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Wang P, Guo J, Zhou Y, Zhu M, Fang S, Sun F, Huang C, Zhu Y, Zhou H, Pan B, Qin Y, Ouyang K, Wei Z, Huang W, García-Sastre A, Chen Y. The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus. Emerg Microbes Infect 2024; 13:2400546. [PMID: 39221898 PMCID: PMC11404376 DOI: 10.1080/22221751.2024.2400546] [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] [Received: 02/14/2024] [Revised: 08/20/2024] [Accepted: 08/30/2024] [Indexed: 09/04/2024]
Abstract
The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-β in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.
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Affiliation(s)
- Pingping Wang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Jianing Guo
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
| | - Yefan Zhou
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Min Zhu
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Senbiao Fang
- Department of Molecular Pharmacology, Tianjin Medical University Cancer Institute & Hospital, Tianjin, People’s Republic of China
- National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin Clinical Research Center for Cancer, Tianjin, People’s Republic of China
| | - Fanyuan Sun
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Chongqiang Huang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Yaohui Zhu
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Huabo Zhou
- Huabo Pet Hospital, Nanning, People’s Republic of China
| | - Boyu Pan
- Department of Molecular Pharmacology, Tianjin Medical University Cancer Institute & Hospital, Tianjin, People’s Republic of China
- National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin Clinical Research Center for Cancer, Tianjin, People’s Republic of China
| | - Yifeng Qin
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Kang Ouyang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Zuzhang Wei
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Weijian Huang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
| | - Adolfo García-Sastre
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Ying Chen
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
- Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
- Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
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Rochman ND, Wolf YI, Koonin EV. Molecular adaptations during viral epidemics. EMBO Rep 2022; 23:e55393. [PMID: 35848484 PMCID: PMC9346483 DOI: 10.15252/embr.202255393] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Revised: 06/18/2022] [Accepted: 06/27/2022] [Indexed: 07/20/2023] Open
Abstract
In 1977, the world witnessed both the eradication of smallpox and the beginning of the modern age of genomics. Over the following half-century, 7 epidemic viruses of international concern galvanized virologists across the globe and led to increasingly extensive virus genome sequencing. These sequencing efforts exerted over periods of rapid adaptation of viruses to new hosts, in particular, humans provide insight into the molecular mechanisms underpinning virus evolution. Investment in virus genome sequencing was dramatically increased by the unprecedented support for phylogenomic analyses during the COVID-19 pandemic. In this review, we attempt to piece together comprehensive molecular histories of the adaptation of variola virus, HIV-1 M, SARS, H1N1-SIV, MERS, Ebola, Zika, and SARS-CoV-2 to the human host. Disruption of genes involved in virus-host interaction in animal hosts, recombination including genome segment reassortment, and adaptive mutations leading to amino acid replacements in virus proteins involved in host receptor binding and membrane fusion are identified as the key factors in the evolution of epidemic viruses.
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Affiliation(s)
- Nash D Rochman
- National Center for Biotechnology InformationNational Library of MedicineBethesdaMDUSA
| | - Yuri I Wolf
- National Center for Biotechnology InformationNational Library of MedicineBethesdaMDUSA
| | - Eugene V Koonin
- National Center for Biotechnology InformationNational Library of MedicineBethesdaMDUSA
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3
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Lubna S, Chinta S, Burra P, Vedantham K, Ray S, Bandyopadhyay D. New substitutions on NS1 protein from influenza A (H1N1) virus: Bioinformatics analyses of Indian strains isolated from 2009 to 2020. Health Sci Rep 2022; 5:e626. [PMID: 35509388 PMCID: PMC9059196 DOI: 10.1002/hsr2.626] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2021] [Revised: 03/21/2022] [Accepted: 03/24/2022] [Indexed: 11/06/2022] Open
Affiliation(s)
- Syeda Lubna
- Birla Institute of Technology and Science, Pilani, Hyderabad Campus Hyderabad Telangana India
| | - Suma Chinta
- Birla Institute of Technology and Science, Pilani, Hyderabad Campus Hyderabad Telangana India
| | - Prakruthi Burra
- Birla Institute of Technology and Science, Pilani, Hyderabad Campus Hyderabad Telangana India
| | - Kiranmayi Vedantham
- Birla Institute of Technology and Science, Pilani, Hyderabad Campus Hyderabad Telangana India
| | | | - Debashree Bandyopadhyay
- Birla Institute of Technology and Science, Pilani, Hyderabad Campus Hyderabad Telangana India
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Trigueiro-Louro J, Santos LA, Almeida F, Correia V, Brito RMM, Rebelo-de-Andrade H. NS1 protein as a novel anti-influenza target: Map-and-mutate antiviral rationale reveals new putative druggable hot spots with an important role on viral replication. Virology 2022; 565:106-116. [PMID: 34773868 DOI: 10.1016/j.virol.2021.11.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Revised: 10/27/2021] [Accepted: 11/01/2021] [Indexed: 01/28/2023]
Abstract
Influenza NS1 is a promising anti-influenza target, considering its conserved and druggable structure, and key function in influenza replication and pathogenesis. Notwithstanding, target identification and validation, strengthened by experimental data, are lacking. Here, we further explored our previously designed structure-based antiviral rationale directed to highly conserved druggable NS1 regions across a broad spectrum of influenza A viruses. We aimed to identify NS1-mutated viruses exhibiting a reduced growth phenotype and/or an altered cell apoptosis profile. We found that NS1 mutations Y171A, K175A (consensus druggable pocket 1), W102A (consensus druggable pocket 3), Q121A and G184P (multiple consensus druggable pockets) - located at hot spots amenable for pharmacological modulation - significantly impaired A(H1N1)pdm09 virus replication, in vitro. This is the first time that NS1-K175A, -W102A, and -Q121A mutations are characterized. Our map-and-mutate strategy provides the basis to establish the NS1 as a promising target using a rationale with a higher resilience to resistance development.
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Affiliation(s)
- João Trigueiro-Louro
- Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal; Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal.
| | - Luís A Santos
- Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal
| | - Filipe Almeida
- Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal; Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal
| | - Vanessa Correia
- Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal
| | - Rui M M Brito
- Chemistry Department and Coimbra Chemistry Centre, Faculty of Science and Technology, University of Coimbra, 3004-535, Coimbra, Portugal
| | - Helena Rebelo-de-Andrade
- Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal; Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal.
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Blaurock C, Blohm U, Luttermann C, Holzerland J, Scheibner D, Schäfer A, Groseth A, Mettenleiter TC, Abdelwhab EM. The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice. Emerg Microbes Infect 2021; 10:1760-1776. [PMID: 34420477 PMCID: PMC8432360 DOI: 10.1080/22221751.2021.1971568] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Avian influenza viruses (AIV) H5N8 clade 2.3.4.4 pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade 2.3.4.4A (H5N8-A) from 2013 to 2015 and clade 2.3.4.4B (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.
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Affiliation(s)
- Claudia Blaurock
- Institute of Molecular Virology and Cell Biology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Ulrike Blohm
- Institute of Immunology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Christine Luttermann
- Institute of Immunology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Julia Holzerland
- Institute of Molecular Virology and Cell Biology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - David Scheibner
- Institute of Molecular Virology and Cell Biology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Alexander Schäfer
- Institute of Immunology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Allison Groseth
- Institute of Molecular Virology and Cell Biology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Thomas C Mettenleiter
- Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
| | - Elsayed M Abdelwhab
- Institute of Molecular Virology and Cell Biology, Federal Research Institute for Animal Health Greifswald-Insel Riems, Germany
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Jahan AS, Biquand E, Muñoz-Moreno R, Le Quang A, Mok CKP, Wong HH, Teo QW, Valkenburg SA, Chin AWH, Man Poon LL, Te Velthuis A, García-Sastre A, Demeret C, Sanyal S. OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1. Cell Rep 2020; 30:1570-1584.e6. [PMID: 32023470 DOI: 10.1016/j.celrep.2020.01.015] [Citation(s) in RCA: 50] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Revised: 10/21/2019] [Accepted: 01/02/2020] [Indexed: 12/16/2022] Open
Abstract
Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.
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Affiliation(s)
- Akhee Sabiha Jahan
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Elise Biquand
- Molecular Genetics of RNA Viruses, CNRS UMR 3569, Université de Paris, Institut Pasteur, Paris, France
| | - Raquel Muñoz-Moreno
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA
| | - Agathe Le Quang
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Chris Ka-Pun Mok
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Ho Him Wong
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Qi Wen Teo
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Sophie A Valkenburg
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Alex W H Chin
- School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Leo Lit Man Poon
- School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
| | - Artejan Te Velthuis
- Division of Virology, Department of Pathology, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK
| | - Adolfo García-Sastre
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA; Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA
| | - Caroline Demeret
- Molecular Genetics of RNA Viruses, CNRS UMR 3569, Université de Paris, Institut Pasteur, Paris, France
| | - Sumana Sanyal
- HKU-Pasteur Research Pole, University of Hong Kong, Hong Kong; School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong; School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong.
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7
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Trigueiro-Louro JM, Correia V, Santos LA, Guedes RC, Brito RMM, Rebelo-de-Andrade H. To hit or not to hit: Large-scale sequence analysis and structure characterization of influenza A NS1 unlocks new antiviral target potential. Virology 2019; 535:297-307. [PMID: 31104825 DOI: 10.1016/j.virol.2019.04.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Revised: 04/22/2019] [Accepted: 04/23/2019] [Indexed: 12/13/2022]
Abstract
Influenza NS1 protein is among the most promising novel druggable anti-influenza target, based on its structure; multiple interactions; and global function in influenza replication and pathogenesis. Notwithstanding, drug development guidance based on NS1 structural biology is lacking. Here, we design a promising strategy directed to highly conserved druggable regions as a result of an exhaustive large-scale sequence analysis and structure characterization of NS1 protein across human-infecting influenza A subtypes, over the past 100 years. We have identified 3 druggable pockets and 8 new potential hot spot residues in the NS1 protein, not described before, additionally to other 16 sites previously identified, which represent attractive targets for pharmacological modulation. This study provides a rationale towards structure-function studies of NS1 druggable sites, which have the potential to accelerate the NS1 target validation. This research also contributes to a deeper comprehension and insight into the evolutionary dynamics of influenza A NS1 protein.
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Affiliation(s)
- João M Trigueiro-Louro
- Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal; Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal.
| | - Vanessa Correia
- Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal
| | - Luís A Santos
- Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal
| | - Rita C Guedes
- Medicinal Chemistry Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal
| | - Rui M M Brito
- Chemistry Department and Coimbra Chemistry Centre, Faculty of Science and Technology, University of Coimbra, 3004-535, Coimbra, Portugal
| | - Helena Rebelo-de-Andrade
- Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003, Lisbon, Portugal; Antiviral Resistance Lab, Research & Development Unit, Infectious Diseases Department, Instituto Nacional de Saúde Doutor Ricardo Jorge, IP, Av. Padre Cruz, 1649-016, Lisbon, Portugal.
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8
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Nogales A, Martinez-Sobrido L, Topham DJ, DeDiego ML. Modulation of Innate Immune Responses by the Influenza A NS1 and PA-X Proteins. Viruses 2018; 10:v10120708. [PMID: 30545063 PMCID: PMC6315843 DOI: 10.3390/v10120708] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Revised: 12/06/2018] [Accepted: 12/08/2018] [Indexed: 12/14/2022] Open
Abstract
Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection.
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Affiliation(s)
- Aitor Nogales
- Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
- Centro de Investigación en Sanidad Animal (CISA)-INIA, Valdeolmos, 28130 Madrid, Spain.
| | - Luis Martinez-Sobrido
- Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
| | - David J Topham
- Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
| | - Marta L DeDiego
- Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA.
- Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain.
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9
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Belser JA, Maines TR, Tumpey TM. Importance of 1918 virus reconstruction to current assessments of pandemic risk. Virology 2018; 524:45-55. [PMID: 30142572 PMCID: PMC9036538 DOI: 10.1016/j.virol.2018.08.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 07/25/2018] [Accepted: 08/09/2018] [Indexed: 01/13/2023]
Abstract
Reconstruction of the 1918 influenza virus has facilitated considerable advancements in our understanding of this extraordinary pandemic virus. However, the benefits of virus reconstruction are not limited to this one strain. Here, we provide an overview of laboratory studies which have evaluated the reconstructed 1918 virus, and highlight key discoveries about determinants of virulence and transmissibility associated with this virus in mammals. We further discuss recent and current pandemic threats from avian and swine reservoirs, and provide specific examples of how reconstruction of the 1918 pandemic virus has improved our ability to contextualize research employing novel and emerging strains. As influenza viruses continue to evolve and pose a threat to human health, studying past pandemic viruses is key to future preparedness efforts.
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Affiliation(s)
- Jessica A Belser
- Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Taronna R Maines
- Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Terrence M Tumpey
- Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
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10
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Mostafa A, Abdelwhab EM, Mettenleiter TC, Pleschka S. Zoonotic Potential of Influenza A Viruses: A Comprehensive Overview. Viruses 2018; 10:v10090497. [PMID: 30217093 PMCID: PMC6165440 DOI: 10.3390/v10090497] [Citation(s) in RCA: 178] [Impact Index Per Article: 25.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2018] [Revised: 08/24/2018] [Accepted: 09/13/2018] [Indexed: 02/06/2023] Open
Abstract
Influenza A viruses (IAVs) possess a great zoonotic potential as they are able to infect different avian and mammalian animal hosts, from which they can be transmitted to humans. This is based on the ability of IAV to gradually change their genome by mutation or even reassemble their genome segments during co-infection of the host cell with different IAV strains, resulting in a high genetic diversity. Variants of circulating or newly emerging IAVs continue to trigger global health threats annually for both humans and animals. Here, we provide an introduction on IAVs, highlighting the mechanisms of viral evolution, the host spectrum, and the animal/human interface. Pathogenicity determinants of IAVs in mammals, with special emphasis on newly emerging IAVs with pandemic potential, are discussed. Finally, an overview is provided on various approaches for the prevention of human IAV infections.
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Affiliation(s)
- Ahmed Mostafa
- Institute of Medical Virology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany.
- Center of Scientific Excellence for Influenza Viruses, National Research Centre (NRC), Giza 12622, Egypt.
| | - Elsayed M Abdelwhab
- Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
| | - Thomas C Mettenleiter
- Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
| | - Stephan Pleschka
- Institute of Medical Virology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany.
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11
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Vermillion MS, Ursin RL, Attreed SE, Klein SL. Estriol Reduces Pulmonary Immune Cell Recruitment and Inflammation to Protect Female Mice From Severe Influenza. Endocrinology 2018; 159:3306-3320. [PMID: 30032246 PMCID: PMC6109301 DOI: 10.1210/en.2018-00486] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Accepted: 07/11/2018] [Indexed: 01/09/2023]
Abstract
Estriol (E3) is an endogenous estrogen in females with broad biological activity within diverse tissue types. In the context of certain T-cell-mediated autoimmune inflammatory diseases, E3 can ameliorate disease severity through immunomodulatory mechanisms that decrease tissue inflammation. Severe disease caused by influenza A virus (IAV) infection is also characterized by aberrant inflammation and immunopathology. How E3 might affect the pathogenesis of IAV infection, however, has not been explored. Gonadally intact female C57BL/6 mice that were treated with exogenous E3 during infection with mouse-adapted 2009 H1N1 had reduced total pulmonary inflammation and improved disease outcomes compared with females that received no hormone. Furthermore, compared with no hormone treatment, E3 treatment reduced the induction of genes associated with proinflammatory cytokine and chemokine responses in the lungs, which preceded clinical disease, reductions in innate immune cell recruitment, altered pulmonary T-cell skewing, and reduced antibody titers during IAV infection. Although E3 treatment was associated with reduced local and systemic anti-influenza adaptive immune responses, there was no effect of E3 on viral replication or clearance. Together, these data suggest that exogenous E3 confers protection during IAV infection through immunomodulatory mechanisms and that E3 may have broad therapeutic potential in the context of both infectious and noninfectious inflammatory diseases.
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Affiliation(s)
- Meghan S Vermillion
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
- Department of Molecular and Comparative Pathobiology, The Johns Hopkins School of Medicine, Baltimore, Maryland
| | - Rebecca L Ursin
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
- Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
| | - Sarah E Attreed
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
| | - Sabra L Klein
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
- Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
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12
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Vermillion MS, Ursin RL, Kuok DIT, Vom Steeg LG, Wohlgemuth N, Hall OJ, Fink AL, Sasse E, Nelson A, Ndeh R, McGrath-Morrow S, Mitzner W, Chan MCW, Pekosz A, Klein SL. Production of amphiregulin and recovery from influenza is greater in males than females. Biol Sex Differ 2018; 9:24. [PMID: 30012205 PMCID: PMC6048771 DOI: 10.1186/s13293-018-0184-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 05/30/2018] [Indexed: 01/07/2023] Open
Abstract
Background Amphiregulin (AREG) is an epidermal growth factor that is a significant mediator of tissue repair at mucosal sites, including in the lungs during influenza A virus (IAV) infection. Previous research illustrates that males of reproductive ages experience less severe disease and recover faster than females following infection with IAV. Methods Whether males and females differentially produce and utilize AREG for pulmonary repair after IAV infection was investigated using murine models on a C57BL/6 background and primary mouse and human epithelial cell culture systems. Results Following sublethal infection with 2009 H1N1 IAV, adult female mice experienced greater morbidity and pulmonary inflammation during the acute phase of infection as well as worse pulmonary function during the recovery phase of infection than males, despite having similar virus clearance kinetics. As compared with females, AREG expression was greater in the lungs of male mice as well as in primary respiratory epithelial cells derived from mouse and human male donors, in response to H1N1 IAVs. Internalization of the epidermal growth factor receptor (EGFR) was also greater in respiratory epithelial cells derived from male than female mice. IAV infection of Areg knock-out (Areg−/−) mice eliminated sex differences in IAV pathogenesis, with a more significant role for AREG in infection of male compared to female mice. Deletion of Areg had no effect on virus replication kinetics in either sex. Gonadectomy and treatment of either wild-type or Areg−/− males with testosterone improved the outcome of IAV as compared with their placebo-treated conspecifics. Conclusions Taken together, these data show that elevated levels of testosterone and AREG, either independently or in combination, improve resilience (i.e., repair and recovery of damaged tissue) and contribute to better influenza outcomes in males compared with females.
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Affiliation(s)
- Meghan S Vermillion
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.,Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Rebecca L Ursin
- Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Denise I T Kuok
- School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | - Landon G Vom Steeg
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Nicholas Wohlgemuth
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Olivia J Hall
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Ashley L Fink
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Eric Sasse
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Andrew Nelson
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Roland Ndeh
- Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Sharon McGrath-Morrow
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.,Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Wayne Mitzner
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Michael C W Chan
- School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | - Andrew Pekosz
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.,Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Sabra L Klein
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. .,Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
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13
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Vermillion MS, Nelson A, Vom Steeg L, Loube J, Mitzner W, Klein SL. Pregnancy preserves pulmonary function following influenza virus infection in C57BL/6 mice. Am J Physiol Lung Cell Mol Physiol 2018; 315:L517-L525. [PMID: 29847990 DOI: 10.1152/ajplung.00066.2018] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Pregnancy is associated with significant anatomic and functional changes to the cardiopulmonary system. Using pregnant C57BL/6 mice, we characterized changes in pulmonary structure and function during pregnancy in healthy animals and following infection with influenza A virus (IAV). We hypothesized that pregnancy-associated alterations in pulmonary physiology would contribute to the more severe outcome of IAV infection. Nonpregnant and pregnant females (at embryonic day 10.5) were either mock-infected or infected with 2009 H1N1 IAV for assessment of pulmonary function, structure, and inflammation at 8 days postinoculation. There were baseline differences in pulmonary function, with pregnant females having greater lung compliance, total lung capacity, and fixed lung volume than nonpregnant females. Following IAV infection, both pregnant and nonpregnant females exhibited reduced circulating progesterone, which in nonpregnant females was associated with increased pulmonary resistance and decreased lung compliance, minute ventilation, and oxygen diffusing capacity compared with uninfected nonpregnant females. In pregnant females, reduced concentrations of progesterone were associated with adverse pregnancy outcomes, but measures of pulmonary function were preserved following IAV infection and were not significantly different from uninfected pregnant mice. Following IAV infection, infectious virus titers and total numbers of pulmonary leukocytes were similar between pregnant and nonpregnant females, but the histological density of pulmonary inflammation was reduced in pregnant animals. These data suggest that pregnancy in mice is associated with significant alterations in pulmonary physiology but that these changes served to preserve lung function during IAV infection. Pregnancy-associated alterations in pulmonary physiology may serve to protect females during severe influenza.
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Affiliation(s)
- Meghan S Vermillion
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland.,Department of Molecular and Comparative Pathobiology, The Johns Hopkins School of Medicine , Baltimore, Maryland
| | - Andrew Nelson
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland
| | - Landon Vom Steeg
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland
| | - Jeffery Loube
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland
| | - Wayne Mitzner
- Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland
| | - Sabra L Klein
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland.,Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland
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14
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A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity. J Virol 2018; 92:JVI.00149-18. [PMID: 29563291 DOI: 10.1128/jvi.00149-18] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 03/14/2018] [Indexed: 01/02/2023] Open
Abstract
Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals.IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.
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15
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Plant EP, Ilyushina NA, Sheikh F, Donnelly RP, Ye Z. Influenza virus NS1 protein mutations at position 171 impact innate interferon responses by respiratory epithelial cells. Virus Res 2017; 240:81-86. [PMID: 28757142 DOI: 10.1016/j.virusres.2017.07.021] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2017] [Revised: 07/21/2017] [Accepted: 07/26/2017] [Indexed: 01/09/2023]
Abstract
The influenza virus NS1 protein interacts with a wide range of proteins to suppress the host cell immune response and facilitate virus replication. The amino acid sequence of the 2009 pandemic virus NS1 protein differed from sequences of earlier related viruses. The functional impact of these differences has not been fully defined. Therefore, we made mutations to the NS1 protein based on these sequence differences, and assessed the impact of these changes on host cell interferon (IFN) responses. We found that viruses with mutations at position 171 replicated efficiently but did not induce expression of interferon genes as effectively as wild-type viruses in A459 lung epithelial cells. The decreased ability of these NS1 mutant viruses to induce IFN gene and protein expression correlated with decreased activation of STAT1 and lower levels of IFN-stimulated gene (ISG) expression. These findings demonstrate that mutations at position 171 in the NS1 protein result in decreased expression of IFN and ISGs by A549 cells. Consequently, these viruses may be more virulent than the parental strains that do not contain mutations at position 171 in the NS1 protein.
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Affiliation(s)
- Ewan P Plant
- Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD, USA.
| | - Natalia A Ilyushina
- Division of Biotechnology Research and Review II, Center for Drug Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD, USA.
| | - Faruk Sheikh
- Division of Biotechnology Research and Review II, Center for Drug Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD, USA.
| | - Raymond P Donnelly
- Division of Biotechnology Research and Review II, Center for Drug Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD, USA.
| | - Zhiping Ye
- Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD, USA.
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16
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Ylösmäki L, Fagerlund R, Kuisma I, Julkunen I, Saksela K. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein. Viruses 2016; 8:101. [PMID: 27092521 PMCID: PMC4848595 DOI: 10.3390/v8040101] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Revised: 03/31/2016] [Accepted: 04/04/2016] [Indexed: 12/18/2022] Open
Abstract
The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.
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Affiliation(s)
- Leena Ylösmäki
- Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland.
| | - Riku Fagerlund
- Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland.
| | - Inka Kuisma
- Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland.
| | - Ilkka Julkunen
- Department of Virology, University of Turku, 20520 Turku, Finland and Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), 00300 Helsinki, Finland.
| | - Kalle Saksela
- Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland.
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17
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Kamal RP, Katz JM, York IA. Molecular determinants of influenza virus pathogenesis in mice. Curr Top Microbiol Immunol 2015; 385:243-74. [PMID: 25038937 DOI: 10.1007/82_2014_388] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.
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Affiliation(s)
- Ram P Kamal
- Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA,
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18
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Kong W, Liu L, Wang Y, He Q, Wu S, Qin Z, Wang J, Sun H, Sun Y, Zhang R, Pu J, Liu J. C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission. J Gen Virol 2015; 96:259-268. [DOI: 10.1099/vir.0.071001-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Affiliation(s)
- Weili Kong
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Lirong Liu
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Yu Wang
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Qiming He
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Sizhe Wu
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Zhihua Qin
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Jinliang Wang
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Honglei Sun
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Yipeng Sun
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Rui Zhang
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Juan Pu
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
| | - Jinhua Liu
- Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
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19
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Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses. Vet Microbiol 2014; 175:17-25. [PMID: 25480165 DOI: 10.1016/j.vetmic.2014.11.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Revised: 11/04/2014] [Accepted: 11/07/2014] [Indexed: 11/21/2022]
Abstract
To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity.
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20
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The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus. Biochem Biophys Res Commun 2014; 449:19-25. [DOI: 10.1016/j.bbrc.2014.04.132] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Accepted: 04/25/2014] [Indexed: 11/19/2022]
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21
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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22
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Piralla A, Pariani E, Campanini G, Rovida F, Ranghiero A, Fiorina L, Amendola A, Zanetti A, Baldanti F. Multiple clusters of A(H1N1)pdm09 virus circulating in severe cases of influenza during the 2010-2011 season: a phylogenetic and molecular analysis of the neuraminidase gene. J Med Virol 2013; 85:944-52. [PMID: 23588719 DOI: 10.1002/jmv.23569] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/27/2012] [Indexed: 02/04/2023]
Abstract
The molecular characterization of circulating influenza A viruses is crucial to detect mutations potentially involved in increased virulence, drug resistance and immune escape. A molecular and phylogenetic analysis of A(H1N1)pdm09 neuraminidase (NA) gene sequences from different patient categories defined according to the severity of influenza infection were analyzed. A total of 126 influenza A(H1N1)pdm09 positive samples from patients with severe infections in comparison with those with moderate and mild infections was performed in Lombardy (Northern Italy, nearly 10 million inhabitants) during the 2010-2011 season. NA sequences included in this study segregated into five distinct clusters. Nineteen amino acid substitutions were detected exclusively in NA sequences of viruses identified in patients with severe or moderate influenza infection. Three of them (F74S, S79P, E287K) were observed in virus strains with the 222G/N hemagglutinin mutation. None of NA sequences under study had mutations related to the resistance to the NA inhibitors. Four out of 126 (3.2%) NA sequences from patients with severe infection lost a N-linked glycosylation site due to the change from N to K at residue 386. Two additional N-linked glycosylation sites in the NA stalk region (residues 42 and 44) were found in 12 (9.5%) NA sequences. Sporadic NA mutations were detected in NA viral sequences from critically ill patients, and no variants with reduced sensitivity to NA inhibitors were observed either in treated or untreated patients.
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Affiliation(s)
- Antonio Piralla
- Molecular Virology Unit, Virology and Microbiology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
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Ramírez-Martínez G, Cruz-Lagunas A, Jiménez-Alvarez L, Espinosa E, Ortíz-Quintero B, Santos-Mendoza T, Herrera MT, Canché-Pool E, Mendoza C, Bañales JL, García-Moreno SA, Morán J, Cabello C, Orozco L, Aguilar-Delfín I, Hidalgo-Miranda A, Romero S, Suratt BT, Selman M, Zúñiga J. Seasonal and pandemic influenza H1N1 viruses induce differential expression of SOCS-1 and RIG-I genes and cytokine/chemokine production in macrophages. Cytokine 2013; 62:151-9. [PMID: 23434273 PMCID: PMC4148900 DOI: 10.1016/j.cyto.2013.01.018] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2012] [Revised: 01/17/2013] [Accepted: 01/19/2013] [Indexed: 12/28/2022]
Abstract
BACKGROUND Infection with pandemic (pdm) A/H1N1 virus induces high levels of pro-inflammatory mediators in blood and lungs of experimental animals and humans. METHODS To compare the involvement of seasonal A/PR/8/34 and pdm A/H1N1 virus strains in the regulation of inflammatory responses, we analyzed the changes in the whole-genome expression induced by these strains in macrophages and A549 epithelial cells. We also focused on the functional implications (cytokine production) of the differential induction of suppressors of cytokine signaling (SOCS)-1, SOCS-3, retinoid-inducible gene (RIG)-I and interferon receptor 1 (IFNAR1) genes by these viral strains in early stages of the infection. RESULTS We identified 130 genes differentially expressed by pdm A/H1N1 and A/PR/8/34 infections in macrophages. mRNA levels of SOCS-1 and RIG-I were up-regulated in macrophages infected with the A/PR/8/34 but not with pdm A/H1N1 virus. mRNA levels of SOCS-3 and IFNAR1 induced by A/PR/8/34 and pdm A/H1N1 strains in macrophages, as well as in A549 cells were similar. We found higher levels of IL-6, TNF-α, IL-10, CCL3, CCL5, CCL4 and CXCL8 (p < 0.05) in supernatants from cultures of macrophages infected with the pdm A/H1N1 virus compared to those infected with the A/PR/8/34 strain, coincident with the lack of SOCS-1 and RIG-I expression. In contrast, levels of INF-α were higher in cultures of macrophages 48h after infection with the A/PR/8/34 strain than with the pdm A/H1N1 virus. CONCLUSIONS These findings suggest that factors inherent to the pdm A/H1N1 viral strain may increase the production of inflammatory mediators by inhibiting SOCS-1 and modifying the expression of antiviral immunity-related genes, including RIG-I, in human macrophages.
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Affiliation(s)
- Gustavo Ramírez-Martínez
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Alfredo Cruz-Lagunas
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Luis Jiménez-Alvarez
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Enrique Espinosa
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Blanca Ortíz-Quintero
- Department of Biochemistry, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Teresa Santos-Mendoza
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - María Teresa Herrera
- Department of Microbiology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Elsy Canché-Pool
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Criselda Mendoza
- Research Unit, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - José L. Bañales
- Research Unit, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Sara A. García-Moreno
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Juan Morán
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Carlos Cabello
- Department of Virology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
- Research Unit, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Lorena Orozco
- Laboratories of Multifactorial Diseases and Cancer Genomics, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Irma Aguilar-Delfín
- Laboratories of Multifactorial Diseases and Cancer Genomics, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Alfredo Hidalgo-Miranda
- Laboratories of Multifactorial Diseases and Cancer Genomics, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Sandra Romero
- Laboratories of Multifactorial Diseases and Cancer Genomics, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Benjamin T. Suratt
- Department of Medicine, University of Vermont College of Medicine, Burlington, VT, USA
| | - Moisés Selman
- Research Unit, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
| | - Joaquín Zúñiga
- Department of Immunology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
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24
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SUMOylation affects the interferon blocking activity of the influenza A nonstructural protein NS1 without affecting its stability or cellular localization. J Virol 2013; 87:5602-20. [PMID: 23468495 DOI: 10.1128/jvi.02063-12] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Our pioneering studies on the interplay between the small ubiquitin-like modifier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ~4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.
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25
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Capua I, Munoz O. Emergence of influenza viruses with zoonotic potential: open issues which need to be addressed. A review. Vet Microbiol 2013; 165:7-12. [PMID: 23567150 DOI: 10.1016/j.vetmic.2013.01.044] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2012] [Revised: 01/22/2013] [Accepted: 01/30/2013] [Indexed: 10/27/2022]
Abstract
The real and perceived impact of influenza infections in animals has changed dramatically over the last 10 years, due mainly to the better understanding of the public health implications of avian and swine influenza viruses. On a number of occasions in the last decade avian-to-human transmissions of H5, H7 and H9 virus subtypes have occurred, and the first influenza pandemic of the new millennium occurred as a result of the emergence and spread of a virus from pigs. Although the mechanisms that allow influenza viruses to jump from one host species to another are not fully understood, several genetic signatures linked to the crossing of species barriers have been identified. This has led to a re-evaluation of the importance of understanding these viruses in the animal reservoir, to the extent that millions of euros have been invested in surveillance, research and capacity building worldwide. This has resulted in an enhanced collaboration with our medical counterparts, leading to many discoveries that will contribute to an understanding of the complex mechanisms that lead to the emergence of a pandemic virus.
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Affiliation(s)
- Ilaria Capua
- Istituto Zooprofilattico Sperimentale delle Venezie, OIE/FAO and National Reference Laboratory for Newcastle Disease and Avian Influenza, OIE Collaborating Centre for Diseases at Human-Animal Interface, Viale dell'Università 10, 35020, Legnaro, Padova, Italy.
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26
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Soubies SM, Hoffmann TW, Croville G, Larcher T, Ledevin M, Soubieux D, Quéré P, Guérin JL, Marc D, Volmer R. Deletion of the C-terminal ESEV domain of NS1 does not affect the replication of a low-pathogenic avian influenza virus H7N1 in ducks and chickens. J Gen Virol 2012; 94:50-58. [PMID: 23052391 DOI: 10.1099/vir.0.045153-0] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.
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Affiliation(s)
- Sébastien M Soubies
- INRA, UMR 1225, Ecole nationale vétérinaire de Toulouse, F-31076 Toulouse, France.,Université de Toulouse, ENVT, UMR 1225, F-31076 Toulouse, France
| | - Thomas W Hoffmann
- Equipe BioVA, INRA UMR1282, Infectiologie et Santé Publique, ISP, F-37380 Nouzilly, France.,Université François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-37000 Tours, France
| | - Guillaume Croville
- INRA, UMR 1225, Ecole nationale vétérinaire de Toulouse, F-31076 Toulouse, France.,Université de Toulouse, ENVT, UMR 1225, F-31076 Toulouse, France
| | - Thibaut Larcher
- INRA UMR 703, APEX, Oniris-La Chantrerie, F-44307 Nantes, France
| | - Mireille Ledevin
- INRA UMR 703, APEX, Oniris-La Chantrerie, F-44307 Nantes, France
| | - Denis Soubieux
- Equipe BioVA, INRA UMR1282, Infectiologie et Santé Publique, ISP, F-37380 Nouzilly, France.,Université François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-37000 Tours, France
| | - Pascale Quéré
- Université François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-37000 Tours, France.,Equipe PIA, INRA UMR1282, Infectiologie et Santé Publique, ISP, F-37380 Nouzilly, France
| | - Jean-Luc Guérin
- INRA, UMR 1225, Ecole nationale vétérinaire de Toulouse, F-31076 Toulouse, France.,Université de Toulouse, ENVT, UMR 1225, F-31076 Toulouse, France
| | - Daniel Marc
- Equipe BioVA, INRA UMR1282, Infectiologie et Santé Publique, ISP, F-37380 Nouzilly, France.,Université François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-37000 Tours, France
| | - Romain Volmer
- INRA, UMR 1225, Ecole nationale vétérinaire de Toulouse, F-31076 Toulouse, France.,Université de Toulouse, ENVT, UMR 1225, F-31076 Toulouse, France
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27
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Moraes MVD, Milanez M, Almada BVP, Sipolatti V, Rebouças MRGO, Nunes VRR, Akel AN, Zatz M, Errera FIV, Louro ID, Paula F. Variable expressivity of osteogenesis imperfecta in a Brazilian family due to p.G1079S mutation in the COL1A1 gene. GENETICS AND MOLECULAR RESEARCH 2012; 11:3246-55. [PMID: 23079818 DOI: 10.4238/2012.september.12.7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Osteogenesis imperfecta (OI) is a Mendelian disease with genetic heterogeneity characterized by bone fragility, recurrent fractures, blue sclerae, and short stature, caused mostly by mutations in COL1A1 or COL1A2 genes, which encode the pro-α1(I) and pro-α2(I) chains of type I collagen, respectively. A Brazilian family that showed variable expression of autosomal dominant OI was identified and characterized. Scanning for mutations was carried out using SSCP and DNA sequence analysis. The missense mutation c.3235G>A was identified within exon 45 of the COL1A1 gene in a 16-year-old girl diagnosed as having OI type I; it resulted in substitution of a glycine residue (G) by a serine (S) at codon 1079 (p.G1079S). The proband's mother had the disease signs, but without bone fractures, as did five of nine uncles and aunts of the patient. All of them carried the mutation, which was excluded in four healthy brothers of the patient's mother. This is the first description in a Brazilian family with OI showing variable expression; only one among seven carriers for the c.3235G>A mutation developed bone fractures, the most striking clinical feature of this disease. This finding has a significant implication for prenatal diagnosis in OI disease.
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Affiliation(s)
- M V D Moraes
- Núcleo de Genética Humana e Molecular, Departamento de Ciências Biológicas, Centro de Ciências Humanas e Naturais, Universidade Federal do Espírito Santo, Vitória, ES, Brasil
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28
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Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses. Proc Natl Acad Sci U S A 2012; 109:15900-5. [PMID: 23019374 DOI: 10.1073/pnas.1205576109] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.
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29
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Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt. Virol J 2012; 9:173. [PMID: 22925485 PMCID: PMC3492205 DOI: 10.1186/1743-422x-9-173] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2012] [Accepted: 08/14/2012] [Indexed: 11/23/2022] Open
Abstract
Background Avian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness. Methods Recently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated. Results Phylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens. Conclusion The study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.
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30
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Hrincius ER, Hennecke AK, Gensler L, Nordhoff C, Anhlan D, Vogel P, McCullers JA, Ludwig S, Ehrhardt C. A Single Point Mutation (Y89F) within the Non-Structural Protein 1 of Influenza A Viruses Limits Epithelial Cell Tropism and Virulence in Mice. THE AMERICAN JOURNAL OF PATHOLOGY 2012; 180:2361-74. [DOI: 10.1016/j.ajpath.2012.02.029] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/04/2011] [Revised: 02/22/2012] [Accepted: 02/28/2012] [Indexed: 12/15/2022]
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31
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Shelton H, Smith M, Hartgroves L, Stilwell P, Roberts K, Johnson B, Barclay W. An influenza reassortant with polymerase of pH1N1 and NS gene of H3N2 influenza A virus is attenuated in vivo. J Gen Virol 2012; 93:998-1006. [PMID: 22323532 PMCID: PMC3541804 DOI: 10.1099/vir.0.039701-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2011] [Accepted: 02/02/2012] [Indexed: 12/12/2022] Open
Abstract
Influenza viruses readily mutate by accumulating point mutations and also by reassortment in which they acquire whole gene segments from another virus in a co-infected host. The NS1 gene is a major virulence factor of influenza A virus. The effects of changes in NS1 sequence depend on the influenza polymerase constellation. Here, we investigated the consequences of a virus with the polymerase of pandemic H1N1 2009 acquiring an NS gene segment derived from a seasonal influenza A H3N2 virus, a combination that might arise during natural reassortment of viruses that currently circulate in humans. We generated recombinant influenza viruses with surface HA and NA genes and matrix M gene segment from A/PR/8/34 virus, but different combinations of polymerase and NS genes. Thus, any changes in phenotype were not due to differences in receptor use, entry, uncoating or virus release. In Madin-Darby canine kidney (MDCK) cells, the virus with the NS gene from the H3N2 parent showed enhanced replication, probably a result of increased control of the interferon response. However, in mice the same virus was attenuated in comparison with the virus containing homologous pH1N1 polymerase and NS genes. Levels of viral RNA during single-cycles of replication were lower for the virus with H3N2 NS, and this virus reached lower titres in the lungs of infected mice. Thus, virus with pH1N1 polymerase genes did not increase its virulence by acquiring the H3N2 NS gene segment, and MDCK cells were a poor predictor of the outcome of infection in vivo.
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Affiliation(s)
- Holly Shelton
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
| | - Matt Smith
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
| | - Lorian Hartgroves
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
| | - Peter Stilwell
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
| | - Kim Roberts
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
| | - Ben Johnson
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
- Health Protection Agency Colindale, 61 Colindale Avenue, London, UK
| | - Wendy Barclay
- Division of Infectious Diseases, Imperial College London, St Mary’s Campus, London, UK
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32
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Wang J, Qi X, Lu C. Mutations in the C-terminal tail of NS1 protein facilitate the replication of classical swine H1N1 influenza A virus in mice. Folia Microbiol (Praha) 2012; 57:169-75. [PMID: 22430886 DOI: 10.1007/s12223-012-0110-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2011] [Accepted: 03/02/2012] [Indexed: 11/26/2022]
Abstract
The NS1 protein of classical swine H1N1 influenza A virus evolved dynamically during the past 80 years, most notable changes happened in the four C-terminal sequences and the C-terminal truncation of 11 amino acids. However, the role of these changes on the virulence of classical swine H1N1 influenza A virus remains unknown. Using reverse genetics, three NS1 mutant viruses (RSEV, GSEI, and EPEV) and a wild-type virus (PEQK) were generated from A/Swine/Shanghai/1/2005 virus and the pathogenicity of the viruses was determined in mice. The results showed that RSEV and PEQK viruses could not infect the mice. By contrast, GSEI and EPEV viruses could replicate in the lungs of mice without prior adaptation. The viral titers in lungs from GSEI and EPEV virus-infected mice were 2,300 and 7 pfu/g at fourth-day post-infection, respectively. Mild-to-moderate alveolitis was observed in the histopathological test of lungs from GSEI and EPEV virus-infected mice. The results indicated that C-terminal GSEI and EPEV motifs of NS1 protein involved in viral virulence and facilitated the A/Swine/Shanghai/1/2005 virus crossing the species barrier from swine to mice.
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Affiliation(s)
- Jinxiang Wang
- Key Laboratory of Animal Bacteriology, Ministry of Agriculture, Nanjing Agricultural University, Jiangsu, People's Republic of China
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33
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Kuiken T, Riteau B, Fouchier RAM, Rimmelzwaan GF. Pathogenesis of influenza virus infections: the good, the bad and the ugly. Curr Opin Virol 2012; 2:276-86. [PMID: 22709515 DOI: 10.1016/j.coviro.2012.02.013] [Citation(s) in RCA: 99] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2012] [Revised: 02/20/2012] [Accepted: 02/24/2012] [Indexed: 12/15/2022]
Abstract
The clinical outcome of different influenza virus infections ranges from subclinical upper respiratory tract disease to fatal lower respiratory tract disease. An important determinant in the pathogenesis of these diseases is the tissue tropism of the influenza virus. Furthermore, virulence is often correlated with virus replication and is regulated by multiple virus genes. Host defense against virus infection consists of both innate and adaptive immune responses. However, excessive or dysbalanced immune response may result in lung tissue damage, reduced respiratory capacity, and severe disease or even death. By interdisciplinary efforts to better understand the intricate interaction between virus, tissue, and immune response, we may be able to find new ways to improve the outcome of influenza virus infections.
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Affiliation(s)
- T Kuiken
- Erasmus Medical Center, Department of Virology, Rotterdam, The Netherlands
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Variability among the neuraminidase, non-structural 1 and PB1-F2 proteins in the influenza A virus genome. Virus Genes 2012; 44:363-73. [PMID: 22261818 DOI: 10.1007/s11262-012-0714-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2011] [Accepted: 01/04/2012] [Indexed: 11/26/2022]
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York I, Donis RO. The 2009 pandemic influenza virus: where did it come from, where is it now, and where is it going? Curr Top Microbiol Immunol 2012; 370:241-57. [PMID: 22638836 DOI: 10.1007/82_2012_221] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Around 2008 or 2009, an influenza A virus that had been circulating undetected in swine entered human population. Unlike most swine influenza infections of humans, this virus established sustained human-to-human transmission, leading to a global pandemic. The virus responsible, 2009 pandemic H1N1 (H1N1pdm), is the result of multiple reassortment events that brought together genomic segments from classical H1N1 swine influenza virus, human seasonal H3N2 influenza virus, North American avian influenza virus, and Eurasian avian-origin swine influenza viruses. Genetically, H1N1pdm possesses a number of unusual features, although the genomic characteristics that permitted sustained human-to-human transmission are yet unclear. Human infection with H1N1pdm has generally resulted in low mortality, although certain subgroups (including pregnant women, people with some chronic medical conditions, morbidly obese individuals, and immunosuppressed people) have significantly higher risk of severe disease. As H1N1pdm has spread throughout the human population it continued to evolve. It has also reentered the swine population as a circulating pathogen, and has been transiently identified in other species such as turkeys, cats, and domestic ferrets. Most genetic changes in H1N1pdm to date have not been clearly linked to changes in antigenicity, disease severity, antiviral drug resistance, or transmission efficiency. However, the rapid evolution rate characteristic of influenza viruses suggests that changes in antigenicity are inevitable in future years. Experience with this first pandemic of twenty-first century reemphasizes the importance of influenza surveillance in animals as well as humans, and offers lessons to develop and enhance our ability to identify potentially pandemic influenza viruses in the future.
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Affiliation(s)
- Ian York
- Molecular Virology and Vaccines Branch, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Road-Mail Stop G-16, Atlanta, GA 30333, USA
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Seyer R, Hrincius ER, Ritzel D, Abt M, Mellmann A, Marjuki H, Kühn J, Wolff T, Ludwig S, Ehrhardt C. Synergistic adaptive mutations in the hemagglutinin and polymerase acidic protein lead to increased virulence of pandemic 2009 H1N1 influenza A virus in mice. J Infect Dis 2011; 205:262-71. [PMID: 22102733 DOI: 10.1093/infdis/jir716] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus.
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Affiliation(s)
- Roman Seyer
- Institute of Molecular Virology, ZMBE, Muenster, Germany
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Bavagnoli L, Dundon WG, Garbelli A, Zecchin B, Milani A, Parakkal G, Baldanti F, Paolucci S, Volmer R, Tu Y, Wu C, Capua I, Maga G. The PDZ-ligand and Src-homology type 3 domains of epidemic avian influenza virus NS1 protein modulate human Src kinase activity during viral infection. PLoS One 2011; 6:e27789. [PMID: 22110760 PMCID: PMC3215730 DOI: 10.1371/journal.pone.0027789] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2011] [Accepted: 10/25/2011] [Indexed: 01/29/2023] Open
Abstract
The Non-structural 1 (NS1) protein of avian influenza (AI) viruses is important for pathogenicity. Here, we identify a previously unrecognized tandem PDZ-ligand (TPL) domain in the extreme carboxy terminus of NS1 proteins from a subset of globally circulating AI viruses. By using protein arrays we have identified several human PDZ-cellular ligands of this novel domain, one of which is the RIL protein, a known regulator of the cellular tyrosine kinase Src. We found that the AI NS1 proteins bind and stimulate human Src tyrosine kinase, through their carboxy terminal Src homology type 3-binding (SHB) domain. The physical interaction between NS1 and Src and the ability of AI viruses to modulate the phosphorylation status of Src during the infection, were found to be influenced by the TPL arrangement. These results indicate the potential for novel host-pathogen interactions mediated by the TPL and SHB domains of AI NS1 protein.
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Affiliation(s)
- Laura Bavagnoli
- Institute of Molecular Genetics National Research Council, Pavia, Italy
| | - William G. Dundon
- World Organization for Animal Health, Food and Agriculture Organization and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy
| | - Anna Garbelli
- Institute of Molecular Genetics National Research Council, Pavia, Italy
| | - Bianca Zecchin
- World Organization for Animal Health, Food and Agriculture Organization and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy
| | - Adelaide Milani
- World Organization for Animal Health, Food and Agriculture Organization and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy
| | - Geetha Parakkal
- Institute of Molecular Genetics National Research Council, Pavia, Italy
| | - Fausto Baldanti
- Molecular Virology Unit, Virology and Microbiology, Fondazione Istituto Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, Pavia, Italy
| | - Stefania Paolucci
- Molecular Virology Unit, Virology and Microbiology, Fondazione Istituto Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, Pavia, Italy
| | - Romain Volmer
- Université de Toulouse, Institut National Polytechnique, Ecole Nationale de Veterinaire, Unitè Mixte de Recherche 1225, Interactions Hotes-Agents Pathogènes, Toulouse, France
| | - Yizeng Tu
- Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Chuanyue Wu
- Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Ilaria Capua
- World Organization for Animal Health, Food and Agriculture Organization and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy
| | - Giovanni Maga
- Institute of Molecular Genetics National Research Council, Pavia, Italy
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Meunier I, Embury-Hyatt C, Stebner S, Gray M, Bastien N, Li Y, Plummer F, Kobinger GP, von Messling V. Virulence differences of closely related pandemic 2009 H1N1 isolates correlate with increased inflammatory responses in ferrets. Virology 2011; 422:125-31. [PMID: 22074911 DOI: 10.1016/j.virol.2011.10.018] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2011] [Revised: 09/01/2011] [Accepted: 10/19/2011] [Indexed: 12/13/2022]
Abstract
Several early pandemic H1N1 influenza isolates cause severe disease in different animals models, while most strains result in mild clinical signs similar to seasonal influenza. In this study, the pathogenesis of the virulent Mexican isolate A/Mexico/InDRE4487/2009 and a mild Canadian isolate A/Canada-AB/RV1532/2009 was compared in ferrets. These viruses differed at nine residues, none of which has been previously identified as virulence factor. The Mexican isolate caused more severe disease and higher mortality, and reached higher peak nasal wash titers. Both viruses grew similarly in the respiratory tract, but only the virulent virus was detected in the gut after day 3. During the acute phase, both strains caused similar lung pathology, however the Mexican isolate induced severe inflammation even after virus clearance. This virus was also associated with a rapid and sustained induction of inflammatory cytokines, indicating that early dysregulation of the host response contributes importantly to the disease outcome.
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Tu J, Guo J, Zhang A, Zhang W, Zhao Z, Zhou H, Liu C, Chen H, Jin M. Effects of the C-terminal truncation in NS1 protein of the 2009 pandemic H1N1 influenza virus on host gene expression. PLoS One 2011; 6:e26175. [PMID: 22022552 PMCID: PMC3192165 DOI: 10.1371/journal.pone.0026175] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2011] [Accepted: 09/21/2011] [Indexed: 11/18/2022] Open
Abstract
The 2009 pandemic H1N1 influenza virus encodes an NS1 protein with 11 amino acids (aa) truncation at the C-terminus. The C-terminal tail of influenza virus NS1 protein constitutes a nucleolar localization signal (NoLS) and is the binding domain of the cellular pre-mRNA processing protein, poly(A)-binding protein II (PABII). Here, our studies showed that the C-terminal-truncated NS1 of the 2009 pandemic virus was inefficient at blocking host gene expression, extension of the truncated NS1 to its full length increased the inhibition of host gene expression. Mechanistically, this increased inhibition of host gene expression by the full-length NS1 was not associated with nucleolar localization, but was due to the restoration of NS1's binding capacity to PABII. Furthermore, in vitro and in vivo characterization of two recombinant viruses encoding either the C-terminal 11-aa truncated or full-length NS1 of the 2009 pandemic virus showed that the C-terminal 11-aa truncation in NS1 did not significantly alter virus replication, but increased virus pathogenicity in mice.
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Affiliation(s)
- Jiagang Tu
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Jing Guo
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Anding Zhang
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Wenting Zhang
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Zongzheng Zhao
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Hongbo Zhou
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Cheng Liu
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Huanchun Chen
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
| | - Meilin Jin
- Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China
- * E-mail:
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Tombari W, Nsiri J, Larbi I, Guerin JL, Ghram A. Genetic evolution of low pathogenecity H9N2 avian influenza viruses in Tunisia: acquisition of new mutations. Virol J 2011; 8:467. [PMID: 21992186 PMCID: PMC3223530 DOI: 10.1186/1743-422x-8-467] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2011] [Accepted: 10/12/2011] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Since the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. To monitor variations of Influenza viruses during the outbreaks, Tunisian H9N2 virus isolates were identified and genetically characterized. METHODS The genomic RNA segments of Tunisian H9N2 strains were subjected to RT-PCR amplifications followed by sequencing analysis. RESULTS Phylogenetic analysis demonstrated that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 viruses represent multiple reassortant lineages, with genes coming from Middle East strains, and share the common ancestor Qa/HK/G1/97 isolate which has contributed internal genes of H5N1 virus circulating in Asia. Some of the internal genes seemed to have undergone broad reassortments with other influenza subtypes. Deduced amino acid sequences of the hemagglutinin (HA) gene showed the presence of additional glycosylation site and Leu at position 234 indicating to binding preference to α (2, 6) sialic acid receptors, indicating their potential to directly infect humans. The Hemagglutinin cleavage site motif sequence is 333 PARSSR*GLF341 which indicates the low pathogenicity nature of the Tunisian H9N2 strains and the potential to acquire the basic amino acids required for the highly pathogenic strains. Their neuraminidase protein (NA) carried substitutions in the hemadsorption (HB) site, similar to those of other avian H9N2 viruses from Asia, Middle Eastern and human pandemic H2N2 and H3N2 that bind to α -2, 6 -linked receptors. Two avian virus-like aa at positions 661 (A) and 702 (K), similar to H5N1 strains, were identified in the polymerase (PB2) protein. Likewise, matrix (M) protein carried some substitutions which are linked with increasing replication in mammals. In addition, H9N2 strain recently circulating carried new polymorphism, "GSEV" PDZ ligand (PL) C-terminal motif in its non structural (NS) protein.Two new aa substitutions (I) and (V), that haven't been previously reported, were identified in the polymerase and matrix proteins, respectively. Nucleoprotein and non-structural protein carried some substitutions similar to H5N1 strains. CONCLUSION Considering these new mutations, the molecular basis of tropism, host responses and enhanced virulence will be defined and studied. Otherwise, Continuous monitoring of viral genetic changes throughout the year is warranted to monitor variations of Influenza viruses in the field.
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Affiliation(s)
- Wafa Tombari
- Veterinary Microbiology laboratory, Pasteur Institute of Tunis, 1002 Tunis-Belvédère, Tunisia
| | - Jihene Nsiri
- Veterinary Microbiology laboratory, Pasteur Institute of Tunis, 1002 Tunis-Belvédère, Tunisia
| | - Imen Larbi
- Veterinary Microbiology laboratory, Pasteur Institute of Tunis, 1002 Tunis-Belvédère, Tunisia
| | - Jean Luc Guerin
- INRA, UMR 1225, Ecole nationale vétérinaire de Toulouse, F-31076 Toulouse, France
| | - Abdeljelil Ghram
- Veterinary Microbiology laboratory, Pasteur Institute of Tunis, 1002 Tunis-Belvédère, Tunisia
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The DBA.2 mouse is susceptible to disease following infection with a broad, but limited, range of influenza A and B viruses. J Virol 2011; 85:12825-9. [PMID: 21917963 DOI: 10.1128/jvi.05930-11] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We assessed the relative susceptibilities to disease of the DBA.2 and C57BL/6 mouse models upon infection with a range of influenza A and B viruses. DBA.2 mice were more susceptible to disease upon inoculation with human H1N1 influenza A virus strains, several swine influenza viruses, and influenza B viruses but were not overtly susceptible to infection with human seasonal H3N2 strains. Hemagglutination inhibition and immunoglobulin isotype profiling indicated that DBA.2 and C57BL/6 mice generate comparable humoral responses upon equivalent 50% mouse lethal dose (MLD(50)) challenges with influenza virus. Our data demonstrate the utility of DBA.2 mice for the elucidation of influenza virus pathogenicity determinants and the testing of influenza vaccines.
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2009 pandemic H1N1 influenza virus causes disease and upregulation of genes related to inflammatory and immune responses, cell death, and lipid metabolism in pigs. J Virol 2011; 85:11626-37. [PMID: 21900171 DOI: 10.1128/jvi.05705-11] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in naïve swine populations, and might reassort with currently circulating swine influenza viruses.
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Abstract
Influenza A viruses are zoonotic pathogens that continuously circulate and change in several animal hosts, including birds, pigs, horses and humans. The emergence of novel virus strains that are capable of causing human epidemics or pandemics is a serious possibility. Here, we discuss the value of surveillance and characterization of naturally occurring influenza viruses, and review the impact that new developments in the laboratory have had on our understanding of the host tropism and virulence of viruses. We also revise the lessons that have been learnt from the pandemic viruses of the past 100 years.
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Affiliation(s)
- Rafael A Medina
- Department of Microbiology, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
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44
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Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses. J Virol 2011; 85:6275-86. [PMID: 21507962 DOI: 10.1128/jvi.02125-10] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.
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Abstract
Please cite this paper as: Neumann G, Kawaoka Y. (2011) The first influenza pandemic of the new millennium. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2011.00202.x. In the spring of 2009, a novel influenza A virus of the H1N1 subtype emerged that transmitted efficiently among humans; by June of 2009, the outbreak reached pandemic status. The pandemic virus possesses six viral RNA segments from so‐called triple reassortant swine viruses that emerged in North American pig populations in the late 1990s and two viral RNA segments from Eurasian avian‐like swine influenza viruses. Most human infections with the virus have been mild; however, severe and fatal infections occurred among certain risk groups, but also among those without any known risk factors. Here, we summarize the evolutionary, epidemiological, clinical, and molecular findings on the pandemic virus. We also discuss the arsenal of antiviral compounds and vaccines available to prevent and treat infections with the virus.
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Affiliation(s)
- Gabriele Neumann
- Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53711, USA
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Octaviani CP, Li C, Noda T, Kawaoka Y. Reassortment between seasonal and swine-origin H1N1 influenza viruses generates viruses with enhanced growth capability in cell culture. Virus Res 2010; 156:147-50. [PMID: 21195732 DOI: 10.1016/j.virusres.2010.12.014] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2010] [Revised: 12/20/2010] [Accepted: 12/23/2010] [Indexed: 12/01/2022]
Abstract
One year after the emergence of the 2009 influenza (H1N1) pandemic, most cases have been relatively mild; however, the possibility of enhanced viral growth ability by reassortment with seasonal viruses cannot be overlooked. Here, we show that reassortant viruses containing a seasonal H1 HA and swine-origin NA and M genes have enhanced virus growth over their wild-type parental viruses. The emergence of such viruses in nature could, therefore, represent a threat.
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Affiliation(s)
- Cássio Pontes Octaviani
- Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Abstract
Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.
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Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding. J Virol 2010; 84:11802-13. [PMID: 20844044 DOI: 10.1128/jvi.01136-10] [Citation(s) in RCA: 169] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
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Intracellular distribution of NS1 correlates with the infectivity and interferon antagonism of an avian influenza virus (H7N1). J Virol 2010; 84:11858-65. [PMID: 20844052 DOI: 10.1128/jvi.01011-10] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Highly pathogenic avian influenza viruses of subtype H7N1 that emerged during an outbreak in 1999 and 2000 in Italy differ from their low-pathogenicity precursor viruses by changes in several genes, including three mutations in the NS1 protein. Two of them involve amino acid exchanges located within or closely adjacent to the nuclear export signal of NS1. The third mutation resulted in a new stop codon and thereby a C-terminal truncation of the NS1 protein of the highly pathogenic viruses. To find out whether these mutations contribute to the phenotypic differences between the highly pathogenic and low pathogenic viruses, we generated recombinants of the highly pathogenic A/ostrich/Italy/984/00 strain that contained the nuclear export signal and/or the extended C terminus of NS1 of a low pathogenic virus (A/chicken/Italy/1082/99). Using these recombinants we could demonstrate that replication rate and spread of infection in chicken fibroblast cultures, as well as infectivity for chicken embryos is reduced, whereas the mean death time for chicken embryos is increased, when the highly pathogenic virus acquires the NS1 motifs of the low pathogenic virus. Analysis of beta interferon transcription in chicken fibroblasts infected with the recombinants revealed that the mutations observed in the nuclear export signal of the highly pathogenic viruses were responsible for the enhanced interferon antagonism of these viruses. Cell fractionation and immunofluorescence studies in chicken fibroblasts showed that the nuclear export signal of the highly pathogenic viruses is responsible for cytoplasmic accumulation of NS1, whereas the C-terminal truncation promotes transport into the nucleoli. Comparative analysis in human A549 cells indicated that intracellular distribution of NS1 is host specific. Taken together, these observations support the concept that compartmentalization of NS1 within the cell contributes to the pathogenicity of avian influenza viruses.
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50
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Virulence determinants of avian H5N1 influenza A virus in mammalian and avian hosts: role of the C-terminal ESEV motif in the viral NS1 protein. J Virol 2010; 84:10708-18. [PMID: 20686040 DOI: 10.1128/jvi.00610-10] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner.
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