Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Fourteen ORFs have been identified in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome. ORF 3a of SARS-CoV codes for a recently identified transmembrane protein, but its function remains unknown. In this study we confirmed the 3a protein expression and investigated its localization at the surface of SARS-CoV-infected or 3a-cDNA-transfected cells. Our experiments showed that recombinant 3a protein can form a homotetramer complex through interprotein disulfide bridges in 3a-cDNA-transfected cells, providing a clue to ion channel function. The putative ion channel activity of this protein was assessed in 3a-complement RNA-injected Xenopus oocytes by two-electrode voltage clamp. The results suggest that 3a protein forms a potassium sensitive channel, which can be efficiently inhibited by barium. After FRhK-4 cells were transfected with an siRNA, which is known to suppress 3a expression, followed by infection with SARS-CoV, the released virus was significantly decreased, whereas the replication of the virus in the infected cells was not changed. Our observation suggests that SARS-CoV ORF 3a functions as an ion channel that may promote virus release. This finding will help to explain the highly pathogenic nature of SARS-CoV and to develop new strategies for treatment of SARS infection.

[1]  A. Maeda,et al.  Release of Coronavirus E Protein in Membrane Vesicles from Virus-Infected Cells and E Protein-Expressing Cells , 1999, Virology.

[2]  W. Hong,et al.  A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, Is Transported to the Cell Surface and Undergoes Endocytosis , 2004, Journal of Virology.

[3]  V. Georgiev.  Virology , 1955, Nature.

[4]  Obi L. Griffith,et al.  The Genome Sequence of the SARS-Associated Coronavirus , 2003, Science.

[5]  J. A. Comer,et al.  A novel coronavirus associated with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[6]  E. Marbán,et al.  Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel. , 2004, Molecular cell.

[7]  M. Montal,et al.  Structure–function correlates of Vpu, a membrane protein of HIV‐1 , 2003, FEBS letters.

[8]  W. Haseltine,et al.  Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Shengqi Wang,et al.  Subcellular localization and membrane association of SARS-CoV 3a protein , 2005, Virus Research.

[10]  Y. Guan,et al.  Synthetic Peptides outside the Spike Protein Heptad Repeat Regions as Potent Inhibitors of Sars-Associated Coronavirus , 2005, Antiviral therapy.

[11]  M. Stocker,et al.  Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier α-subunits , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Hay,et al.  Structural characteristics of the M2 protein of influenza a viruses: Evidence that it forms a tetrameric channe , 1991, Virology.

[13]  Y. Wang,et al.  Characterization of the 3a Protein of SARS-associated Coronavirus in Infected Vero E6 Cells and SARS Patients , 2004, Journal of Molecular Biology.

[14]  Christian Drosten,et al.  Identification of a novel coronavirus in patients with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[15]  Lawrence H. Pinto,et al.  Influenza virus M2 protein has ion channel activity , 1992, Cell.

[16]  K. Baltensperger,et al.  Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers. , 2005, Cellular signalling.

[17]  L. Pinto,et al.  Demonstrating the intrinsic ion channel activity of virally encoded proteins , 2003, FEBS letters.

[18]  C. Hsiao,et al.  Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus , 2004, FEBS Letters.

[19]  A. Reynolds,et al.  Rational siRNA design for RNA interference , 2004, Nature Biotechnology.

[20]  ヤクルト本社.  Cell Signalling , 1998, The Journal of physiology.

[21]  Y. Guan,et al.  Prophylactic and Therapeutic Effects of Small Interfering Rna Targeting Sars-Coronavirus , 2003, Antiviral therapy.

[22]  W. Hong,et al.  The severe acute respiratory syndrome coronavirus 3a is a novel structural protein , 2005, Biochemical and Biophysical Research Communications.

[23]  Krishna Shankara Narayanan,et al.  Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is a Viral Structural Protein , 2005, Journal of Virology.

[24]  W. Hong,et al.  Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway , 2004, Journal of Virology.

[25]  M. Saraste,et al.  FEBS Lett , 2000 .

[26]  J. Sung,et al.  The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells. , 2005, The Journal of general virology.

[27]  Sheng Ye,et al.  Atomic structure of a Na+- and K+-conducting channel , 2006, Nature.

[28]  P. Gage,et al.  Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Krishna Shankara Narayanan,et al.  Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is Released in Membranous Structures from 3a Protein-Expressing Cells and Infected Cells , 2006, Journal of Virology.

[30]  P. Gage,et al.  The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels , 1996, Journal of virology.

[31]  D. Cmarko,et al.  Influenza virus M2 protein and haemagglutinin conformation changes during intracellular transport. , 1995, Acta virologica.

[32]  J. V. Van Etten,et al.  A potassium channel protein encoded by chlorella virus PBCV-1. , 2000, Science.

[33]  C. Machamer,et al.  Infectious Bronchitis Virus E Protein Is Targeted to the Golgi Complex and Directs Release of Virus-Like Particles , 2000, Journal of Virology.

[34]  P. Henklein,et al.  Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV‐1‐infected cells , 1996, FEBS letters.

[35]  J. Sodroski,et al.  Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. , 1991, Proceedings of the National Academy of Sciences of the United States of America.