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      Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

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          Abstract

          Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na +/K + ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

          Author Summary

          Several highly pathogenic viruses encode small transmembrane proteins with ion-conduction properties named viroporins. Viroporins are generally involved in virus production and maturation processes, which many times are achieved by altering the ion homeostasis of cell organelles. Cells have evolved mechanisms to sense these imbalances in ion concentrations as a danger signal, and consequently trigger the innate immune system. Recently, it has been demonstrated that viroporins are inducers of cytosolic macromolecular complexes named inflammasomes that trigger the activation of key inflammatory cytokines such as IL-1β. The repercussions of this system in viral pathogenesis or disease outcome are currently being explored. SARS-CoV infection induces an uncontrolled inflammatory response leading to pulmonary damage, edema accumulation, severe hypoxemia and eventually death. In this study, we report that SARS-CoV E protein ion channel activity is a determinant of virulence, as the elimination of this function attenuated the virus, reducing the harmful inflammatory cytokine burst produced after infection, in which inflammasome activation plays a critical role. This led to less pulmonary damage and to disease resolution. These novel findings may be of relevance for other viral infections and can possibly be translated in order to find therapies for their associated diseases.

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          Most cited references47

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          Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.

          A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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            Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

            The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society
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              Characterization of a novel coronavirus associated with severe acute respiratory syndrome.

              P Rota (2003)
              In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                May 2014
                1 May 2014
                : 10
                : 5
                : e1004077
                Affiliations
                [1 ]Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain
                [2 ]Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castellón, Spain
                [3 ]School of Biological Sciences, Division of Structural and Computational Biology, Nanyang Technological University, Singapore, Singapore
                Vanderbilt University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JLNT LE MLD. Performed the experiments: JLNT MLD CVB JMJG JARN RFD. Analyzed the data: JLNT LE MLD CVB JMJG JARN RFD CCR AA VMA. Contributed reagents/materials/analysis tools: JT. Wrote the paper: JLNT LE MLD. Revised the manuscript: JLNT MLD CVB JMJG JARN RFD CCR AA JT VMA LE.

                [¤]

                Current address: David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America

                Article
                PPATHOGENS-D-13-03381
                10.1371/journal.ppat.1004077
                4006877
                24788150
                377cc6af-0330-499a-bb30-792fd4390133
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 December 2013
                : 5 March 2014
                Page count
                Pages: 19
                Funding
                This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699 and 0258-3413/HHSN266200700010C). Financial support from Generalitat Valenciana (Prometeu 2012/069) and Fundacion Caixa Castello-Bancaixa (Project No. P1-1B2012-03) is also acknowledged. JLN received a contract from NIH. JAR and CCR received fellowships from Fundacion La Caixa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Histology
                Biochemistry
                Proteins
                Protein Structure
                Computational Biology
                Synthetic Biology
                Developmental Biology
                Molecular Development
                Cytokines
                Genetics
                Molecular Genetics
                Immunology
                Immune System
                Innate Immune System
                Immunity
                Microbiology
                Animal Models of Infection
                Virology
                Molecular Biology
                Macromolecular Structure Analysis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Virulence Factors
                Pathogenesis

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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