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Kitagawa Y, Yamaguchi M, Kohno M, Sakai M, Itoh M, Gotoh B. Respirovirus C protein inhibits activation of type I interferon receptor-associated kinases to block JAK-STAT signaling. FEBS Lett 2019; 594:864-877. [PMID: 31705658 DOI: 10.1002/1873-3468.13670] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 10/22/2019] [Accepted: 10/31/2019] [Indexed: 12/31/2022]
Abstract
Respirovirus C protein blocks the type I interferon (IFN)-stimulated activation of the JAK-STAT pathway. It has been reported that C protein inhibits IFN-α-stimulated tyrosine phosphorylation of STATs, but the underlying mechanism is poorly understood. Here, we show that the C protein of Sendai virus (SeV), a member of the Respirovirus genus, binds to the IFN receptor subunit IFN-α/β receptor subunit (IFNAR)2 and inhibits IFN-α-stimulated tyrosine phosphorylation of the upstream receptor-associated kinases, JAK1 and TYK2. Analysis of various SeV C mutant (Cm) proteins demonstrates the importance of the inhibitory effect on receptor-associated kinase phosphorylation for blockade of JAK-STAT signaling. Furthermore, this inhibitory effect and the IFNAR2 binding capacity are observed for all the respirovirus C proteins examined. Our results suggest that respirovirus C protein inhibits activation of the receptor-associated kinases JAK1 and TYK2 possibly through interaction with IFNAR2.
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Affiliation(s)
- Yoshinori Kitagawa
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Japan
| | - Mayu Yamaguchi
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Japan
| | - Miki Kohno
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Japan.,Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
| | - Madoka Sakai
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Japan.,Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
| | - Masae Itoh
- Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
| | - Bin Gotoh
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Japan
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2
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Oda K, Oda T, Matoba Y, Sato M, Irie T, Sakaguchi T. Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein-mediated blockade of type 1 interferon signaling. J Biol Chem 2017; 292:19752-19766. [PMID: 28978648 DOI: 10.1074/jbc.m117.786285] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2017] [Revised: 09/28/2017] [Indexed: 11/06/2022] Open
Abstract
Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity. We previously demonstrated by using protein crystallography that two molecules of Y3 (the C-terminal half of the C protein) can bind to the homodimer of the N-terminal domain of STAT1 (STAT1ND), elucidating the mechanism of inhibition of IFN-γ signal transduction. SeV C protein also blocks the signal transduction of IFN-α/β by inhibiting the phosphorylation of STAT1 and STAT2, although the mechanism for the inhibition is unclear. Therefore, we sought to elucidate the mechanism of inhibition of the IFN signal transduction via STAT1 and STAT2. Small angle X-ray scattering analysis indicated that STAT1ND associates with the N-terminal domain of STAT2 (STAT2ND) with the help of a Gly-rich linker. We generated a linker-less recombinant protein possessing a STAT1ND:STAT2ND heterodimeric structure via an artificial disulfide bond. Analytical size-exclusion chromatography and surface plasmon resonance revealed that one molecule of Y3 can associate with a linker-less recombinant protein. We propose that one molecule of C protein associates with the STAT1:STAT2 heterodimer, inducing a conformational change to an antiparallel form, which is easily dephosphorylated. This suggests that association of C protein with the STAT1ND:STAT2ND heterodimer is an important factor to block the IFN-α/β signal transduction.
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Affiliation(s)
| | - Takashi Oda
- the Structural Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan
| | - Yasuyuki Matoba
- Microbiology, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8551 and
| | - Mamoru Sato
- the Structural Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan
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Bedsaul JR, Zaritsky LA, Zoon KC. Type I Interferon-Mediated Induction of Antiviral Genes and Proteins Fails to Protect Cells from the Cytopathic Effects of Sendai Virus Infection. J Interferon Cytokine Res 2016; 36:652-665. [PMID: 27508859 DOI: 10.1089/jir.2016.0051] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Sendai virus (SeV), a murine paramyxovirus, has been used to study the induction of type I interferon (IFN) subtypes in robust quantities. Few studies have measured whether the IFN that SeV induces actually fulfills its intended purpose of interfering with virus-mediated effects in the cells in which it is produced. We determined the effects of IFN on SeV-mediated cytopathic effects (CPE) and the ability of IFN to protect against virus infection. SeV-induced biologically active IFN resulted in Jak/STAT activation and the production of a number of interferon-stimulated genes (ISGs). However, these responses did not inhibit SeV replication or CPE. This observation was not due to SeV effects on canonical IFN signaling. Furthermore, pretreating cells with type I IFN and establishing an antiviral state before infection did not mediate SeV effects. Therefore, the induction of canonical IFN signaling pathways and ISGs does not always confer protection against the IFN-inducing virus. Because type I IFNs are approved to treat various infections, our findings suggest that typical markers of IFN activity may not be indicative of a protective antiviral response and should not be used alone to determine whether an antiviral state against a particular virus is achieved.
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Affiliation(s)
- Jacquelyn R Bedsaul
- Cytokine Biology Section, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) , Bethesda, Maryland
| | - Luna A Zaritsky
- Cytokine Biology Section, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) , Bethesda, Maryland
| | - Kathryn C Zoon
- Cytokine Biology Section, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) , Bethesda, Maryland
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4
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Liu LW, Nishikawa T, Kaneda Y. An RNA Molecule Derived From Sendai Virus DI Particles Induces Antitumor Immunity and Cancer Cell-selective Apoptosis. Mol Ther 2015; 24:135-45. [PMID: 26548591 DOI: 10.1038/mt.2015.201] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2015] [Accepted: 10/23/2015] [Indexed: 12/12/2022] Open
Abstract
Inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) induces anticancer immunity and cancer cell-selective apoptosis through the recognition of viral RNA genome fragments by retinoic acid-inducible gene-I (RIG-I). Here, we discovered that the "copy-back" type of defective-interfering (DI) particles that exist in the Cantell strain of HVJ induced the human PC3 prostate cancer cell death more effectively than the Sendai/52 strain or Cantell strain, which contain fewer DI particles. DI particle genomic RNA (~550 bases) activated proapoptotic genes such as Noxa and/or TNF-related apoptosis-inducing ligand (TRAIL) in human prostate cancer cells to induce cancer cell-selective apoptosis. DI particle-derived RNA was synthesized by in vitro transcription (in vitro transcribed (IVT)-B2). IVT-B2 RNA, which has a double-stranded region in its secondary structure, promoted a stronger anticancer effect than IVT-HN RNA, which does not have a double-stranded region in its secondary structure. The intratumoral transfection of IVT-B2 significantly reduced the volume of a human prostate tumor and induced tumor cell apoptosis in the xenograft mouse model. Moreover, the involvement of natural killer (NK) cells in IVT-B2-RNA-induced anticancer effects was also suggested. These findings provide a novel nucleic acid medicine for the treatment of cancer.
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Affiliation(s)
- Li-Wen Liu
- Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Tomoyuki Nishikawa
- Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Yasufumi Kaneda
- Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan
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5
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Forero A, Giacobbi NS, McCormick KD, Gjoerup OV, Bakkenist CJ, Pipas JM, Sarkar SN. Simian virus 40 large T antigen induces IFN-stimulated genes through ATR kinase. THE JOURNAL OF IMMUNOLOGY 2014; 192:5933-42. [PMID: 24799566 DOI: 10.4049/jimmunol.1303470] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Polyomaviruses encode a large T Ag (LT), a multifunctional protein essential for the regulation of both viral and host cell gene expression and productive viral infection. Previously, we have shown that stable expression of LT protein results in upregulation of genes involved in the IFN induction and signaling pathway. In this study, we focus on the cellular signaling mechanism that leads to the induction of IFN responses by LT. Our results show that ectopic expression of SV40 LT results in the induction of IFN-stimulated genes (ISGs) in human fibroblasts and confers an antiviral state. We describe a LT-initiated DNA damage response (DDR) that activates IFN regulatory factor 1, causing IFN-β production and consequent ISG expression in human cells. This IFN-β and ISG induction is dependent on ataxia-telangiectasia mutated and Rad3-related (ATR) kinase, but independent of ATM. ATR kinase inhibition using a selective kinase inhibitor (ETP-46464) caused a decrease in IFN regulatory factor 1 stabilization and ISG expression. Furthermore, expression of a mutant LT that does not induce DDR also does not induce IFN-β and ISGs. These results show that, in the absence of viral infection, LT-initiated activation of ATR-dependent DDR is sufficient for the induction of an IFN-β-mediated innate immune response in human cells. Thus, we have uncovered a novel and critical role for ATR as a mediator of antiviral responses utilizing LT.
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Affiliation(s)
- Adriana Forero
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Nicholas S Giacobbi
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15213; and
| | - Kevin D McCormick
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Ole V Gjoerup
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Christopher J Bakkenist
- Department of Radiation Oncology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - James M Pipas
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15213; and
| | - Saumendra N Sarkar
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Zimmermann M, Armeanu-Ebinger S, Bossow S, Lampe J, Smirnow I, Schenk A, Lange S, Weiss TS, Neubert W, Lauer UM, Bitzer M. Attenuated and protease-profile modified sendai virus vectors as a new tool for virotherapy of solid tumors. PLoS One 2014; 9:e90508. [PMID: 24598703 PMCID: PMC3944018 DOI: 10.1371/journal.pone.0090508] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2013] [Accepted: 02/01/2014] [Indexed: 12/03/2022] Open
Abstract
Multiple types of oncolytic viruses are currently under investigation in clinical trials. To optimize therapeutic outcomes it is believed that the plethora of different tumor types will require a diversity of different virus types. Sendai virus (SeV), a murine parainfluenza virus, displays a broad host range, enters cells within minutes and already has been applied safely as a gene transfer vector in gene therapy patients. However, SeV spreading naturally is abrogated in human cells due to a lack of virus activating proteases. To enable oncolytic applications of SeV we here engineered a set of novel recombinant vectors by a two-step approach: (i) introduction of an ubiquitously recognized cleavage-motive into SeV fusion protein now enabling continuous spreading in human tissues, and (ii) profound attenuation of these rSeV by the knockout of viral immune modulating accessory proteins. When employing human hepatoma cell lines, newly generated SeV variants now reached high titers and induced a profound tumor cell lysis. In contrast, virus release from untransformed human fibroblasts or primary human hepatocytes was found to be reduced by about three log steps in a time course experiment which enables the cumulation of kinetic differences of the distinct phases of viral replication such as primary target cell infection, target cell replication, and progeny virus particle release. In a hepatoma xenograft animal model we found a tumor-specific spreading of our novel recombinant SeV vectors without evidence of biodistribution into non-malignant tissues. In conclusion, we successfully developed novel tumor-selective oncolytic rSeV vectors, constituting a new tool for virotherapy of solid tumors being ready for further preclinical and clinical development to address distinct tumor types.
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Affiliation(s)
- Martina Zimmermann
- Department of Internal Medicine I, Medical University Hospital, Tübingen, Germany
| | | | - Sascha Bossow
- Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany
| | - Johanna Lampe
- Institute for Molecular Medicine Finland FIMM, Helsinki, Finland
| | - Irina Smirnow
- Department of Internal Medicine I, Medical University Hospital, Tübingen, Germany
| | - Andrea Schenk
- Department of Internal Medicine I, Medical University Hospital, Tübingen, Germany
| | - Sebastian Lange
- Medizinische Klinik, Klinikum rechts der Isar, Technische Universität, München, Germany
| | - Thomas S. Weiss
- Center for Liver Cell Research, Department of Pediatrics and Adolescent Medicine, University of Regensburg Hospital, Regensburg, Germany
| | - Wolfgang Neubert
- Max-Planck-Institute for Biochemistry, Department Molecular Virology, Martinsried Germany
| | - Ulrich M. Lauer
- Department of Internal Medicine I, Medical University Hospital, Tübingen, Germany
| | - Michael Bitzer
- Department of Internal Medicine I, Medical University Hospital, Tübingen, Germany
- * E-mail:
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Lo MK, Søgaard TM, Karlin DG. Evolution and structural organization of the C proteins of paramyxovirinae. PLoS One 2014; 9:e90003. [PMID: 24587180 PMCID: PMC3934983 DOI: 10.1371/journal.pone.0090003] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2013] [Accepted: 01/24/2014] [Indexed: 12/21/2022] Open
Abstract
The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.
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Affiliation(s)
- Michael K. Lo
- Centers for Disease Control and Prevention, Viral Special Pathogens Branch, Atlanta, Georgia, United States of America
| | - Teit Max Søgaard
- Division of Structural Biology, Oxford University, Oxford, United Kingdom
| | - David G. Karlin
- Division of Structural Biology, Oxford University, Oxford, United Kingdom
- Department of Zoology, University of Oxford, Oxford, United Kingdom
- * E-mail:
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Yamaguchi M, Kitagawa Y, Zhou M, Itoh M, Gotoh B. An anti-interferon activity shared by paramyxovirus C proteins: inhibition of Toll-like receptor 7/9-dependent alpha interferon induction. FEBS Lett 2013; 588:28-34. [PMID: 24269682 DOI: 10.1016/j.febslet.2013.11.015] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Revised: 10/31/2013] [Accepted: 11/06/2013] [Indexed: 10/26/2022]
Abstract
Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.
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Affiliation(s)
- Mayu Yamaguchi
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
| | - Yoshinori Kitagawa
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
| | - Min Zhou
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
| | - Masae Itoh
- Department of Microbiology, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan
| | - Bin Gotoh
- Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan.
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9
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Paramyxovirus activation and inhibition of innate immune responses. J Mol Biol 2013; 425:4872-92. [PMID: 24056173 DOI: 10.1016/j.jmb.2013.09.015] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 09/12/2013] [Accepted: 09/12/2013] [Indexed: 12/18/2022]
Abstract
Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.
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10
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Clustered basic amino acids of the small sendai virus C protein Y1 are critical to its RAN GTPase-mediated nuclear localization. PLoS One 2013; 8:e73740. [PMID: 23951363 PMCID: PMC3739745 DOI: 10.1371/journal.pone.0073740] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2013] [Accepted: 07/26/2013] [Indexed: 12/15/2022] Open
Abstract
The Sendai virus (SeV) C proteins are shown to exert multiple functions during the course of infection. Perhaps reflecting their many functions, they occur at multiple sites of the cell. In this study, we focused on the nuclear-localizing ability of the smaller C protein, Y1, and found that this translocation is mediated by Ran GTPase but not by passive diffusion, and that basic residues within the 149-157 amino acid region are critical for that. The mechanism of inhibition of interferon (IFN)-signaling seemed to differ between the C and Y1 proteins, since deletion of 12 C-terminal amino acids resulted in a loss of the function for the C but not for the Y1 protein. The ability of Y1 mutants to inhibit IFN-α-induced, ISRE-driven expression of a reporter gene almost paralleled with that to localize in the nucleus. These results suggest that nuclear localization of the Y1 protein might be important for the inhibitory effect on type-I IFN-stimulated gene expression.
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11
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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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12
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Wells G, Addington-Hall M, Malur AG. Mutations within the human parainfluenza virus type 3 (HPIV 3) C protein affect viral replication and host interferon induction. Virus Res 2012; 167:385-90. [PMID: 22634035 DOI: 10.1016/j.virusres.2012.05.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2012] [Revised: 05/09/2012] [Accepted: 05/15/2012] [Indexed: 10/28/2022]
Abstract
Human parainfluenza virus type 3 (HPIV 3) encodes a multifunctional C protein that is capable of inhibiting viral replication and counteracting the host interferon (IFN) signaling pathway. We recently demonstrated that the C protein is phosphorylated both in vitro and in vivo and mutations within the phosphorylation sites exhibit differential inhibitory activities in vitro. In this study, we report for the first time the successful recovery of mutant HPIV 3 viruses containing mutations within the C protein. Three mutant viruses, Cm-1, Cm-3 and Cm-4, harboring individual mutations of S7, S47T48 and S81 residues, respectively, were examined for their replication profiles and their ability to abrogate host IFN induction. Viral transcription was similar for all viruses; however Cm-3 displayed a relatively higher replication. Infection of cells with Cm-1 and Cm-3 led to the activation of IFN regulatory transcription factor 3 (IRF-3) and subsequent increase in IFN-β mRNA levels as determined by immunofluorescence assay and RT-PCR analyses, respectively. Moreover, Cm-3 was able to partially resist the interferon induced antiviral state in Vero cells. Taken together, these results suggest that mutations within the C protein differentially affect viral replication and host interferon induction.
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Affiliation(s)
- Greg Wells
- Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA
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13
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Sakaguchi T, Irie T, Kuwayama M, Ueno T, Yoshida A, Kawabata R. Analysis of interaction of Sendai virus V protein and melanoma differentiation-associated gene 5. Microbiol Immunol 2012; 55:760-7. [PMID: 21851384 DOI: 10.1111/j.1348-0421.2011.00379.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Sendai virus (SeV), a pneumotropic virus of rodents, has an accessory protein, V, and the V protein has been shown to interact with MDA5, inhibiting IRF3 activation and interferon-β production. In the present study, interaction of the V protein with various IRF3-activating proteins including MDA5 was investigated in a co-immunoprecipitation assay. We also investigated interaction of mutant V proteins from SeVs of low pathogenicity with MDA5. The V protein interacted with at least retinoic acid inducible gene I, inhibitor of κB kinase epsilon and IRF3 other than MDA5. However, only MDA5 interacted with the V protein dependently on the C-terminal V unique (Vu) region, inhibiting IRF3 reporter activation. The Vu region has been shown to be important for viral pathogenicity. We thus focused on interaction of the V protein with MDA5. Point mutations in the Vu region destabilized the V protein or abolished the interaction with MDA5 when the V protein was stable. The V-R₃₂₀G protein was highly stable and interacted with MDA5, but did not inhibit activation of IRF3 induced by MDA5. Viral pathogenicity of SeV is related to the inhibitory effect of the V protein on MDA5, but is not always related to the binding of V protein with MDA5.
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Affiliation(s)
- Takemasa Sakaguchi
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
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14
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The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome. PLoS One 2012; 7:e28382. [PMID: 22355301 PMCID: PMC3280236 DOI: 10.1371/journal.pone.0028382] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2011] [Accepted: 11/07/2011] [Indexed: 02/06/2023] Open
Abstract
Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.
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15
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Expression of the Sendai (murine parainfluenza) virus C protein alleviates restriction of measles virus growth in mouse cells. Proc Natl Acad Sci U S A 2011; 108:15384-9. [PMID: 21896767 DOI: 10.1073/pnas.1107382108] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Measles virus (MV), a human pathogen, uses the signaling lymphocyte activation molecule (SLAM) or CD46 as an entry receptor. Although several transgenic mice expressing these receptors have been generated as small animal models for measles, these mice usually have to be made defective in IFN-α/β signaling to facilitate MV replication. Similarly, when functional receptors are expressed by transfection, mouse cells do not allow MV growth as efficiently as primate cells. In this study, we demonstrate that MV efficiently grows in SLAM-expressing mouse cells in which the Sendai virus (SeV) C protein is transiently expressed. We developed a SLAM-expressing mouse cell line whose genome also encodes the SeV C protein downstream of the sequence flanked with loxP sequences. When this cell line was infected with the recombinant MV expressing the Cre recombinase, the SeV C protein was readily expressed. Importantly, the Cre recombinase-encoding MV grew in this cell line much more efficiently than it did in the parental cell. The minigenome assay demonstrated that the SeV C protein does not modulate MV RNA synthesis. Analyses using the mutant proteins with the defined functional defects revealed that the IFN-antagonist function, but not the budding-accelerating function, of the SeV C protein was critical for supporting efficient MV growth in mouse cells. Our results indicate that insufficient IFN antagonism can be an important determinant of the host range of viruses, and the system described here may be useful to overcome the species barrier of other human viruses.
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16
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Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells. J Virol 2010; 84:11718-28. [PMID: 20810726 DOI: 10.1128/jvi.00798-10] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.
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17
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Irie T, Nagata N, Igarashi T, Okamoto I, Sakaguchi T. Conserved charged amino acids within Sendai virus C protein play multiple roles in the evasion of innate immune responses. PLoS One 2010; 5:e10719. [PMID: 20502666 PMCID: PMC2873429 DOI: 10.1371/journal.pone.0010719] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2010] [Accepted: 04/27/2010] [Indexed: 12/24/2022] Open
Abstract
One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C′/C(−), 4C(−), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-α-mediated anti-viral state. Infection by the virus (Cm2′) containing mutations at K77 and D80 induced significant IFN-β production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2′ virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-β and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.
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Affiliation(s)
- Takashi Irie
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
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18
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Abstract
Regulation of protein synthesis by viruses occurs at all levels of translation. Even prior to protein synthesis itself, the accessibility of the various open reading frames contained in the viral genome is precisely controlled. Eukaryotic viruses resort to a vast array of strategies to divert the translation machinery in their favor, in particular, at initiation of translation. These strategies are not only designed to circumvent strategies common to cell protein synthesis in eukaryotes, but as revealed more recently, they also aim at modifying or damaging cell factors, the virus having the capacity to multiply in the absence of these factors. In addition to unraveling mechanisms that may constitute new targets in view of controlling virus diseases, viruses constitute incomparably useful tools to gain in-depth knowledge on a multitude of cell pathways.
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19
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Mao H, Chattopadhyay S, Banerjee AK. N-terminally truncated C protein, CNDelta25, of human parainfluenza virus type 3 is a potent inhibitor of viral replication. Virology 2009; 394:143-8. [PMID: 19747707 DOI: 10.1016/j.virol.2009.08.026] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2009] [Revised: 07/06/2009] [Accepted: 08/18/2009] [Indexed: 11/27/2022]
Abstract
The C protein of human parainfluenza virus type 3 (HPIV3) is a multifunctional accessory protein that inhibits viral transcription and interferon (IFN) signaling. In the present study, we found that removal of N-terminal 25 or 50 amino acid residues from the C protein (CNDelta25 or CNDelta50) totally abolished viral RNA synthesis in the HPIV3 minigenome system. Further N-terminal or C-terminal deletion impaired the inhibitory ability of CNDelta25 and CNDelta50. Subsequent mutagenesis analysis suggested that the N-terminal-charged amino acid residues (K3, K6, K12, E16, and R24) contribute to the higher inhibition caused by CNDelta25 than the C protein. Consistent with viral RNA synthesis inhibition, the growth of HPIV3 was significantly decreased by 5 logs in HeLa-derived cell line expressing CNDelta25. Interestingly, replication of respiratory syncytial virus (RSV), another important respiratory tract pathogen, was also strongly inhibited in the presence of CNDelta25. These findings provide a promising potential to use CNDelta25 as an antiviral agent against the clinically important respiratory tract diseases caused by HPIV3 and RSV.
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Affiliation(s)
- Hongxia Mao
- Virology Section, Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, NN1-06, 9500 Euclid Avenue, Cleveland, OH 44195, USA
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20
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Malur AG, Wells G, McCoy A, Banerjee AK. Evidence for phosphorylation of human parainfluenza virus type 3 C protein: mutant C proteins exhibit variable inhibitory activities in vitro. Virus Res 2009; 144:180-7. [PMID: 19410612 PMCID: PMC2736354 DOI: 10.1016/j.virusres.2009.04.022] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2008] [Revised: 04/16/2009] [Accepted: 04/26/2009] [Indexed: 11/16/2022]
Abstract
The P mRNA of human parainfluenza virus type 3, like other members of the subfamily Paramyxovirinae, gives rise to several polypeptides, one amongst them, the C protein, which is involved in inhibition of viral RNA synthesis as well as counteracting the host interferon signaling pathway. As a further step towards characterizing the function of C protein we present evidence to demonstrate the phosphorylation of C protein. Evidence for this observation emerged from deletion mapping studies coupled with mass spectroscopy analysis confirming residues S7, S22, S47T48 and S81 residues as the phosphorylation sites within the NH(2)-terminus of C protein. Here, we utilized a HPIV 3 minigenome replication assay and real time RT-PCR analysis to measure the relative RNA levels synthesized in the presence of mutant C proteins. Mutants S7A and S81A displayed low levels of RNA while mutant 5A that was devoid of all these phosphorylation sites exhibited high RNA level in comparison to wild type C during transcription. Interestingly, high levels of RNA were observed in the presence of S81A and mutant 5A during replication. Taken together, our results indicate that phosphorylation may differentially affect the inhibitory activity of C protein thereby regulating viral RNA synthesis.
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Affiliation(s)
- Achut G Malur
- Department of Microbiology and Immunology, Brody School of Medicine, Biotech 124, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA.
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21
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Chambers R, Takimoto T. Host specificity of the anti-interferon and anti-apoptosis activities of parainfluenza virus P/C gene products. J Gen Virol 2009; 90:1906-1915. [PMID: 19423549 DOI: 10.1099/vir.0.011700-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Human parainfluenza virus type 1 (HPIV-1) and Sendai virus (SeV) are highly homologous in structure and sequence, whilst maintaining distinct host ranges. These viruses express accessory proteins from their P/C gene that are known to have activities against innate immunity. The accessory proteins expressed from the P/C gene of these viruses are different. In addition to the nested set of C proteins, SeV expresses V protein from edited P mRNA, which is not expressed by HPIV-1. This study evaluated the host specificity and role of the P/C gene products in anti-interferon (IFN) and anti-apoptosis activity by characterizing a recombinant SeV, rSeVhP, in which the SeV P/C gene was replaced with that of HPIV-1. Unlike SeV, rSeVhP infection strongly activated IFN regulatory transcription factor (IRF)-3 and nuclear factor-kappaB, resulting in an increased level of IFN-beta induction compared with SeV in murine cells. In contrast, activation of IRF-3 was not observed in rSeVhP-infected human A549 cells. rSeVhPSV, which expressed SeV V protein from an inserted gene in rSeVhP, induced less IFN-beta than rSeVhP, suggesting that V contributes to the suppression of IFN production in murine cells. Furthermore, rSeVhP induced apoptotic cell death in murine but not in A549 cells. These data indicate the functional difference in P/C gene products from SeV and HPIV-1 in antagonizing IFN induction and apoptosis, which is likely to be one of the major factors for pathogenicity in specific hosts.
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Affiliation(s)
- Raychel Chambers
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA
| | - Toru Takimoto
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA
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22
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The C proteins of human parainfluenza virus type 1 (HPIV1) control the transcription of a broad array of cellular genes that would otherwise respond to HPIV1 infection. J Virol 2008; 83:1892-910. [PMID: 19052086 DOI: 10.1128/jvi.01373-08] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role beta interferon played in this response. We similarly evaluated HPIV1-P(C-), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-C(F170S), a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used these data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2,000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-kappaB were overrepresented in some of the C protein-targeted pathways, but other pathways were dominated by less-known factors, such as forkhead transcription factor FOXD1. Surprisingly, the host responses to the P(C-) and C(F170S) mutants were very similar, and only subtle differences in the expression kinetics of caspase 3 and TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses.
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23
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Sleeman K, Bankamp B, Hummel KB, Lo MK, Bellini WJ, Rota PA. The C, V and W proteins of Nipah virus inhibit minigenome replication. J Gen Virol 2008; 89:1300-1308. [PMID: 18420809 DOI: 10.1099/vir.0.83582-0] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.
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Affiliation(s)
- Katrina Sleeman
- Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Bettina Bankamp
- Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Kimberly B Hummel
- Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Michael K Lo
- Emory University, Atlanta, GA, USA.,The Research Institute, Nationwide Children's Hospital, Columbus, OH, USA.,Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - William J Bellini
- Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Paul A Rota
- Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
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24
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IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence. J Virol 2008; 82:3500-8. [PMID: 18216110 DOI: 10.1128/jvi.02536-07] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Here, we report that specific manipulations of the cellular response to virus infection can cause prevention of apoptosis and consequent establishment of persistent infection. Infection of several human cell lines with Sendai virus (SeV) or human parainfluenza virus 3, two prototypic paramyxoviruses, caused slow apoptosis, which was markedly accelerated upon blocking the action of phosphatidylinositol 3-kinases (PI3 kinases) in the infected cells. The observed apoptosis required viral gene expression and the action of the caspase 8 pathway. Although virus infection activated PI3 kinase, as indicated by AKT activation, its blockage did not inhibit JNK activation or IRF-3 activation. The action of neither the Jak-STAT pathway nor the NF-kappaB pathway was required for apoptosis. In contrast, IRF-3 activation was essential, although induction of the proapototic protein TRAIL by IRF-3 was not required. When IRF-3 was absent or its activation by the RIG-I pathway was blocked, SeV established persistent infection, as documented by viral protein production and infectious virus production. Introduction of IRF-3 in the persistently infected cells restored the cells' ability to undergo apoptosis. These results demonstrated that in our model system, IRF-3 controlled the fate of the SeV-infected cells by promoting apoptosis and preventing persistence.
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25
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Sakaguchi T, Kato A, Kiyotani K, Yoshida T, Nagai Y. Studies on the paramyxovirus accessory genes by reverse genetics in the Sendai virus-mouse system. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2008; 84:439-451. [PMID: 19075516 PMCID: PMC3720547 DOI: 10.2183/pjab.84.439] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/29/2008] [Accepted: 10/20/2008] [Indexed: 05/27/2023]
Abstract
Nucleotide sequencing of the entire genomes was completed in the 1980s for most members of the Paramyxoviridae. It then became a new common task with challenge for researchers in the field to establish a system to recover the virus entirely from cDNA, thereby allowing reverse genetics (free manipulation of the viral genome). Using Sendai virus, we established a system of incomparable virus recovery efficiency early on. This technology was then fully exploited in answering a series of long-held questions. In particular, two accessory genes whose functions had remained enigmatic were demonstrated to encode special functions critical in viral in vivo pathogenesis producing fatal pneumonia in mice, although dispensable in virus replication at the in vitro cellular level. Their in vivo functions were found to counteract the two respective facets of the antiviral state induced by interferons and an interferon regulatory factor 3-dependent but yet unknown effector. These achievements appear to have facilitated a scientific trend where the accessory genes are a focus of active investigation in studies on other paramyxoviruses and opened up a new common ground shared between virology and immunology.
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Affiliation(s)
- Takemasa Sakaguchi
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University,
Japan
| | - Atsushi Kato
- Department of Virology 3, National Institute of Infectious Diseases,
Japan
| | - Katsuhiro Kiyotani
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University,
Japan
| | - Tetsuya Yoshida
- Department of Clinical Engineering, Faculty of Health Sciences, Hiroshima International University,
Japan
| | - Yoshiyuki Nagai
- Center of Research Network for Infectious Diseases, RIKEN,
Japan
- Recipient of
Japan Academy Prize in 2008
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26
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Dibben O, Thorpe LC, Easton AJ. Roles of the PVM M2-1, M2-2 and P gene ORF 2 (P-2) proteins in viral replication. Virus Res 2007; 131:47-53. [PMID: 17881076 DOI: 10.1016/j.virusres.2007.08.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2007] [Revised: 08/11/2007] [Accepted: 08/12/2007] [Indexed: 10/22/2022]
Abstract
A plasmid-based reverse genetics system for pneumonia virus of mice (PVM) using a synthetic minigenome is described. The system was used to investigate the functions of several viral proteins. The M2-1 protein of PVM was shown to enhance reporter gene expression when present at low levels, similar to the situation for the equivalent respiratory syncytial virus (RSV) M2-1 protein, but at high levels was shown to reduce gene expression from the minigenome activity, which differs significantly form the situation with RSV. Analysis of levels of nucleocapsid complex RNA showed that high levels of the PVM M2-1 protein inhibits RNA replication rather than transcription. In contrast, expression of the PVM M2-2 protein in conjunction with the polymerase proteins in a minigenome assay greatly reduced the levels of CAT reporter protein. This is similar to the situation with the RSV M2-2 protein although there is no significant sequence identity between the M2-2 proteins of the pneumoviruses. A significant difference between the genome organisations of RSV and PVM is that the P gene of PVM contains a second open reading frame, encoding the P-2 protein, which has no counterpart in the RSV P gene. Co-expression of the PVM P-2 protein with the minigenome inhibited virus gene expression. This resembles the situation seen with the accessory proteins expressed from alternate reading frames of the P gene of other paramyxoviruses. Analysis of levels of antigenome RNA and CAT mRNA produced by the minigenome in the presence of the P2 protein indicated that the protein inhibits viral transcription in a dose-dependent fashion.
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Affiliation(s)
- Oliver Dibben
- Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
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27
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Magoffin DE, Mackenzie JS, Wang LF. Genetic analysis of J-virus and Beilong virus using minireplicons. Virology 2007; 364:103-11. [PMID: 17397895 DOI: 10.1016/j.virol.2007.01.045] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2006] [Revised: 01/02/2007] [Accepted: 01/31/2007] [Indexed: 11/16/2022]
Abstract
J-virus (JPV), isolated from wild mice in Australia, and Beilong virus (BeiPV), originally isolated from human mesangial cells in China and subsequently detected in rat mesangial cells, represent a new group of paramyxoviruses which have exceptionally large genomes (>19 kb) and contain more than six transcriptional units. In this study, minireplicons were employed to assess the taxonomic status of JPV and BeiPV. Our results demonstrated that, whilst the genome replication machineries of JPV and BeiPV can be interchanged, they were not functional when exchanged with that of Nipah virus. These studies indicate that JPV and BeiPV are closely related to each other and support the classification of these two viruses into a separate genus. In addition, the minireplicons were also used to demonstrate that these large-genome viruses also comply with the 'rule of six' and that over-expression of the C protein has a detrimental effect on minigenome replication.
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Affiliation(s)
- Danielle E Magoffin
- CSIRO Livestock Industries, CSIRO Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia
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28
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Chelbi-Alix MK, Vidy A, El Bougrini J, Blondel D. Rabies viral mechanisms to escape the IFN system: the viral protein P interferes with IRF-3, Stat1, and PML nuclear bodies. J Interferon Cytokine Res 2007; 26:271-80. [PMID: 16689655 DOI: 10.1089/jir.2006.26.271] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative, and immunomodulatory activities. The different biologic actions of IFN are believed to be mediated by the products of specifically IFN-stimulated genes (ISG) in the target cells. The IFN response is the first line of defense against viral infections. Viruses, which require the cellular machinery for their replication, have evolved different ways to counteract the action of IFN by inhibiting IFN production or Jak-Stat signaling or by altering ISG products. This review focuses on the role of viral proteins from the RNA virus family, particularly rabies P protein. P protein mediates inhibition of the IFN system by different pathways: it inhibits IFN production by impairing IFN regulatory factor-3 (IRF-3) phosphorylation and IFN signaling by blocking nuclear transport of Stat1 and alters promyelocytic leukemia (PML) nuclear bodies by retaining PML in the cytoplasm.
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29
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Affiliation(s)
- Andrea Paun
- The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, USA
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30
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Kato A, Kiyotani K, Kubota T, Yoshida T, Tashiro M, Nagai Y. Importance of the anti-interferon capacity of Sendai virus C protein for pathogenicity in mice. J Virol 2007; 81:3264-71. [PMID: 17215288 PMCID: PMC1866026 DOI: 10.1128/jvi.02590-06] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K(151)A, E(153)A, and R(154)A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K(151)A, E(153)K, and R(157)L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1(-/-) mice but was cleared from those of STAT1(+/+) mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.
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Affiliation(s)
- Atsushi Kato
- Department of Virology 3, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan.
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Rivals JP, Plattet P, Currat-Zweifel C, Zurbriggen A, Wittek R. Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins. Virology 2006; 359:6-18. [PMID: 17046044 DOI: 10.1016/j.virol.2006.07.054] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2006] [Revised: 04/27/2006] [Accepted: 07/17/2006] [Indexed: 11/16/2022]
Abstract
The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.
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Affiliation(s)
- Jean-Paul Rivals
- Institut de Biotechnologie, Bâtiment de Biologie, University of Lausanne, CH-1015 Lausanne, Switzerland
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Kiyotani K, Sakaguchi T, Kato A, Nagai Y, Yoshida T. Paramyxovirus Sendai virus V protein counteracts innate virus clearance through IRF-3 activation, but not via interferon, in mice. Virology 2006; 359:82-91. [PMID: 17027894 DOI: 10.1016/j.virol.2006.08.053] [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] [Received: 06/29/2006] [Revised: 08/07/2006] [Accepted: 08/29/2006] [Indexed: 10/24/2022]
Abstract
The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-beta induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-alpha/beta receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN.
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Affiliation(s)
- Katsuhiro Kiyotani
- Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
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Faísca P, Desmecht D. Sendai virus, the mouse parainfluenza type 1: a longstanding pathogen that remains up-to-date. Res Vet Sci 2006; 82:115-25. [PMID: 16759680 DOI: 10.1016/j.rvsc.2006.03.009] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2004] [Revised: 01/12/2006] [Accepted: 03/08/2006] [Indexed: 11/20/2022]
Abstract
Biologically speaking, Sendai virus (SeV), the murine parainfluenza virus type 1, is perceived as a common respiratory pathogen that is endemic in many rodent colonies throughout the world. Currently it is believed that SeV is the leading cause of pneumonia in mice and together with the mouse hepatitis viruses, is the most prevalent and important of the naturally occurring infections of mice. The scientific community also considers SeV as the archetype organism of the Paramyxoviridae family because most of the basic biochemical, molecular and biologic properties of the whole family were derived from its own characteristics. Recently, scientific interest for this old pathogen has re-emerged, this time because of its potential value as a vector for gene transfer. This review aimed at drawing an exhaustive picture of this multifaceted pathogen.
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Affiliation(s)
- P Faísca
- Department of Pathology, Faculty of Veterinary Medicine, University of Liège, Sart Tilman Faculty of Veterinary Medicine B43, B-4000 Liège, Belgium.
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Bousse T, Chambers RL, Scroggs RA, Portner A, Takimoto T. Human parainfluenza virus type 1 but not Sendai virus replicates in human respiratory cells despite IFN treatment. Virus Res 2006; 121:23-32. [PMID: 16677733 DOI: 10.1016/j.virusres.2006.03.012] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2005] [Revised: 03/14/2006] [Accepted: 03/20/2006] [Indexed: 11/27/2022]
Abstract
Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-beta into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-beta, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-beta pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV.
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Affiliation(s)
- Tatiana Bousse
- Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 672, NY 14642, USA
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Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006; 441:101-5. [PMID: 16625202 DOI: 10.1038/nature04734] [Citation(s) in RCA: 2956] [Impact Index Per Article: 155.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2006] [Accepted: 03/20/2006] [Indexed: 12/22/2022]
Abstract
The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.
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Affiliation(s)
- Hiroki Kato
- Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Kash JC, Mühlberger E, Carter V, Grosch M, Perwitasari O, Proll SC, Thomas MJ, Weber F, Klenk HD, Katze MG. Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence. J Virol 2006; 80:3009-20. [PMID: 16501110 PMCID: PMC1395418 DOI: 10.1128/jvi.80.6.3009-3020.2006] [Citation(s) in RCA: 146] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We studied the effect of filovirus infection on host cell gene expression by characterizing the regulation of gene expression responses in human liver cells infected with Zaire Ebolavirus (ZEBOV), Reston Ebolavirus (REBOV), and Marburgvirus (MARV), using transcriptional profiling and bioinformatics. Expression microarray analysis demonstrated that filovirus infection resulted in the up-regulation of immune-related genes and the down-regulation of many coagulation and acute-phase proteins. These studies further revealed that a common feature of filovirus virulence is suppression of key cellular antiviral responses, including TLR-, interferon (IFN) regulatory factor 3-, and PKR-related pathways. We further showed that ZEBOV and MARV were more potent antagonists of the IFN response and inhibited the expression of most of the IFN-stimulated genes (ISGs) observed in mock-infected IFN-alpha-2b treated cells, compared to REBOV infection, which activated more than 20% of these ISGs. Finally, we examined IFN-related gene expression in filovirus-infected cells treated with IFN-alpha-2b. These experiments revealed that a majority of genes induced in mock-infected cells treated with type I IFN were antagonized in treated ZEBOV- and MARV-infected cells, while in contrast, REBOV infection resulted in a significant increase in ISG expression. Analysis of STAT1 and -2 phosphorylation following IFN treatment showed a significant reduction of STAT phosphorylation for MARV but not for ZEBOV and REBOV, indicating that different mechanisms might be involved in antagonizing IFN signaling pathways by the different filovirus species. Taken together, these studies showed a correlation between antagonism of type I IFN responses and filovirus virulence.
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Affiliation(s)
- John C Kash
- Department of Microbiology, School of Medicine, University of Washington, Box 358070, Seattle, Washington 98195-8070, USA.
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Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, Miyagishi M, Taira K, Foy E, Loo YM, Gale M, Akira S, Yonehara S, Kato A, Fujita T. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2005; 175:2851-8. [PMID: 16116171 DOI: 10.4049/jimmunol.175.5.2851] [Citation(s) in RCA: 1273] [Impact Index Per Article: 63.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.
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Affiliation(s)
- Mitsutoshi Yoneyama
- Antiviral Innate Immunity Project, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Honkomagome, Tokyo, Japan
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Bartlett EJ, Amaro-Carambot E, Surman SR, Newman JT, Collins PL, Murphy BR, Skiadopoulos MH. Human parainfluenza virus type I (HPIV1) vaccine candidates designed by reverse genetics are attenuated and efficacious in African green monkeys. Vaccine 2005; 23:4631-46. [PMID: 15951066 DOI: 10.1016/j.vaccine.2005.04.035] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2004] [Revised: 03/02/2005] [Accepted: 04/29/2005] [Indexed: 11/16/2022]
Abstract
A set of recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) vaccine candidates was evaluated for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). Temperature sensitive (ts) and non-ts attenuating (att) mutations in the P/C and L genes were introduced individually or in various combinations into rHPIV1, including the C(R84G) and HN(T553A) mutations identified in the present work and the C(F170S), L(Y942A), and L(L992C) mutations identified previously. The rHPIV1 vaccine candidates exhibited a spectrum of attenuation in AGMs. One genetically and phenotypically stable vaccine candidate, rC(R84G/F170S)L(Y942A/L992C), was attenuated and efficacious in AGMs and is a promising live attenuated intranasal HPIV1 vaccine candidate suitable for clinical evaluation.
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Affiliation(s)
- Emmalene J Bartlett
- Laboratory of Infectious Diseases, Respiratory Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 50, Room 6511, 50 South Drive MSC 8007, Bethesda, MD 20892-8007, USA.
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Bankamp B, Wilson J, Bellini WJ, Rota PA. Identification of naturally occurring amino acid variations that affect the ability of the measles virus C protein to regulate genome replication and transcription. Virology 2005; 336:120-9. [PMID: 15866077 DOI: 10.1016/j.virol.2005.03.009] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2005] [Revised: 02/17/2005] [Accepted: 03/10/2005] [Indexed: 11/24/2022]
Abstract
The C protein of measles virus (MV C) is a basic protein of 186 amino acids (aa) that plays at least two roles in infected cells, interference with the innate immune response and modulation of viral polymerase activity. In this study, Northern blots were used to demonstrate that C proteins from three vaccine strains and three wild-type isolates of MV downregulated both mRNA transcription and genome replication in a plasmid-based mini-genome assay. The effect on transcription always paralleled the effect on replication; however, the six MV C proteins varied considerably in their ability to inhibit polymerase activity. Though the amino-terminal 45 aa of the C protein are more variable among different MV strains than the remaining 75% of the protein, the ability of the MV C proteins to inhibit polymerase activity was not regulated by substitutions in the amino terminus, but rather by the more conserved region containing aa 46-167. Naturally occurring substitutions at positions 147 and 166, but not 88 and 186, were found to regulate MV C protein activity. Deletion of the carboxyl-terminal 19 aa did not affect the polymerase-modulating activity. Though we did not find a link between the aa changes in MV C and attenuation, these data provide new information regarding the functions of this non-structural protein.
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Affiliation(s)
- Bettina Bankamp
- Measles, Mumps, Rubella and Herpes Virus Team, Centers for Disease Control and Prevention, Mail-stop C-22, 1600 Clifton Road, Atlanta, GA 30333, USA
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Malur AG, Chattopadhyay S, Maitra RK, Banerjee AK. Inhibition of STAT 1 phosphorylation by human parainfluenza virus type 3 C protein. J Virol 2005; 79:7877-82. [PMID: 15919942 PMCID: PMC1143680 DOI: 10.1128/jvi.79.12.7877-7882.2005] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The P mRNA of the viruses belonging to the subfamily Paramyxovirinae possesses a unique property of giving rise to several accessory proteins by a process that involves the utilization of overlapping open reading frames (the C proteins) and by an "RNA-editing" mechanism (the V proteins). Although these proteins are considered accessory, numerous studies have highlighted the importance of these proteins in virus transcription and interferon signaling, including our previous observation on the role of human parainfluenza virus type 3 (HPIV 3) C protein in the transcription of viral genome (Malur et al., Virus Res. 99:199-204, 2004). In this report, we have addressed its role in interferon signaling by generating a stable cell line, L-C6, by using the lentiviral expression system which expresses HPIV 3 C protein. The L-C6 cells were efficient in abrogating both alpha and gamma interferon-induced antiviral states and demonstrated a drastic reduction in the formation of gamma-activated factor complexes in the cell extracts. Western blot analysis subsequently revealed a defect in the phosphorylation of STAT 1 in these cells. Taken together, our results indicate that HPIV 3 C protein is capable of counteracting the interferon signaling pathway by specifically inhibiting the activation of STAT 1.
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Affiliation(s)
- Achut G Malur
- Cleveland Clinic Foundation, Section Virology NN-10, Department of Molecular Biology, 9500 Euclid Avenue, Cleveland, OH 44195-5178, 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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Kato A. [Sendai virus proteins counteracting the host innate immunity]. Uirusu 2005; 54:179-88. [PMID: 15745155 DOI: 10.2222/jsv.54.179] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The nucleotide sequence of Sendai virus (SeV) genome was determined in the 1980's. During the analysis of its cDNA, two mRNAs were found to be transcribed from the P gene; one encoding P protein, the other encoding V protein. In addition, C protein was found to be translated from both/ mRNAs. Though the function of V and C proteins was being unknown for a while, the reverse-genetic technique of paramyxoviruses developed at the latter half of the 1990's gave the light on studying them. The V or C protein-knockout-SeV can be made successfully, indicating that the V and C proteins are nonessential for virus growth, However, V knockout-SeV was cleared from the mouse lungs at the one day post inoculation, and C knockout-SeV was cleared immediately after the inoculation. Both V and C proteins were thus appeared to be important for counteracting host innate immunity generated in the early phase of viral infection.
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Affiliation(s)
- Atsushi Kato
- Department of Virology 3, National Institute of Infectious Diseases 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan.
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