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      Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability

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          Abstract

          To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in Escherichia coli. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in E. coli cells.

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          Most cited references18

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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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            Mechanisms and enzymes involved in SARS coronavirus genome expression.

            A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and post-translational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries.
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              The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine.

              Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.
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                Author and article information

                Contributors
                Journal
                Biochem Biophys Res Commun
                Biochem. Biophys. Res. Commun
                Biochemical and Biophysical Research Communications
                Elsevier
                0006-291X
                1090-2104
                30 October 2004
                3 December 2004
                30 October 2004
                : 325
                : 1
                : 374-380
                Affiliations
                [a ]School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
                [b ]Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
                Author notes
                [* ]Corresponding author. Fax: +65 67791117 dxliu@ 123456imcb.a-star.edu.sg
                Article
                S0006-291X(04)02295-8
                10.1016/j.bbrc.2004.10.050
                7092894
                15522242
                75f86c29-9f59-4873-b793-37191098ac28
                Copyright © 2004 Elsevier Inc. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 27 September 2004
                Categories
                Article

                Biochemistry
                severe acute respiratory syndrome coronavirus,envelope protein,membrane permeability,escherichia coli,e protein,oligomerization,viroporin

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