuV virulence in an in vivo model. Here, using a clone of a wild type MuV strain expressing the E95D mutant V protein, we present data linking inhibition of STAT3 targeting with the accelerated clearance of the virus and reduced neurovirulence in vivo, suggesting its role in promoting antiviral responses. These data suggest a rational approach to virus attenuation that could be exploited for future vaccine development.
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Affiliation(s)
- Tahir Malik
- DVP/Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
| | - Laurie Ngo
- DVP/Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
| | - Trent Bosma
- DVP/Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
| | - Steven Rubin
- GlaxoSmithKline, 14200 Shady Grove Rd, Rockville, MD 20850, USA.
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HBx regulates transcription factor PAX8 stabilization to promote the progression of hepatocellular carcinoma. Oncogene 2019; 38:6696-6710. [PMID: 31391550 DOI: 10.1038/s41388-019-0907-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2018] [Revised: 05/29/2019] [Accepted: 05/30/2019] [Indexed: 02/07/2023]
Abstract
Transcription factor PAX8 expression is upregulated in several types of cancers. However, little is known about the function of PAX8 in the progression of hepatoma and its regulatory mechanisms. Here, we show that PAX8 silencing inhibits the proliferation and clonogenicity of hepatoma cells and its growth in vivo. The HBV X protein (HBx) does not directly interacts, but stabilizes PAX8 by inhibiting proteasome-dependent ubiquitination and degradation. Furthermore, the E3 ubiquitin ligase complex component Skp2 through its LRR domain directly interacts with the Prd domain of PAX8 and targets PAX8 by recognizing its lysine 275 for ubiquitination and degradation in hepatoma cells. In addition, HBx directly interacts and is colocalized with Skp2 to inhibit its recognition and subsequent ubiquitination and degradation of PAX8 in hepatoma cells. Moreover, HBx upregulates the expression and phosphorylation of Aurora A, a serine-threonine kinase, which interacts with and phosphorylates PAX8 at S209 and T277, compromising the Skp2-recognized PAX8 ubiquitination and destabilization. Thus, HBx stabilizes PAX8 protein by inhibiting the Skp2 targeted PAX8 ubiquitination and enhancing the Aurora A-mediated its phosphorylation, contributing to the progression of hepatoma. Our findings suggest that PAX8 may a new target for design of therapies and uncover new insights into the pathogenesis of hepatoma.
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10
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Becker T, Le-Trilling VTK, Trilling M. Cellular Cullin RING Ubiquitin Ligases: Druggable Host Dependency Factors of Cytomegaloviruses. Int J Mol Sci 2019; 20:E1636. [PMID: 30986950 PMCID: PMC6479302 DOI: 10.3390/ijms20071636] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Revised: 03/27/2019] [Accepted: 03/28/2019] [Indexed: 12/20/2022] Open
Abstract
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that frequently causes morbidity and mortality in individuals with insufficient immunity, such as transplant recipients, AIDS patients, and congenitally infected newborns. Several antiviral drugs are approved to treat HCMV infections. However, resistant HCMV mutants can arise in patients receiving long-term therapy. Additionally, side effects and the risk to cause birth defects limit the use of currently approved antivirals against HCMV. Therefore, the identification of new drug targets is of clinical relevance. Recent work identified DNA-damage binding protein 1 (DDB1) and the family of the cellular cullin (Cul) RING ubiquitin (Ub) ligases (CRLs) as host-derived factors that are relevant for the replication of human and mouse cytomegaloviruses. The first-in-class CRL inhibitory compound Pevonedistat (also called MLN4924) is currently under investigation as an anti-tumor drug in several clinical trials. Cytomegaloviruses exploit CRLs to regulate the abundance of viral proteins, and to induce the proteasomal degradation of host restriction factors involved in innate and intrinsic immunity. Accordingly, pharmacological blockade of CRL activity diminishes viral replication in cell culture. In this review, we summarize the current knowledge concerning the relevance of DDB1 and CRLs during cytomegalovirus replication and discuss chances and drawbacks of CRL inhibitory drugs as potential antiviral treatment against HCMV.
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Affiliation(s)
- Tanja Becker
- Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany.
| | | | - Mirko Trilling
- Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany.
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11
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Ohta K, Matsumoto Y, Ito M, Nishio M. Tetherin antagonism by V proteins is a common trait among the genus Rubulavirus. Med Microbiol Immunol 2017; 206:319-326. [PMID: 28466381 DOI: 10.1007/s00430-017-0509-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2017] [Accepted: 04/26/2017] [Indexed: 12/27/2022]
Abstract
Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae, was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus.
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Affiliation(s)
- Keisuke Ohta
- Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1, Kimiidera, Wakayama, 641-8509, Japan
| | - Yusuke Matsumoto
- Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1, Kimiidera, Wakayama, 641-8509, Japan
| | - Morihiro Ito
- Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi, Japan
| | - Machiko Nishio
- Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1, Kimiidera, Wakayama, 641-8509, Japan.
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12
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Slagle BL, Bouchard MJ. Hepatitis B Virus X and Regulation of Viral Gene Expression. Cold Spring Harb Perspect Med 2016; 6:a021402. [PMID: 26747833 DOI: 10.1101/cshperspect.a021402] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The efficient replication of hepatitis B virus (HBV) requires the HBV regulatory hepatitis B virus X (HBx) protein. The exact contributions of HBx are not fully understood, in part because of the limitations of the assays used for its study. When HBV replication is driven from a plasmid DNA, the contribution of HBx is modest. However, there is an absolute requirement for HBx in assays that recapitulate the infectious virus life cycle. There is much evidence that HBx can contribute directly to HBV replication by acting on viral promoters embedded within protein coding sequences. In addition, HBx may also contribute indirectly by modulating cellular pathways to benefit virus replication. Understanding the mechanism(s) of HBx action during virus replication may provide insight into novel ways to disrupt chronic HBV replication.
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Affiliation(s)
- Betty L Slagle
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030
| | - Michael J Bouchard
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102
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Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5. J Virol 2015; 89:11845-57. [PMID: 26378167 DOI: 10.1128/jvi.01832-15] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 09/06/2015] [Indexed: 02/08/2023] Open
Abstract
UNLABELLED Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs.
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14
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Hannah J, Zhou P. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B. Gene 2015; 573:33-45. [PMID: 26344709 DOI: 10.1016/j.gene.2015.08.064] [Citation(s) in RCA: 91] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Revised: 08/03/2015] [Accepted: 08/27/2015] [Indexed: 01/29/2023]
Abstract
The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention.
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Affiliation(s)
- Jeffrey Hannah
- Department of Pathology, Weill Cornell Medical College, 1300 York Ave. NY, NY 10065, United States.
| | - Pengbo Zhou
- Department of Pathology, Weill Cornell Medical College, 1300 York Ave. NY, NY 10065, United States.
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15
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Liu Y, Huang X, He X, Zhou Y, Jiang X, Chen-Kiang S, Jaffrey SR, Xu G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. FASEB J 2015; 29:4829-39. [PMID: 26231201 DOI: 10.1096/fj.15-274050] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 07/27/2015] [Indexed: 12/21/2022]
Abstract
The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.
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Affiliation(s)
- Yaobin Liu
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Xiangao Huang
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Xian He
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Yanqing Zhou
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Xiaogang Jiang
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Selina Chen-Kiang
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Samie R Jaffrey
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
| | - Guoqiang Xu
- *Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China; and Department of Pathology and Laboratory Medicine and Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA
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16
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Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation. Biochem J 2015; 467:365-86. [PMID: 25886174 PMCID: PMC4403949 DOI: 10.1042/bj20141450] [Citation(s) in RCA: 178] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.
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17
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Hoffmann HH, Schneider WM, Rice CM. Interferons and viruses: an evolutionary arms race of molecular interactions. Trends Immunol 2015; 36:124-38. [PMID: 25704559 DOI: 10.1016/j.it.2015.01.004] [Citation(s) in RCA: 308] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Revised: 01/16/2015] [Accepted: 01/16/2015] [Indexed: 12/24/2022]
Abstract
Over half a century has passed since interferons (IFNs) were discovered and shown to inhibit virus infection in cultured cells. Since then, researchers have steadily brought to light the molecular details of IFN signaling, catalogued their pleiotropic effects on cells, and harnessed their therapeutic potential for a variety of maladies. While advances have been plentiful, several fundamental questions have yet to be answered and much complexity remains to be unraveled. We explore the current knowledge surrounding four main questions: are type I IFN subtypes differentially produced in response to distinct pathogens? How are IFN subtypes distinguished by cells? What are the mechanisms and consequences of viral antagonism? Lastly, how can the IFN response be harnessed to improve vaccine efficacy?
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Affiliation(s)
- Hans-Heinrich Hoffmann
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
| | - William M Schneider
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
| | - Charles M Rice
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.
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18
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Hepatitis B virus HBx protein interactions with the ubiquitin proteasome system. Viruses 2014; 6:4683-702. [PMID: 25421893 PMCID: PMC4246244 DOI: 10.3390/v6114683] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 11/16/2014] [Accepted: 11/20/2014] [Indexed: 01/04/2023] Open
Abstract
The hepatitis B virus (HBV) causes acute and chronic hepatitis, and the latter is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes a 17-kDa regulatory protein, HBx, which is required for virus replication. Although the precise contribution(s) of HBx to virus replication is unknown, many viruses target cellular pathways to create an environment favorable for virus replication. The ubiquitin proteasome system (UPS) is a major conserved cellular pathway that controls several critical processes in the cell by regulating the levels of proteins involved in cell cycle, DNA repair, innate immunity, and other processes. We summarize here the interactions of HBx with components of the UPS, including the CUL4 adaptor DDB1, the cullin regulatory complex CSN, and the 26S proteasome. Understanding how these protein interactions benefit virus replication remains a challenge due to limited models in which to study HBV replication. However, studies from other viral systems that similarly target the UPS provide insight into possible strategies used by HBV.
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19
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Xu G, Jiang X, Jaffrey SR. A mental retardation-linked nonsense mutation in cereblon is rescued by proteasome inhibition. J Biol Chem 2013; 288:29573-85. [PMID: 23983124 DOI: 10.1074/jbc.m113.472092] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
A nonsense mutation in cereblon (CRBN) causes autosomal recessive nonsyndromic mental retardation. Cereblon is a substrate receptor for the Cullin-RING E3 ligase complex and couples the ubiquitin ligase to specific ubiquitination targets. The CRBN nonsense mutation (R419X) results in a protein lacking 24 amino acids at its C terminus. Although this mutation has been linked to mild mental retardation, the mechanism by which the mutation affects CRBN function is unknown. Here, we used biochemical and mass spectrometric approaches to explore the function of this mutant. We show that the protein retains its ability to assemble into a Cullin-RING E3 ligase complex and catalyzes the ubiquitination of CRBN-target proteins. However, we find that this mutant exhibits markedly increased levels of autoubiquitination and is more readily degraded by the proteasome than the wild type protein. We also show that the level of the mutant protein can be restored by a treatment of cells with a clinically utilized proteasome inhibitor, suggesting that this agent may be useful for the treatment of mental retardation associated with the CRBN R419X mutation. These data demonstrate that enhanced autoubiquitination and degradation account for the defect in CRBN activity that leads to mental retardation.
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Affiliation(s)
- Guoqiang Xu
- From the Department of Pharmacology, College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Translational Research for Neuro-Psycho-Diseases, Soochow University, Suzhou, Jiangsu 215123, China and
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20
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Audsley MD, Moseley GW. Paramyxovirus evasion of innate immunity: Diverse strategies for common targets. World J Virol 2013; 2:57-70. [PMID: 24175230 PMCID: PMC3785049 DOI: 10.5501/wjv.v2.i2.57] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 02/14/2013] [Accepted: 04/10/2013] [Indexed: 02/05/2023] Open
Abstract
The paramyxoviruses are a family of > 30 viruses that variously infect humans, other mammals and fish to cause diverse outcomes, ranging from asymptomatic to lethal disease, with the zoonotic paramyxoviruses Nipah and Hendra showing up to 70% case-fatality rate in humans. The capacity to evade host immunity is central to viral infection, and paramyxoviruses have evolved multiple strategies to overcome the host interferon (IFN)-mediated innate immune response through the activity of their IFN-antagonist proteins. Although paramyxovirus IFN antagonists generally target common factors of the IFN system, including melanoma differentiation associated factor 5, retinoic acid-inducible gene-I, signal transducers and activators of transcription (STAT)1 and STAT2, and IFN regulatory factor 3, the mechanisms of antagonism show remarkable diversity between different genera and even individual members of the same genus; the reasons for this diversity, however, are not currently understood. Here, we review the IFN antagonism strategies of paramyxoviruses, highlighting mechanistic differences observed between individual species and genera. We also discuss potential sources of this diversity, including biological differences in the host and/or tissue specificity of different paramyxoviruses, and potential effects of experimental approaches that have largely relied on in vitro systems. Importantly, recent studies using recombinant virus systems and animal infection models are beginning to clarify the importance of certain mechanisms of IFN antagonism to in vivo infections, providing important indications not only of their critical importance to virulence, but also of their potential targeting for new therapeutic/vaccine approaches.
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21
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Caignard G, Lucas-Hourani M, Dhondt KP, Labernardière JL, Petit T, Jacob Y, Horvat B, Tangy F, Vidalain PO. The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells. PLoS One 2013; 8:e53881. [PMID: 23342031 PMCID: PMC3544715 DOI: 10.1371/journal.pone.0053881] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2012] [Accepted: 12/04/2012] [Indexed: 12/17/2022] Open
Abstract
The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host.
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Affiliation(s)
- Grégory Caignard
- Unité de Génomique Virale et Vaccination, Centre National de la Recherche Scientifique, URA-3015, Virology Department, Institut Pasteur, Paris, France
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22
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Hodgson AJ, Hyser JM, Keasler VV, Cang Y, Slagle BL. Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication. Virology 2012; 426:73-82. [PMID: 22342275 DOI: 10.1016/j.virol.2012.01.021] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2011] [Revised: 12/25/2011] [Accepted: 01/14/2012] [Indexed: 12/20/2022]
Abstract
Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx(69), HBx(90/91), HBx(R96E)) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.
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Affiliation(s)
- Amanda J Hodgson
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
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23
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Identification of DNA-damage DNA-binding protein 1 as a conditional essential factor for cytomegalovirus replication in interferon-γ-stimulated cells. PLoS Pathog 2011; 7:e1002069. [PMID: 21698215 PMCID: PMC3116810 DOI: 10.1371/journal.ppat.1002069] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2010] [Accepted: 03/29/2011] [Indexed: 01/17/2023] Open
Abstract
The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2 (STAT2)-mediated interferon signal transduction. We wished to explore by which molecular mechanism pM27 accomplishes this effect. We demonstrate that pM27 is essential and sufficient to curtail the protein half-life of STAT2 molecules. Pharmacologic inhibition of the proteasome restored STAT2 amounts, leading to poly-ubiquitin-conjugated STAT2 forms. PM27 was found in complexes with an essential host ubiquitin ligase complex adaptor protein, DNA-damage DNA-binding protein (DDB) 1. Truncation mutants of pM27 showed a strict correlation between DDB1 interaction and their ability to degrade STAT2. SiRNA-mediated knock-down of DDB1 restored STAT2 in the presence of pM27 and strongly impaired viral replication in interferon conditioned cells, thus phenocopying the growth attenuation of M27-deficient virus. In a constructive process, pM27 recruits DDB1 to exploit ubiquitin ligase complexes catalyzing the obstruction of the STAT2-dependent antiviral state of cells to permit viral replication.
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24
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Schaap-Nutt A, D'Angelo C, Amaro-Carambot E, Nolan SM, Davis S, Wise SM, Higgins C, Bradley K, Kim O, Mayor R, Skiadopoulos MH, Collins PL, Murphy BR, Schmidt AC. Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates. Virology 2010; 406:65-79. [PMID: 20667570 PMCID: PMC2932766 DOI: 10.1016/j.virol.2010.07.011] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2010] [Revised: 06/15/2010] [Accepted: 07/06/2010] [Indexed: 02/06/2023]
Abstract
The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.
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Affiliation(s)
- Anne Schaap-Nutt
- Laboratory of Infectious Diseases, RNA Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
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25
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Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference. J Virol 2010; 84:11152-63. [PMID: 20719949 DOI: 10.1128/jvi.01375-10] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.
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26
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Ramachandran A, Horvath CM. Paramyxovirus disruption of interferon signal transduction: STATus report. J Interferon Cytokine Res 2010; 29:531-7. [PMID: 19694544 DOI: 10.1089/jir.2009.0070] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
RNA viruses in the paramyxovirus family have evolved a number of strategies to escape host cell surveillance and antiviral responses. One mechanism exploited by a number of viruses in this family is direct targeting of cytokine-inducible transcription regulators in the STAT family. Diverse members of this large virus family effectively suppress STAT signaling by the actions of their V proteins, or the related proteins derived from alternate viral mRNAs. These viral proteins have distinct means of targeting STATs, resulting in a variety of negative effects on STATs and their signal transduction. Recent developments in understanding STAT targeting will be reviewed.
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Affiliation(s)
- Aparna Ramachandran
- Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA
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27
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Complete genome sequence and pathogenicity of two swine parainfluenzavirus 3 isolates from pigs in the United States. J Virol 2009; 84:686-94. [PMID: 19906928 DOI: 10.1128/jvi.00847-09] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3' leader and 5' trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3' untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.
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A point mutation, E95D, in the mumps virus V protein disengages STAT3 targeting from STAT1 targeting. J Virol 2009; 83:6347-56. [PMID: 19386700 DOI: 10.1128/jvi.00596-09] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Mumps virus, like other paramyxoviruses in the Rubulavirus genus, encodes a V protein that can assemble a ubiquitin ligase complex from cellular components, leading to the destruction of cellular signal transducer and activator of transcription (STAT) proteins. While many V proteins target the interferon-activated STAT1 or STAT2 protein, mumps virus V protein is unique in its ability to also target STAT3 for ubiquitin modification and proteasome-mediated degradation. Here we report that a single amino acid substitution in the mumps virus V protein, E95D, results in defective STAT3 targeting while maintaining the ability to target STAT1. Results indicate that the E95D mutation disrupts the ability of the V protein to associate with STAT3. A recombinant mumps virus carrying the E95D mutation in its P and V proteins replicates normally in cultured cells but fails to induce targeting of STAT3. Infection with the recombinant virus results in the differential regulation of a number of cellular genes compared to wild-type mumps virus and increases cell death in infected cells, producing a large-plaque phenotype.
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Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells. J Virol 2008; 82:8124-37. [PMID: 18524825 DOI: 10.1128/jvi.00430-08] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV) oncoprotein, mimics a constitutively activated tumor necrosis factor receptor and activates various signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt. LMP1 is essential for EBV-mediated B-cell transformation and is sufficient to transform several cell lines. Cellular transformation has been associated strongly with genomic instability, while DNA repair plays an important role in maintaining genomic stability. Previously, we have shown that LMP1 represses DNA repair by the C-terminal activating region 1 (CTAR1) in human epithelial cells. In the present study, we demonstrate that the PI3K/Akt pathway is required for LMP1-mediated repression of DNA repair. Through the LMP1/PI3K/Akt pathway, FOXO3a, which can induce DNA repair, is inactivated because of phosphorylation and relocalization. Expression of a constitutively active FOXO3a mutant can rescue LMP1-mediated repression of DNA repair. Furthermore, LMP1 can decrease the expression of DNA damage-binding protein 1 (DDB1), which functions in nucleotide excision repair, through the PI3K/Akt/FOXO3a pathway. LMP1-mediated repression of DNA repair is restored by DDB1, although only partially. These results suggest that LMP1 triggers the PI3K/Akt pathway to inactivate FOXO3a and decrease DDB1, which can lead to repression of DNA repair and may contribute to genomic instability in human epithelial cells.
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Kraus TA, Garza L, Horvath CM. Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection. Virology 2007; 371:196-205. [PMID: 17964629 DOI: 10.1016/j.virol.2007.10.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2007] [Revised: 08/13/2007] [Accepted: 10/01/2007] [Indexed: 11/25/2022]
Abstract
Parainfluenza virus 5 (PIV5 or SV5) infects several mammalian species but is restricted from efficient replication in mice. In humans, PIV5 evades IFN signaling by targeting STAT1 for proteasomal degradation in a STAT2-dependent reaction. In contrast, cell culture experiments have demonstrated that the divergent murine STAT2 protein fails to support STAT1 targeting. Expression of human STAT2 in mouse cells can overcome the species restriction to enable PIV5-induced STAT1 degradation and subsequent IFN antagonism. Here, we describe a transgenic mouse that ubiquitously expresses human STAT2. PIV5 infection induces STAT1 degradation leading to enhanced virus replication and protein expression in the cells from the transgenic mouse but not from the non-transgenic littermates. Importantly, intranasal inoculation with PIV5 results in increased viral load in the lungs of the transgenic mice compared to wild-type littermates. These transgenic mice provide a small animal model to study the role of innate immune evasion in paramyxovirus pathogenesis.
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Affiliation(s)
- Thomas A Kraus
- Department of Medicine, Northwestern University, Evanston, IL 60208, USA.
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31
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Lin Y, Sun M, Fuentes SM, Keim CD, Rothermel T, He B. Inhibition of interleukin-6 expression by the V protein of parainfluenza virus 5. Virology 2007; 368:262-72. [PMID: 17692882 PMCID: PMC2100396 DOI: 10.1016/j.virol.2007.07.009] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2007] [Revised: 06/29/2007] [Accepted: 07/06/2007] [Indexed: 01/22/2023]
Abstract
The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-beta production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-beta promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-alpha and IL-1beta, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-alpha, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VDeltaC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VDeltaC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VDeltaC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-beta even though rPIV5VDeltaC-infected cells produced IFN-beta. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-kappaB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5.
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Affiliation(s)
- Yuan Lin
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA 16802
| | - Minghao Sun
- Graduate Program in Pathobiology, Pennsylvania State University, University Park, PA 16802
| | - Sandra M. Fuentes
- Graduate Program in Pathobiology, Pennsylvania State University, University Park, PA 16802
| | - Celia D. Keim
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA 16802
| | - Terri Rothermel
- Graduate Program in Pathobiology, Pennsylvania State University, University Park, PA 16802
| | - Biao He
- Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA 16802
- Graduate Program in Pathobiology, Pennsylvania State University, University Park, PA 16802
- The Huck Institutes of Life sciences, Pennsylvania State University, University Park, PA 16802
- Center of Molecular Immunology and Infectious Disease, Pennsylvania State University, University Park, PA 16802
- *Corresponding author: Biao He, Center of Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 115 Henning Bldg., University Park, PA 16802, Phone: 814 863 8533, Fax: 814 863 6140,
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Hagmaier K, Stock N, Precious B, Childs K, Wang LF, Goodbourn S, Randall RE. Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs. J Gen Virol 2007; 88:956-966. [PMID: 17325370 PMCID: PMC2884952 DOI: 10.1099/vir.0.82579-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2006] [Accepted: 10/10/2006] [Indexed: 01/30/2023] Open
Abstract
Mapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.
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Affiliation(s)
- K. Hagmaier
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - N. Stock
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - B. Precious
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - K. Childs
- Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK
| | - L.-F. Wang
- CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia
| | - S. Goodbourn
- Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK
| | - R. E. Randall
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
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33
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Schröfelbauer B, Hakata Y, Landau NR. HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc Natl Acad Sci U S A 2007; 104:4130-5. [PMID: 17360488 PMCID: PMC1820720 DOI: 10.1073/pnas.0610167104] [Citation(s) in RCA: 162] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G(2) arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G(2) arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.
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Affiliation(s)
- Bärbel Schröfelbauer
- *Infectious Disease Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099; and
- Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, A-1180 Vienna, Austria
| | - Yoshiyuki Hakata
- *Infectious Disease Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099; and
| | - Nathaniel R. Landau
- *Infectious Disease Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099; and
- To whom correspondence should be sent at the present address:
New York University School of Medicine, Department of Microbiology, 550 First Avenue, New York, NY 10016. E-mail:
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34
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Nishio M, Tsurudome M, Ishihara H, Ito M, Ito Y. The conserved carboxyl terminus of human parainfluenza virus type 2 V protein plays an important role in virus growth. Virology 2007; 362:85-98. [PMID: 17250865 DOI: 10.1016/j.virol.2006.12.017] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2006] [Revised: 10/18/2006] [Accepted: 12/18/2006] [Indexed: 10/23/2022]
Abstract
Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth.
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Affiliation(s)
- Machiko Nishio
- Department of Microbiology, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu, Mie, 514-8507, Japan.
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35
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Prost S, Lu P, Caldwell H, Harrison D. E2F regulates DDB2: consequences for DNA repair in Rb-deficient cells. Oncogene 2006; 26:3572-81. [PMID: 17173070 DOI: 10.1038/sj.onc.1210151] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
DDB2, a gene mutated in XPE patients, is involved in global genomic repair especially the repair of cyclobutane pyrimidine dimers (CPDs), and is regulated by p53 in human cells. We show that DDB2 is expressed in mouse tissues and demonstrate, using primary mouse epithelial cells, that mouse DDB2 is regulated by E2F transcription factors. Retinoblastoma (Rb), a tumor suppressor critical for the control of cell cycle progression, regulates E2F activity. Using Cre-Lox technology to delete Rb in primary mouse hepatocytes, we show that DDB2 gene expression increases, leading to elevated DDB2 protein levels. Furthermore, we show that endogenous E2F1 and E2F3 bind to DDB2 promoter and that treatment with E2F1-antisense or E2F1-small interfering RNA (siRNA) decreases DDB2 transcription, demonstrating that E2F1 is a transcriptional regulator for DDB2. This has consequences for global genomic repair: in Rb-null cells, where E2F activity is elevated, global DNA repair is increased and removal of CPDs is more efficient than in wild-type cells. Treatment with DDB2-siRNA decreases DDB2 expression and abolishes the repair phenotype of Rb-null cells. In summary, these results identify a new regulatory pathway for DDB2 by E2F, which does not require but is potentiated by p53, and demonstrate that DDB2 is involved in global repair in mouse epithelial cells.
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Affiliation(s)
- S Prost
- Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.
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36
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Chen M, Gerlier D. Viral hijacking of cellular ubiquitination pathways as an anti-innate immunity strategy. Viral Immunol 2006; 19:349-62. [PMID: 16987055 DOI: 10.1089/vim.2006.19.349] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Viruses are obligate parasites of host cells. Virus-host coevolution has selected virus for growth despite antiviral defenses set up by hosting cells and organisms. Ubiquitin conjugation onto proteins, through a cascade of reactions mediated by E1 (ubiquitin-activating enzyme) and E2 and E3 (ubiquitin- conjugating ligases), is one of the major regulatory systems that, in particular, tightly controls the concentration of cellular proteins by sorting them for degradation. The combined diversity of E2 and E3 ligases ensures the selective/specific ubiquitination of a large number of protein substrates within the cell interior. Therefore it is not surprising that several viruses encode proteins with E3 ubiquitin ligase activities that target cellular proteins playing a key role in innate antiviral mechanisms.
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Affiliation(s)
- Mingzhou Chen
- CNRS, Université de Lyon, UMR5537, Laboratoire de Virologie et Pathogenèse Virale, IFR Laennec, Lyon, France
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37
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Nanda SK, Baron MD. Rinderpest virus blocks type I and type II interferon action: role of structural and nonstructural proteins. J Virol 2006; 80:7555-68. [PMID: 16840335 PMCID: PMC1563703 DOI: 10.1128/jvi.02720-05] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2005] [Accepted: 05/15/2006] [Indexed: 12/18/2022] Open
Abstract
Rinderpest virus (RPV) is a paramyxovirus closely related to the human pathogen Measles virus. It causes severe disease in cattle, buffalo, and some wild animals; although it can infect humans, it does not cause disease. Here, we demonstrate that RPV blocks the action of both type I (alpha) and type II (gamma) interferons (IFNs) by blocking the phosphorylation and nuclear translocation of STAT1 and STAT2 and that this block is not related to species specificity. In addition, both wild-type virulent and vaccine strains of the virus blocked IFN action. Unlike the case with some other paramyxoviruses, neither STAT1 nor STAT2 is degraded upon virus infection. STAT1 is bound by both the viral structural protein P, and thereby recruited to concentrations of viral protein in the cell, and the nonstructural protein V. Although both P and V proteins bind to STAT1 and can block IFN action when expressed in transfected cells, the IFN antagonist activity of the P protein is weaker than that of the V protein. The viral C protein also seems to weakly block IFN-induced activation of STAT1 in transfection experiments. However, studies with knockout viruses showed that the viral V protein appears to be the dominant inhibitor of IFN signaling in the context of virus infection, since prevention of viral V expression restored the IFN sensitivity of infected cells. Although a change in the distribution pattern of STAT2 was observed in virus-infected cells, STAT2 was not bound by any viral protein.
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Affiliation(s)
- Sambit K Nanda
- Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, United Kingdom
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38
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Sugasawa K. UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair. J Mol Histol 2006; 37:189-202. [PMID: 16858626 DOI: 10.1007/s10735-006-9044-7] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2006] [Accepted: 06/21/2006] [Indexed: 12/31/2022]
Abstract
The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.
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Affiliation(s)
- Kaoru Sugasawa
- Genome Damage Response Research Unit, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
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39
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Cruz CD, Palosaari H, Parisien JP, Devaux P, Cattaneo R, Ouchi T, Horvath CM. Measles virus V protein inhibits p53 family member p73. J Virol 2006; 80:5644-50. [PMID: 16699046 PMCID: PMC1472123 DOI: 10.1128/jvi.02400-05] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2005] [Accepted: 03/11/2006] [Indexed: 01/23/2023] Open
Abstract
Paramyxovirus V proteins function as host interference factors that inactivate antiviral responses, including interferon. Characterization of cellular proteins that copurify with ectopically expressed measles virus V protein has revealed interactions with DNA binding domains of p53 family proteins, p53 and p73. Specific transcriptional assays reveal that expression of measles virus V cDNA inhibits p73, but not p53. Expression of measles virus V cDNA can delay cell death induced by genotoxic stress and also can decrease the abundance of the proapoptotic factor PUMA, a p73 target. Recombinant measles virus with an engineered deficiency in V protein is capable of inducing more severe cytopathic effects than the wild type, implicating measles virus V protein as an inhibitor of cell death. These findings also suggest that p73-PUMA signaling may be a previously unrecognized arm of cellular innate antiviral immunity.
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Affiliation(s)
- Cristian D Cruz
- Pancoe-ENH Research Pavilion, Northwestern University, 2200 Campus Drive, Evanston, IL 60208, USA
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40
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Zhang X, Zhang H, Ye L. Effects of hepatitis B virus X protein on the development of liver cancer. ACTA ACUST UNITED AC 2006; 147:58-66. [PMID: 16459163 DOI: 10.1016/j.lab.2005.10.003] [Citation(s) in RCA: 156] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2005] [Revised: 10/08/2005] [Accepted: 10/20/2005] [Indexed: 02/08/2023]
Abstract
Hepatitis B virus (HBV) infections play an important role in the development of cirrhosis and hepatocellular carcinoma (HCC). The pathogenesis of HBV-related HCC, however, has not been fully described. Evidence suggests that the HBV X protein (HBx) plays a crucial role in the pathogenesis of HCC. The high occurrence of anti-HBx antibody in the serum of HCC patients indicates that it could be a prognostic marker of HBV infection and HCC. HBx stimulates and influences signal transduction pathways within cells. HBx also binds to such protein targets as p53, proteasome subunits, and UV-damaged DNA binding proteins. It also interacts with the cyclic AMP-responsive element binding protein, ATF-2, NFkappaB, and basal transcription factors. HBx is primarily localized to the cytoplasm, where it interacts with and stimulates protein kinases, including protein kinase C, Janus kinase/STAT, IKK, PI-3-K, stress-activated protein kinase/Jun N-terminal kinase, and protein kinase B/Akt. It is also found in the mitochondrion, where it influences the Bcl-2 family. This review examines the role of HBx in the life cycle of HBV as well as the various signal transduction pathways involved in the pathogenesis of HBV-induced hepatocarcinogenesis.
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Affiliation(s)
- Xiaodong Zhang
- Department of Cancer Research, Institute for Molecular Biology, Nankai University, Tianjin, P. R. China.
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41
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Witko SE, Kotash C, Sidhu MS, Udem SA, Parks CL. Inhibition of measles virus minireplicon-encoded reporter gene expression by V protein. Virology 2006; 348:107-19. [PMID: 16445957 DOI: 10.1016/j.virol.2005.12.019] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2005] [Revised: 09/15/2005] [Accepted: 12/14/2005] [Indexed: 10/25/2022]
Abstract
Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110-131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription.
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Affiliation(s)
- Susan E Witko
- Wyeth Vaccines Research, 401 North Middletown Road, Pearl River, NY 10965, USA
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42
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Li T, Chen X, Garbutt KC, Zhou P, Zheng N. Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell 2006; 124:105-17. [PMID: 16413485 DOI: 10.1016/j.cell.2005.10.033] [Citation(s) in RCA: 203] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2005] [Revised: 08/16/2005] [Accepted: 10/11/2005] [Indexed: 01/28/2023]
Abstract
The DDB1-Cul4A ubiquitin ligase complex promotes protein ubiquitination in diverse cellular functions and is reprogrammed by the V proteins of paramyxoviruses to degrade STATs and block interferon signaling. Here we report the crystal structures of DDB1 alone and in complex with the simian virus 5 V protein. The DDB1 structure reveals an intertwined three-propeller cluster, which contains two tightly coupled beta propellers with a large pocket in between and a third beta propeller flexibly attached on the side. The rigid double-propeller fold of DDB1 is targeted by the viral V protein, which inserts an entire helix into the double-propeller pocket, whereas the third propeller domain docks DDB1 to the N terminus of the Cul4A scaffold. Together, these results not only provide structural insights into how the virus hijacks the DDB1-Cul4A ubiquitin ligase but also establish a structural framework for understanding the multiple functions of DDB1 in the uniquely assembled cullin-RING E3 machinery.
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Affiliation(s)
- Ti Li
- Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
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Nishio M, Tsurudome M, Ito M, Ito Y. Human parainfluenza virus type 4 is incapable of evading the interferon-induced antiviral effect. J Virol 2006; 79:14756-68. [PMID: 16282476 PMCID: PMC1287573 DOI: 10.1128/jvi.79.23.14756-14768.2005] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The V proteins of some paramyxoviruses have developed the ability to efficiently inactivate STAT protein function as a countermeasure for evading interferon (IFN) responses. Human parainfluenza virus type 4 (hPIV4) is one of the rubulaviruses, which are members of the family Paramyxoviridae, and has a V protein with a highly conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. In order to study the function of the hPIV4 V protein, we established HeLa cells expressing the hPIV4A V protein (HeLa/FlagPIV4V). The hPIV4 V protein had no ability to reduce the level of STAT1 or STAT2, although it associated with STAT1, STAT2, DDB1, and Cul4A. It interfered with neither STAT1 and STAT2 tyrosine phosphorylation nor IFN-induced STAT nuclear accumulation. In addition, HeLa/FlagPIV4V cells are fully sensitive to both beta interferon (IFN-beta) and IFN-gamma, indicating that the hPIV4 V protein has no ability to block IFN-induced signaling. We further established HeLa cells expressing various chimeric proteins between the hPIV2 and hPIV4A V proteins. The lack of IFN-antagonistic activity of the hPIV4 V protein is caused by both the P/V common and V-specific domains. At least two regions (amino acids [aa] 32 to 45 and aa 143 to 164) of hPIV4 V in the P/V common domain and one region (aa 200 to 212) of the C terminus are involved in the inability to evade the IFN-induced signaling. Moreover, we established HeLa cells persistently infected with hPIV4 to make sure of the inability to escape IFN and confirmed that hPIV4 is the only paramyxovirus analyzed to date that can't evade the IFN-induced antiviral responses.
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Affiliation(s)
- Machiko Nishio
- Department of Microbiology, Mie University School of Medicine, 2-174, Edobashi, Tsu-shi, Mie Prefecture, 514-8507 Japan.
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Precious B, Childs K, Fitzpatrick-Swallow V, Goodbourn S, Randall RE. Simian virus 5 V protein acts as an adaptor, linking DDB1 to STAT2, to facilitate the ubiquitination of STAT1. J Virol 2005; 79:13434-41. [PMID: 16227264 PMCID: PMC1262611 DOI: 10.1128/jvi.79.21.13434-13441.2005] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.
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Affiliation(s)
- B Precious
- School of Biology, University of St. Andrews, Fife KY16 9TS, United Kingdom
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Nishio M, Tsurudome M, Ito M, Garcin D, Kolakofsky D, Ito Y. Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication. J Virol 2005; 79:8591-601. [PMID: 15956600 PMCID: PMC1143765 DOI: 10.1128/jvi.79.13.8591-8601.2005] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.
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Affiliation(s)
- Machiko Nishio
- Department of Microbiology, Mie University School of Medicine, Tsu-shi, Mie Prefecture, Japan.
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Ulane CM, Kentsis A, Cruz CD, Parisien JP, Schneider KL, Horvath CM. Composition and assembly of STAT-targeting ubiquitin ligase complexes: paramyxovirus V protein carboxyl terminus is an oligomerization domain. J Virol 2005; 79:10180-9. [PMID: 16051811 PMCID: PMC1182666 DOI: 10.1128/jvi.79.16.10180-10189.2005] [Citation(s) in RCA: 91] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2005] [Accepted: 05/09/2005] [Indexed: 01/17/2023] Open
Abstract
Transcription regulators STAT1 and STAT2 are key components of the interferon signaling system leading to innate antiviral immunity. The related STAT3 protein is a regulator of interleukin-6-type cytokine signals and can contribute to both cell growth and death important for cancer gene regulation and tumor survival. These three STAT proteins are targeted for proteasome-mediated degradation by RNA viruses in the Rubulavirus genus of the Paramyxoviridae. A single viral protein, the V protein, assembles STAT-specific ubiquitin ligase complexes from cellular components. Simian virus 5 (SV5) targets STAT1, human parainfluenza virus 2 targets STAT2, and mumps virus targets both STAT1 and STAT3. Analysis of the V-dependent degradation complex (VDC) composition and assembly revealed several features contributing to targeting specificity. SV5 and mumps V proteins require STAT2 to recruit the STAT1 target, yet mumps V protein binds STAT3 independent of STAT1 and STAT2. All Rubulavirus V proteins tested require cellular DDB1 to target STATs for degradation but differ in the use of Roc1, which is essential for mumps V STAT3 targeting. Protein interaction analysis reveals that paramyxovirus V proteins can homo- and heterooligomerize and that the conserved cysteine-rich zinc-binding C-terminal domain is necessary and sufficient for oligomerization. Purified SV5 V protein spontaneously assembles into spherical macromolecular particles, and similar particles constitute SV5 and mumps VDC preparations.
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Abstract
The signal transducer and activator of transcription (STAT) family of proteins function to activate gene transcription downstream of myriad cytokine and growth factor signals. The prototype STAT proteins, STAT1 and STAT2, are required for innate and adaptive antimicrobial immune responses that result from interferon signal transduction. While many viruses have evolved the ability to avoid these antiviral cytokines, the Paramyxoviruses are distinct in their abilities to interfere directly with STAT proteins. Individual paramyxovirus species differ greatly in their precise mechanism of STAT signaling evasion, but a virus-encoded protein called V plays a central role in this process. The theme of V-dependent interferon evasion and its variations provide significant insights into virus-host interactions and viral immune evasion that can help define targets for antiviral drug design. Exposure of the viral weapons of STAT destruction may also be instructive for application to STAT-directed therapeutics for diseases characterized by STAT hyperactivity.
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Affiliation(s)
- Curt M Horvath
- Department of Medicine, Evanston Northwestern Healthcare Research Institute, Northwestern University, Evanston, IL, USA.
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Wansley EK, Dillon PJ, Gainey MD, Tam J, Cramer SD, Parks GD. Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells. Virology 2005; 335:131-44. [PMID: 15823612 DOI: 10.1016/j.virol.2005.02.004] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2005] [Revised: 01/24/2005] [Accepted: 02/04/2005] [Indexed: 10/25/2022]
Abstract
A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed.
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Affiliation(s)
- Elizabeth K Wansley
- Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1064, USA
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Kubota T, Yokosawa N, Yokota SI, Fujii N, Tashiro M, Kato A. Mumps virus V protein antagonizes interferon without the complete degradation of STAT1. J Virol 2005; 79:4451-9. [PMID: 15767445 PMCID: PMC1061565 DOI: 10.1128/jvi.79.7.4451-4459.2005] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.
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Affiliation(s)
- Toru Kubota
- Department of Virology III, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo 208-0011, Japan.
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50
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Precious B, Young DF, Andrejeva L, Goodbourn S, Randall RE. In vitro and in vivo specificity of ubiquitination and degradation of STAT1 and STAT2 by the V proteins of the paramyxoviruses simian virus 5 and human parainfluenza virus type 2. J Gen Virol 2005; 86:151-158. [PMID: 15604442 DOI: 10.1099/vir.0.80263-0] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.
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Affiliation(s)
- B Precious
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - D F Young
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - L Andrejeva
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
| | - S Goodbourn
- Department of Biochemistry and Immunology, St George's Hospital Medical School, University of London, London SW17 0RE, UK
| | - R E Randall
- School of Biology, University of St Andrews, Fife KY16 9TS, UK
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