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Fox CR, Yousef NN, Varudkar N, Shiffer EM, Aquino JR, Kedarinath K, Parks GD. Resistance to complement-mediated lysis of parainfluenza virus 5-infected cells is acquired after transition from acute to persistent infection. J Virol 2025; 99:e0189524. [PMID: 39791880 PMCID: PMC11852780 DOI: 10.1128/jvi.01895-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Accepted: 12/13/2024] [Indexed: 01/12/2025] Open
Abstract
Persistent viral infections can be an important medical problem, with persistently infected (PI) cells extending viral shedding, maintaining inflammation, and providing potential sources for new viral variants. Given that PI cells can acquire resistance to some innate immune pathways, we tested the hypothesis that complement (C')-mediated lysis of parainfluenza virus 5 (PIV5)-infected cells would differ between acute-infected and PI cells. Biochemical and real-time cell viability assays showed effective C'-mediated lysis of A549 lung cells acutely infected with PIV5, through pathways that depended on C3 and C5, but largely independent of C6. A PIV5 PI cell line established by long-term culturing of acutely infected A549 cells showed a high-level persistent expression of PIV5 proteins and infectious virus. Under conditions that led to effective lysis of acute PIV5-infected cells, the PI cells were nearly completely resistant to C'-mediated killing. This lack of C' killing was not due to failure to activate C', since C'-treated PIV5 PI cells had extensive C3 and membrane attack complex deposition, as well as production of C3a and C5a. Transcriptomics analysis revealed the C' cascade as the most significantly upregulated pathway in PIV5 PI cells versus acute infection. Biochemical analyses showed that resistance to C' killing correlated with increased expression in PI cells of two major C' inhibitors: complement factor H and Vitronectin. The finding of acquisition of C' resistance after the transition from acute PIV5 infection to PI cells raises the potential to inform therapeutics for PIs based on modulating C' pathways. IMPORTANCE A persistent infection (PI) with RNA viruses can extend virus shedding, prolong inflammation, and be a source of new viral variants. Since profound changes to innate immune pathways can occur in PI cells, it was important to test PI cells for changes in sensitivity to the complement (C') system, powerful innate immune pathways capable of lysing infected cells. Using parainfluenza virus 5 (PIV5) as a model system, we show that PI cells are nearly completely resistant to C'-mediated lysis, in stark contrast to high sensitivity of acute PIV5-infected cells to C' killing. A key finding was the upregulated expression in PI cells of two C' inhibitors: Vitronectin and complement factor H. These are important results with strong potential to inform therapeutics, given that polymorphisms in C' genes can correlate with severity of viral infections, and clinical trials are underway with new drugs that modulate C' responses.
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Affiliation(s)
- Candace R. Fox
- University of Central Florida, College of Medicine, Orlando, Florida, USA
| | - Nasser N. Yousef
- University of Central Florida, College of Medicine, Orlando, Florida, USA
| | - Namita Varudkar
- University of Central Florida, College of Medicine, Orlando, Florida, USA
| | | | - Jenna R. Aquino
- University of Central Florida, College of Medicine, Orlando, Florida, USA
| | - Kritika Kedarinath
- University of Central Florida, College of Medicine, Orlando, Florida, USA
| | - Griffith D. Parks
- University of Central Florida, College of Medicine, Orlando, Florida, USA
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2
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Randall RE, Young D, Pisliakova M, Andrejeva J, West L, Rossler L, Morath V, Hughes D, Goodbourn S. Single-cycle parainfluenza virus type 5 vectors for producing recombinant proteins, including a humanized anti-V5 tag antibody. J Gen Virol 2025; 106. [PMID: 39786361 DOI: 10.1099/jgv.0.002061] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2025] Open
Abstract
Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here, we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors, we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody, with Chinese hamster ovary cells expressing 20-50 mg l-1 after 5 days when cells were grown to a density of ~1×106 cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo.
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Affiliation(s)
- Richard E Randall
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Dan Young
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Maria Pisliakova
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Jelena Andrejeva
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Lynsey West
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Luis Rossler
- Department of Nuclear Medicine, University Clinic rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Volker Morath
- Department of Nuclear Medicine, University Clinic rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - David Hughes
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
| | - Steve Goodbourn
- Section for Pathogen Research, Institute for Infection and Immunity, St George's, University of London, London, SW17 0RE, UK
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Abstract
Viral quasispecies are dynamic distributions of nonidentical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition, and selection that may act as a unit of selection. The quasispecies concept owes its theoretical origins to a model for the origin of life as a collection of mutant RNA replicators. Independently, experimental evidence for the quasispecies concept was obtained from sampling of bacteriophage clones, which revealed that the viral populations consisted of many mutant genomes whose frequency varied with time of replication. Similar findings were made in animal and plant RNA viruses. Quasispecies became a theoretical framework to understand viral population dynamics and adaptability. The evidence came at a time when mutations were considered rare events in genetics, a perception that was to change dramatically in subsequent decades. Indeed, viral quasispecies was the conceptual forefront of a remarkable degree of biological diversity, now evident for cell populations and organisms, not only for viruses. Quasispecies dynamics unveiled complexities in the behavior of viral populations,with consequences for disease mechanisms and control strategies. This review addresses the origin of the quasispecies concept, its major implications on both viral evolution and antiviral strategies, and current and future prospects.
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Affiliation(s)
- Esteban Domingo
- Department of Interactions with the Environment, Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; .,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, 28029 Madrid, Spain
| | - Carlos García-Crespo
- Department of Interactions with the Environment, Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain;
| | - Celia Perales
- Department of Interactions with the Environment, Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; .,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, 28029 Madrid, Spain.,Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain
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4
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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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5
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Young DF, Wignall-Fleming EB, Busse DC, Pickin MJ, Hankinson J, Randall EM, Tavendale A, Davison AJ, Lamont D, Tregoning JS, Goodbourn S, Randall RE. The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit. PLoS Pathog 2019; 15:e1007561. [PMID: 30742688 PMCID: PMC6386407 DOI: 10.1371/journal.ppat.1007561] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2018] [Revised: 02/22/2019] [Accepted: 01/04/2019] [Indexed: 12/24/2022] Open
Abstract
Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.
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Affiliation(s)
- Dan F. Young
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, United Kingdom
| | - Elizabeth B. Wignall-Fleming
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, United Kingdom
- MRC–University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - David C. Busse
- Mucosal Infection and Immunity Group, Section of Virology, Imperial College London, London, United Kingdom
| | - Matthew J. Pickin
- Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom
| | - Jack Hankinson
- Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom
| | - Elizabeth M. Randall
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, United Kingdom
| | - Amy Tavendale
- School of Life Sciences, University of Dundee, Dundee, United Kingdom
| | - Andrew J. Davison
- MRC–University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Douglas Lamont
- School of Life Sciences, University of Dundee, Dundee, United Kingdom
| | - John S. Tregoning
- Mucosal Infection and Immunity Group, Section of Virology, Imperial College London, London, United Kingdom
| | - Steve Goodbourn
- Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom
| | - Richard E. Randall
- School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, United Kingdom
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6
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Developing a platform system for gene delivery: amplifying virus-like particles (AVLP) as an influenza vaccine. NPJ Vaccines 2017; 2:32. [PMID: 29263887 PMCID: PMC5696535 DOI: 10.1038/s41541-017-0031-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2017] [Revised: 09/19/2017] [Accepted: 09/26/2017] [Indexed: 11/29/2022] Open
Abstract
Delivery of a gene of interest to target cells is highly desirable for translational medicine, such as gene therapy, regenerative medicine, vaccine development, and studies of gene function. Parainfluenza virus 5 (PIV5), a paramyxovirus with a negative-sense RNA genome, normally infects cells without causing obvious cytopathic effect, and it can infect many cell types. To exploit these features of PIV5, we established a system generating self-amplifying, virus-like particles (AVLP). Using enhanced green fluorescent protein (EGFP) as a reporter, AVLP encoding EGFP (AVLP–EGFP) successfully delivered and expressed the EGFP gene in primary human cells, including stem cells, airway epithelial cells, monocytes, and T cells. To demonstrate the application of this system for vaccine development, we generated AVLPs to express the HA and M1 antigens from the influenza A virus strain H5N1 (AVLP–H5 and AVLP–M1H5). Immunization of mice with AVLP–H5 and AVLP–M1H5 generated robust antibody and cellular immune responses. Vaccination with a single dose of AVLP–H5 and M1H5 completely protected mice against lethal H5N1 challenge, suggesting that the AVLP-based system is a promising platform for delivery of desirable genes. An ‘imitation virus’ can be used to deliver genetic material to target cells, with farreaching potential for medical application. The capacity to safely and affordably introduce genes into cells is highly-sought. A team led by the University of Georgia’s Biao He created a protein shell using parainfluenza virus 5 proteins, with the resultant particles possessing the ability to infect multiple types of cell and deliver desired genetic material. The team proved the utility of their system by using it to express immunity-promoting components of avian influenza virus in live mice—successfully vaccinating the animals, and enabling them to survive a subsequent lethal infection. His group also showed that their system is also able to deliver and express genes in human cells, prompting further research into this useful tool.
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7
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Thibault PA, Watkinson RE, Moreira-Soto A, Drexler JF, Lee B. Zoonotic Potential of Emerging Paramyxoviruses: Knowns and Unknowns. Adv Virus Res 2017; 98:1-55. [PMID: 28433050 PMCID: PMC5894875 DOI: 10.1016/bs.aivir.2016.12.001] [Citation(s) in RCA: 66] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The risk of spillover of enzootic paramyxoviruses and the susceptibility of recipient human and domestic animal populations are defined by a broad collection of ecological and molecular factors that interact in ways that are not yet fully understood. Nipah and Hendra viruses were the first highly lethal zoonotic paramyxoviruses discovered in modern times, but other paramyxoviruses from multiple genera are present in bats and other reservoirs that have unknown potential to spillover into humans. We outline our current understanding of paramyxovirus reservoir hosts and the ecological factors that may drive spillover, and we explore the molecular barriers to spillover that emergent paramyxoviruses may encounter. By outlining what is known about enzootic paramyxovirus receptor usage, mechanisms of innate immune evasion, and other host-specific interactions, we highlight the breadth of unexplored avenues that may be important in understanding paramyxovirus emergence.
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Affiliation(s)
| | - Ruth E Watkinson
- Icahn School of Medicine at Mount Sinai, New York, NY, United States
| | | | - Jan F Drexler
- Institute of Virology, University of Bonn Medical Centre, Bonn, Germany; German Centre for Infection Research (DZIF), Partner Site Bonn-Cologne, Bonn, Germany
| | - Benhur Lee
- Icahn School of Medicine at Mount Sinai, New York, NY, United States.
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8
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Martínez Gómez JM, Ong LC, Lam JH, Binte Aman SA, Libau EA, Lee PX, St. John AL, Alonso S. Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage. PLoS Negl Trop Dis 2016; 10:e0004536. [PMID: 27007501 PMCID: PMC4805191 DOI: 10.1371/journal.pntd.0004536] [Citation(s) in RCA: 21] [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: 10/03/2015] [Accepted: 02/22/2016] [Indexed: 11/18/2022] Open
Abstract
Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals' death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates.
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Affiliation(s)
- Julia María Martínez Gómez
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Immunology programme, Life Sciences Institute, National University of Singapore, Singapore
| | - Li Ching Ong
- Infectious Disease programme, Singapore-MIT alliance for Research and Technology (SMART), National University of Singapore, Singapore
| | - Jian Hang Lam
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Immunology programme, Life Sciences Institute, National University of Singapore, Singapore
| | | | - Eshele Anak Libau
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Immunology programme, Life Sciences Institute, National University of Singapore, Singapore
| | - Pei Xuan Lee
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Immunology programme, Life Sciences Institute, National University of Singapore, Singapore
| | | | - Sylvie Alonso
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Immunology programme, Life Sciences Institute, National University of Singapore, Singapore
- Infectious Disease programme, Singapore-MIT alliance for Research and Technology (SMART), National University of Singapore, Singapore
- * E-mail:
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9
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Chen Z, Gupta T, Xu P, Phan S, Pickar A, Yau W, Karls RK, Quinn FD, Sakamoto K, He B. Efficacy of parainfluenza virus 5 (PIV5)-based tuberculosis vaccines in mice. Vaccine 2015; 33:7217-7224. [PMID: 26552000 DOI: 10.1016/j.vaccine.2015.10.124] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2015] [Revised: 09/24/2015] [Accepted: 10/28/2015] [Indexed: 01/07/2023]
Abstract
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is an important human pathogen. Bacillus Calmette-Guérin (BCG), a live, attenuated variant of Mycobacterium bovis, is currently the only available TB vaccine despite its low efficacy against the infectious pulmonary form of the disease in adults. Thus, a more-effective TB vaccine is needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, has several characteristics that make it an attractive vaccine vector. It is safe, inexpensive to produce, and has been previously shown to be efficacious as the backbone of vaccines for influenza, rabies, and respiratory syncytial virus. In this work, recombinant PIV5 expressing M. tuberculosis antigens 85A (PIV5-85A) and 85B (PIV5-85B) have been generated and their immunogenicity and protective efficacy evaluated in a mouse aerosol infection model. In a long-term protection study, a single dose of PIV5-85A was found to be most effective in reducing M. tuberculosis colony forming units (CFU) in lungs when compared to unvaccinated, whereas the BCG vaccinated animals had similar numbers of CFUs to unvaccinated animals. BCG-prime followed by a PIV5-85A or PIV5-85B boost produced better outcomes highlighted by close to three-log units lower lung CFUs compared to PBS. The results indicate that PIV5-based M. tuberculosis vaccines are promising candidates for further development.
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Affiliation(s)
- Zhenhai Chen
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Tuhina Gupta
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Pei Xu
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Shannon Phan
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Adrian Pickar
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Wilson Yau
- Department of Pathology, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Russell K Karls
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Frederick D Quinn
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Kaori Sakamoto
- Department of Pathology, University of Georgia College of Veterinary Medicine, Athens, GA, USA
| | - Biao He
- Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA, USA.
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10
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Ng JKW, Zhang SL, Tan HC, Yan B, Maria Martinez Gomez J, Tan WY, Lam JH, Tan GKX, Ooi EE, Alonso S. First experimental in vivo model of enhanced dengue disease severity through maternally acquired heterotypic dengue antibodies. PLoS Pathog 2014; 10:e1004031. [PMID: 24699622 PMCID: PMC3974839 DOI: 10.1371/journal.ppat.1004031] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2013] [Accepted: 02/11/2014] [Indexed: 11/18/2022] Open
Abstract
Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations. Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.
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Affiliation(s)
- Jowin Kai Wei Ng
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
| | | | - Hwee Cheng Tan
- Progamme in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
| | - Benedict Yan
- Department of Pathology, National University Health System and National University of Singapore, Singapore
| | - Julia Maria Martinez Gomez
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
| | - Wei Yu Tan
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
| | - Jian Hang Lam
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
| | - Grace Kai Xin Tan
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
| | - Eng Eong Ooi
- Progamme in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
| | - Sylvie Alonso
- Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- Immunology Programme, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore
- * E-mail:
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11
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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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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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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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Meng C, Qiu X, Jin S, Yu S, Chen H, Ding C. Whole genome sequencing and biological characterization of Duck/JS/10, a new lentogenic class I Newcastle disease virus. Arch Virol 2012; 157:869-80. [PMID: 22310996 DOI: 10.1007/s00705-012-1248-4] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2011] [Accepted: 01/06/2012] [Indexed: 11/25/2022]
Abstract
A lentogenic Newcastle disease virus (NDV), Duck/JS/10 (JS10), was isolated from an unvaccinated duck in China. The complete genome of the virus contained 15,198 nucleotides. Based on length of the genome and a partial sequence of the F gene, the virus was classified as a class I genotype 4 NDV. The antigenicity of the virus was compared with that of NDV strain La Sota via hemagglutination inhibition (HI), virus neutralization (VN) assay and animal experiments. Our results show that JS10 generates higher HI and VN titers than La Sota against both class I and II virulent NDV strains. Experiments on animals demonstrate that virus shedding from chickens vaccinated with JS10 is significantly reduced when compared to those vaccinated with La Sota. Overall, this study strongly suggests that JS10 may qualify as a new vaccine candidate against Newcastle disease.
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Affiliation(s)
- Chunchun Meng
- Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, People's Republic of China
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Basler CF. Nipah and hendra virus interactions with the innate immune system. Curr Top Microbiol Immunol 2012; 359:123-52. [PMID: 22491899 DOI: 10.1007/82_2012_209] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Nipah virus and Hendra virus are related, highly pathogenic paramyxoviruses with unusually broad host ranges. Henipaviruses encode several proteins that block innate immune responses, and these are likely to serve as virulence factors. Specfically, four virus-encoded proteins, the phosphoprotein (P), the V protein, the W protein, and the C protein have each been demonstrated to counteract aspects of the interferon (IFN)-α/β response, a key component of the innate immune response to virus infection. The available data indicate that V and W can inhibit the production of IFNα/β in response to various stimuli, while the P, V, and W proteins also block the ability of IFNs to signal and induce an antiviral state in cells. The C protein also inhibits the antiviral effects of IFNα/β by a poorly characterized mechanism. Reverse genetics systems, which allow the generation of recombinant viruses bearing specific mutations, have demonstrated the importance of the viral IFN-antagonists for replication. With these systems in hand, the field is now poised to define how specific viral IFN-antagonist functions influence viral pathogenesis.
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Affiliation(s)
- Christopher F Basler
- Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
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Rosas-Murrieta NH, Herrera-Camacho I, Palma-Ocampo H, Santos-López G, Reyes-Leyva J. Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies. Virol J 2010; 7:263. [PMID: 20937132 PMCID: PMC2958915 DOI: 10.1186/1743-422x-7-263] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2010] [Accepted: 10/11/2010] [Indexed: 12/24/2022] Open
Abstract
Background Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant. Results VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2. Conclusions Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.
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Affiliation(s)
- Nora H Rosas-Murrieta
- Laboratorio de Bioquímica y Biología Molecular, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif, 103 H, CU-BUAP, San Manuel, CP 72550, Puebla, México.
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Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import. J Virol 2010; 84:6328-43. [PMID: 20427537 DOI: 10.1128/jvi.01878-09] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.
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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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19
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Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe 2010; 7:128-39. [PMID: 20153282 PMCID: PMC2824513 DOI: 10.1016/j.chom.2010.01.004] [Citation(s) in RCA: 283] [Impact Index Per Article: 18.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2009] [Revised: 12/29/2009] [Accepted: 01/12/2010] [Indexed: 01/12/2023]
Abstract
Dengue virus (DENV) causes disease ranging from dengue fever (DF), a self-limited febrile illness, to the potentially lethal dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). DHF/DSS usually occurs in patients who have acquired DENV-reactive antibodies prior to infection, either from a previous infection with a heterologous DENV serotype or from an immune mother. Hence, it has been hypothesized that subneutralizing levels of antibodies exacerbate disease, a phenomenon termed antibody-dependent enhancement (ADE). However, given the lack of suitable animal models for DENV infection, the mechanism of ADE and its contribution to pathology remain elusive. Here we demonstrate in mice that DENV-specific antibodies can sufficiently increase severity of disease so that a mostly nonlethal illness becomes a fatal disease resembling human DHF/DSS. Antibodies promote massive infection of liver sinusoidal endothelial cells (LSECs), resulting in increased systemic levels of virus. Thus, a subprotective humoral response may, under some circumstances, have pathological consequences.
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Chambers R, Takimoto T. Antagonism of innate immunity by paramyxovirus accessory proteins. Viruses 2009; 1:574-593. [PMID: 21994561 PMCID: PMC3185518 DOI: 10.3390/v1030574] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2009] [Revised: 10/22/2009] [Accepted: 10/26/2009] [Indexed: 12/15/2022] Open
Abstract
Paramyxovirinae, a subfamily of Paramyxoviridae, are negative strand RNA viruses comprised of many important human and animal pathogens, which share a high degree of genetic and structural homology. The accessory proteins expressed from the P/V/C gene are major factors in the pathogenicity of the viruses, because of their ability to abrogate various facets of type I interferon (IFN) induction and signaling. Most of the paramyxoviruses exhibit a commonality in their ability to antagonize innate immunity by blocking IFN induction and the Jak/STAT pathway. However, the manner in which the accessory proteins inhibit the pathway differs among viruses. Similarly, there are variations in the capability of the viruses to counteract intracellular detectors (RNA helicases, mda-5 and RIG-I). Furthermore, a functional specificity in the antagonism of the IFN response has been reported, suggesting that specificity in the circumvention of innate immunity restricts viral host range. Available evidence indicates that paramyxoviruses employ specific strategies to antagonize the IFN response of their specific hosts, which is one of the major factors that determine viral pathogenicity and host range.
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Affiliation(s)
| | - Toru Takimoto
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-585-273-2856; Fax: +1-585-473-9573
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21
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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: 20] [Impact Index Per Article: 1.3] [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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Capraro GA, Johnson JB, Kock ND, Parks GD. Virus growth and antibody responses following respiratory tract infection of ferrets and mice with WT and P/V mutants of the paramyxovirus Simian Virus 5. Virology 2008; 376:416-28. [PMID: 18456301 PMCID: PMC2574746 DOI: 10.1016/j.virol.2008.03.034] [Citation(s) in RCA: 26] [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: 03/01/2008] [Revised: 03/19/2008] [Accepted: 03/27/2008] [Indexed: 11/29/2022]
Abstract
P/V gene substitutions convert the non-cytopathic paramyxovirus Simian Virus 5 (SV5), which is a poor inducer of host cell responses in human tissue culture cells, into a mutant (P/V-CPI-) that induces high levels of apoptosis, interferon (IFN)-beta, and proinflammatory cytokines. However, the effect of SV5-P/V gene mutations on virus growth and adaptive immune responses in animals has not been determined. Here, we used two distinct animal model systems to test the hypothesis that SV5-P/V mutants which are more potent activators of innate responses in tissue culture will also elicit higher antiviral antibody responses. In mouse cells, in vitro studies identified a panel of SV5-P/V mutants that ranged in their ability to limit IFN responses. Intranasal infection of mice with these WT and P/V mutant viruses elicited equivalent anti-SV5 IgG responses at all doses tested, and viral titers recovered from the respiratory tract were indistinguishable. In primary cultures of ferret lung fibroblasts, WT rSV5 and P/V-CPI- viruses had phenotypes similar to those established in human cell lines, including differential induction of IFN secretion, IFN signaling and apoptosis. Intranasal infection of ferrets with a low dose of WT rSV5 elicited approximately 500 fold higher anti-SV5 serum IgG responses compared to the P/V-CPI- mutant, and this correlated with overall higher viral titers for the WT virus in tracheal tissues. There was a dose-dependent increase in antibody response to infection of ferrets with P/V-CPI-, but not with WT rSV5. Together our data indicate that WT rSV5 and P/V mutants can elicit distinct innate and adaptive immunity phenotypes in the ferret animal model system, but not in the mouse system. We present a model for the effect of P/V gene substitutions on SV5 growth and immune responses in vivo.
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Affiliation(s)
- Gerald A Capraro
- Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1064, USA
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Randall RE, Goodbourn S. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 2008; 89:1-47. [PMID: 18089727 DOI: 10.1099/vir.0.83391-0] [Citation(s) in RCA: 1233] [Impact Index Per Article: 72.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most, if not all, virus infections in the absence of adaptive immunity. However, viruses can still replicate and cause disease in vivo, because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn, S., Didcock, L. & Randall, R. E. (2000). J Gen Virol 81, 2341-2364] but, since then, a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest, but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review, we describe how viruses activate the IFN system, how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part, we describe how viruses circumvent the IFN response. Here, we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections), whether perturbation of the IFN response by persistent infections can lead to chronic disease, and the importance of the IFN system as a species barrier to virus infections. Lastly, we briefly describe applied aspects that arise from an increase in our knowledge in this area, including vaccine design and manufacture, the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer.
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Affiliation(s)
- Richard E Randall
- School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK
| | - Stephen Goodbourn
- Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK
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24
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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: 18] [Impact Index Per Article: 1.0] [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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25
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Cho SH, Kim SJ, Kwon HJ. Genomic sequence of an antigenic variant Newcastle disease virus isolated in Korea. Virus Genes 2007; 35:293-302. [PMID: 17318427 DOI: 10.1007/s11262-007-0078-z] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2006] [Accepted: 01/04/2007] [Indexed: 11/25/2022]
Abstract
In Korea, extensive Newcastle disease (ND) vaccine programs have been implemented, but ND outbreaks continue to occur occasionally, even in well-vaccinated farms. KBNP-4152 is a virulent ND virus, which has been isolated from vaccinated chickens in Korea. In this study, we conducted a comparison of the antigenicity of KBNP-4152 with that of a vaccine strain, La Sota, via virus-neutralization (VN) and cross haemagglutination-inhibition (HI) tests, and analyzed the genomic sequences. The antigenicity of KBNP-4152 was distinct from La Sota, and the expected genome size was 15,192 nt, as was the case with other recent virulent ND viruses analyzed. Based on the partial F gene, the strain was phylogenetically classified into the VIId genotype, but was distinct from other VII viruses due to amino acid changes at (E347K) and proximal to (M354K), the major linear epitope of HN, as well as relatively low amino acid similarity of the V protein, and a truncated W protein (203 aa vs. 227 aa). Therefore, KBNP-4152 is unique among genotype VII.
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Affiliation(s)
- Sun-Hee Cho
- Laboratory of Avian Diseases, College of Veterinary Medicine and BK21 for Veterinary Science, Seoul National University, Seoul 151-742, Korea
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26
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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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27
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Hagmaier K, Stock N, Goodbourn S, Wang LF, Randall R. A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species. J Gen Virol 2006; 87:3649-3653. [PMID: 17098981 PMCID: PMC2884973 DOI: 10.1099/vir.0.82261-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein, referred to as V(AAHL), that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V, but not V(AAHL), was functional as an IFN antagonist in human, monkey, rabbit, dog, horse, pig and bat cells, which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.
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Affiliation(s)
- Kathrin Hagmaier
- Centre for Biomolecular Sciences, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK
| | - Nicola Stock
- Centre for Biomolecular Sciences, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK
| | - Steve Goodbourn
- Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK
| | - Lin-Fa Wang
- CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia
| | - Richard Randall
- Centre for Biomolecular Sciences, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK
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28
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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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29
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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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30
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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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31
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Arimilli S, Alexander-Miller MA, Parks GD. A simian virus 5 (SV5) P/V mutant is less cytopathic than wild-type SV5 in human dendritic cells and is a more effective activator of dendritic cell maturation and function. J Virol 2006; 80:3416-27. [PMID: 16537609 PMCID: PMC1440371 DOI: 10.1128/jvi.80.7.3416-3427.2006] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI-) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI- infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI- mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-alpha and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.
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Affiliation(s)
- Subhashini Arimilli
- Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1064, USA
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32
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Haller O, Kochs G, Weber F. The interferon response circuit: induction and suppression by pathogenic viruses. Virology 2006; 344:119-30. [PMID: 16364743 PMCID: PMC7125643 DOI: 10.1016/j.virol.2005.09.024] [Citation(s) in RCA: 527] [Impact Index Per Article: 27.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2005] [Accepted: 09/10/2005] [Indexed: 12/14/2022]
Abstract
Type I interferons (IFN-α/β) are potent antiviral cytokines and modulators of the adaptive immune system. They are induced by viral infection or by double-stranded RNA (dsRNA), a by-product of viral replication, and lead to the production of a broad range of antiviral proteins and immunoactive cytokines. Viruses, in turn, have evolved multiple strategies to counter the IFN system which would otherwise stop virus growth early in infection. Here we discuss the current view on the balancing act between virus-induced IFN responses and the viral counterplayers.
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Affiliation(s)
- Otto Haller
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany.
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33
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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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34
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Young VA, Dillon PJ, Parks GD. Variants of the paramyxovirus Simian virus 5 with accelerated or delayed viral gene expression activate proinflammatory cytokine synthesis. Virology 2006; 350:90-102. [PMID: 16480754 DOI: 10.1016/j.virol.2006.01.006] [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: 11/30/2005] [Revised: 12/22/2005] [Accepted: 01/04/2006] [Indexed: 11/30/2022]
Abstract
Our previous results have shown that the parainfluenza virus SV5 is a poor inducer of proinflammatory cytokines interleukin-8 (IL-8) and macrophage chemoattractant protein 1 (MCP-1). By contrast, an engineered P/V mutant rSV5-P/V-CPI- and a naturally-occurring variant WF-PIV (Wake Forest-Parainfluenza Virus) are both potent activators of IL-8 and MCP-1. In the present study, we addressed the question of why rSV5-WT is such a poor inducer of host cytokine responses relative to the two SV5 variants, and we used the CC chemokine RANTES as a measure of host responses. Time course experiments showed high-level secretion of IL-6 and RANTES following infections of human A549 lung epithelial cells with the P/V-CPI- mutant and WF-PIV. By contrast, SV5-WT induced very low cytokine responses, with the notable exception of moderate induction of RANTES. The mechanism of RANTES induction by the two SV5 variants shared common properties, since RANTES secretion from infected cells had similar kinetics, depended on virus replication, correlated with increased RANTES mRNA levels and promoter activation, and was reduced by inhibitors of the p38 MAPK, ERK, and PI3K pathways. Despite the similar mechanisms of RANTES induction, the two SV5 variants differed dramatically in their growth and gene expression kinetics. By comparison to the P/V mutant rSV5-P/V-CPI- which has accelerated viral gene expression, WF-PIV infection showed a prolonged delay in viral replication, and infected cells did not show high-level viral RNA and protein expression until approximately 12-24 hpi. Sequence analysis revealed that the N, P, V, and M genes from WF-PIV differed by 3, 8, 5, and 10 amino acids compared to rSV5-WT, respectively. Chimeric viruses harboring the WF-PIV P/V or M genes in the context of the other rSV5 genes had growth properties similar to rSV5-WT but had a RANTES-inducing phenotype similar to that of the bone fide WF-PIV virus. Our data indicate a role for both the P/V and the M gene products as determinants of RANTES induction in response to SV5 infection.
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Affiliation(s)
- Virginia A Young
- Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1064, USA
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35
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Abstract
Hendra virus and Nipah virus are highly pathogenic paramyxoviruses that have recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These features led to their classification into the new genus Henipavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. This review provides an overview of henipaviruses and the types of infection they cause, and describes how studies on the structure and function of henipavirus proteins expressed from cloned genes have provided insights into the unique biological properties of these emerging human pathogens.
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Affiliation(s)
- Bryan T Eaton
- Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organization, 5 Portarlington Road, Geelong, Victoria 3220, Australia.
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36
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Lin Y, Horvath F, Aligo JA, Wilson R, He B. The role of simian virus 5 V protein on viral RNA synthesis. Virology 2005; 338:270-80. [PMID: 15950997 DOI: 10.1016/j.virol.2005.05.014] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2005] [Revised: 03/10/2005] [Accepted: 05/06/2005] [Indexed: 12/01/2022]
Abstract
The paramyxovirus simian virus 5 (SV5) has seven genes but encodes eight known viral proteins. The V/P gene is transcribed into two mRNA species: V mRNA from a faithful transcription of the gene and P mRNA from transcription with addition of two G residues at a specific site of the gene. V, a 222-amino acid (AA) residue protein, and P, a 392 AA residue protein, share an identical N-terminus domain of 164 amino acid residues. P is essential for SV5 RNA replication and transcription. Whereas it is known that V plays important roles in virus pathogenesis, the role of V in SV5 replication and transcription is not clear. A mini-genome system, free of vaccinia virus gene expression system, consisting of plasmids expressing NP, P, and L, as well as a plasmid encoding a reporter gene, chloramphenicol acetyltransferase (CAT) flanked by SV5 trailer and leader sequences under control of a bacteriophage T7 RNA polymerase promoter, has been established to examine the role of V in SV5 RNA transcription and replication. Addition of V-expressing plasmid in the mini-genome system caused inhibition of the reporter gene expression, suggesting that V plays a role in regulating SV5 gene expression. By examining the amount of encapsidated viral RNA genome using reverse transcription with primer annealing to viral anti-genome RNA and PCR, it was found that expression of V reduced the amount of viral RNA genome in the mini-genome system, suggesting that V inhibits viral RNA replication. To examine whether the V protein inhibits viral RNA transcription as well, a mini-genome system with a defective anti-genome promoter (AGP) such that a reporter gene (luciferase, Luc) expression is only derived from transcription of newly produced mini-genome and not from de novo replicated viral genome due to the defect in replication element has been utilized. The V protein inhibited luciferase expression from the mini-genome with a defective AGP, suggesting V inhibits SV5 transcription. Thus, SV5 V inhibits both SV5 RNA replication and transcription.
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Affiliation(s)
- Yuan Lin
- Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802, USA
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37
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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: 62] [Impact Index Per Article: 3.1] [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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38
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Reid SP, Cárdenas WB, Basler CF. Homo-oligomerization facilitates the interferon-antagonist activity of the ebolavirus VP35 protein. Virology 2005; 341:179-89. [PMID: 16095644 PMCID: PMC3955989 DOI: 10.1016/j.virol.2005.06.044] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2005] [Revised: 04/14/2005] [Accepted: 06/23/2005] [Indexed: 11/30/2022]
Abstract
We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP35 protein. Cross-linking studies demonstrated the ability of VP35 to form trimers, consistent with the presence of a functional coiled-coil motif. VP35 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP35 inhibits signaling that activates interferon regulatory factor 3 (IRF-3) and inhibits (IFN)-alpha/beta production. Experiments comparing the ability of VP35 mutants to block IFN responses demonstrated that the VP35 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP35 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP35, suggesting that an oligomerization function at the amino-terminus facilitates an "IFN-antagonist" function exerted by the carboxy-terminal half of VP35.
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Affiliation(s)
| | | | - Christopher F. Basler
- Corresponding Author: Christopher F. Basler, PhD, Assistant Professor, Dept. Microbiology, Box 1124, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, Tel (212) 241-4847, Fax (212) 534-1684,
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39
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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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40
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Pejawar SS, Parks GD, Alexander-Miller MA. Abortive versus productive viral infection of dendritic cells with a paramyxovirus results in differential upregulation of select costimulatory molecules. J Virol 2005; 79:7544-57. [PMID: 15919909 PMCID: PMC1143689 DOI: 10.1128/jvi.79.12.7544-7557.2005] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.
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Affiliation(s)
- Sharmila S Pejawar
- Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA
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41
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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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42
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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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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: 42] [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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Andrejeva J, Childs KS, Young DF, Carlos TS, Stock N, Goodbourn S, Randall RE. The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter. Proc Natl Acad Sci U S A 2004; 101:17264-9. [PMID: 15563593 PMCID: PMC535396 DOI: 10.1073/pnas.0407639101] [Citation(s) in RCA: 804] [Impact Index Per Article: 38.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2004] [Indexed: 01/20/2023] Open
Abstract
Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN-beta promoter in reporter gene assays and significantly enhanced the activation of the IFN-beta promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN-beta promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN-beta promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.
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Affiliation(s)
- J Andrejeva
- School of Biology, Biomolecular Sciences Building, North Haugh, University of St. Andrews, Fife KY16 9TS, United Kingdom
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Liu WJ, Chen HB, Wang XJ, Huang H, Khromykh AA. Analysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon promoter-driven transcription. J Virol 2004; 78:12225-35. [PMID: 15507609 PMCID: PMC525072 DOI: 10.1128/jvi.78.22.12225-12235.2004] [Citation(s) in RCA: 131] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-alpha/beta); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in approximately 15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-beta promoter resulted in approximately 6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing individual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-beta promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-beta promoter-driven transcription, showed approximately 7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-beta promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.
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Affiliation(s)
- Wen Jun Liu
- Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia
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Ohno S, Ono N, Takeda M, Takeuchi K, Yanagi Y. Dissection of measles virus V protein in relation to its ability to block alpha/beta interferon signal transduction. J Gen Virol 2004; 85:2991-2999. [PMID: 15448362 DOI: 10.1099/vir.0.80308-0] [Citation(s) in RCA: 116] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Interferon (IFN)-alpha and -beta are the main cytokines for innate immune responses against viral infections. To replicate efficiently in the hosts, viruses have evolved various countermeasures to the IFN response. The V protein of measles virus (MV) has been shown to block IFN-alpha/beta signalling. Here, the wild-type IC-B strain of MV was shown to grow comparably in the presence and absence of IFN-alpha, whereas replication of the Edmonston tag strain recovered from cloned DNA was strongly suppressed in its presence. The V protein of the IC-B strain, but not the Edmonston tag strain, blocked IFN-alpha signalling. The V protein of the Edmonston strain from the ATCC also inhibited IFN-alpha signalling. There were three amino acid differences between the V proteins of the Edmonston ATCC and tag strains, and substitutions of both residues at positions 110 and 272 were required for the Edmonston ATCC V protein to lose IFN-antagonist activity. The P protein of the IC-B strain, which shares the N-terminal 231 aa residues with the V protein, also inhibited IFN-alpha signalling. Indeed, fragments comprising only those 231 residues of the IC-B and Edmonston ATCC V proteins, but not the Edmonston tag V protein, were able to block IFN-alpha signalling. However, the N-terminal region of the Edmonston tag V protein, when attached to the C-terminal region of the Edmonston ATCC V protein, inhibited IFN-alpha signalling. Taken together, our results indicate that both the N- and C-terminal regions contribute to the IFN-antagonist activity of the MV V protein.
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Affiliation(s)
- Shinji Ohno
- Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan
| | - Nobuyuki Ono
- Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan
| | - Makoto Takeda
- Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan
| | - Kaoru Takeuchi
- Department of Infection Biology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
| | - Yusuke Yanagi
- Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan
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Nagai Y, Kato A. Accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, as a focus of active investigation by reverse genetics. Curr Top Microbiol Immunol 2004; 283:197-248. [PMID: 15298171 DOI: 10.1007/978-3-662-06099-5_6] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/19/2023]
Abstract
The Paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, comprises several genera each containing important human and animal pathogens. They possess in common six basal genes essential for viral replication and, in addition, a subset of accessory genes that are largely unique to each genus. These accessory genes are either encoded in one or more alternative overlapping frames of a basal gene, which are accessed transcriptionally or translationally, or inserted before or between the basal genes as one or more extra genes. However, the question of how the individual accessory genes contribute to actual viral replication and pathogenesis remained unanswered. It was not even established whether they are dispensable or indispensable for the viral life cycle. The plasmid-based reverse genetics of the full-length viral genome has now come into wide use to demonstrate that most, if not all, of these putative accessory genes can be disrupted without destroying viral infectivity, conclusively defining them as indeed dispensable accessory genes. Studies on the phenotypes of the resulting gene knockout viruses have revealed that the individual accessory genes greatly contribute specifically and additively to the overall viral fitness both in vitro and in vivo.
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Affiliation(s)
- Y Nagai
- Toyama Institute of Health, 17-1 Nakataikouyama, Kosugi-machi, 939-0363, Toyama, Japan.
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Peeters B, Verbruggen P, Nelissen F, de Leeuw O. The P gene of Newcastle disease virus does not encode an accessory X protein. J Gen Virol 2004; 85:2375-2378. [PMID: 15269379 DOI: 10.1099/vir.0.80160-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Many paramyxoviruses encode non-essential accessory proteins that are involved in the regulation of virus replication and inhibition of cellular antiviral responses. It has been suggested that the P gene mRNA of Newcastle disease virus (NDV) encodes an accessory protein – the so-called X protein – by translation initiation at a conserved in-frame AUG codon at position 120. Using a monoclonal antibody that specifically detected the P and X proteins, it was shown that an accessory X protein was not expressed in NDV-infected cells. Recombinant NDV strains in which the AUG was changed into a GCC (Ala) or GUC (Val) codon were viable but showed a reduction in virulence, probably because the amino acid change affected the function of the P and/or V protein.
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Affiliation(s)
- Ben Peeters
- Division of Infectious Diseases, Animal Sciences Group, Wageningen University and Research Centre, PO Box 65, NL-8200 AB Lelystad, The Netherlands
| | - Paul Verbruggen
- Division of Infectious Diseases, Animal Sciences Group, Wageningen University and Research Centre, PO Box 65, NL-8200 AB Lelystad, The Netherlands
| | - Frank Nelissen
- Division of Infectious Diseases, Animal Sciences Group, Wageningen University and Research Centre, PO Box 65, NL-8200 AB Lelystad, The Netherlands
| | - Olav de Leeuw
- Division of Infectious Diseases, Animal Sciences Group, Wageningen University and Research Centre, PO Box 65, NL-8200 AB Lelystad, The Netherlands
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Shaw ML, García-Sastre A, Palese P, Basler CF. Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively. J Virol 2004; 78:5633-41. [PMID: 15140960 PMCID: PMC415790 DOI: 10.1128/jvi.78.11.5633-5641.2004] [Citation(s) in RCA: 187] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-alpha/beta receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol. 76:11476-11483, 2002; M. S. Park et al., J. Virol. 77:1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products.
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Affiliation(s)
- Megan L Shaw
- Department of Microbiology, Box 1124, Mount Sinai School of Medicine, One Gustave L. Levy Pl., New York, NY 10029, USA
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50
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Rodriguez JJ, Cruz CD, Horvath CM. Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion. J Virol 2004; 78:5358-67. [PMID: 15113915 PMCID: PMC400366 DOI: 10.1128/jvi.78.10.5358-5367.2004] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.
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Affiliation(s)
- Jason J Rodriguez
- Immunobiology Center, The Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA
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