The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death (cid:1) ‡

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

[1]  R. Johnston,et al.  Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-κB Signaling , 2009, Journal of Virology.

[2]  Abraham J Koster,et al.  SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum , 2008, PLoS biology.

[3]  S. Jameel,et al.  Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation. , 2008, The Journal of general virology.

[4]  M. Lenardo,et al.  Photoconversion of Lysotracker Red to a green fluorescent molecule , 2007, Cell Research.

[5]  J. Peiris,et al.  Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. , 2007, The Journal of general virology.

[6]  A. Pekosz,et al.  Severe Acute Respiratory Syndrome Coronavirus Gene 7 Products Contribute to Virus-Induced Apoptosis , 2007, Journal of Virology.

[7]  David Franco,et al.  Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication , 2007, Journal of Virology.

[8]  Caroline C. Friedel,et al.  Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome , 2007, PloS one.

[9]  Julian Druce,et al.  Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein , 2007, Journal of Virology.

[10]  Ralph Baric,et al.  A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice , 2007, PLoS pathogens.

[11]  J. Lippincott-Schwartz,et al.  Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA , 2006, Journal of Virology.

[12]  Wanjin Hong,et al.  Over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells , 2006, Virus Research.

[13]  Wei Lu,et al.  Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release , 2006, Proceedings of the National Academy of Sciences.

[14]  J. Onderwater,et al.  Ultrastructure and Origin of Membrane Vesicles Associated with the Severe Acute Respiratory Syndrome Coronavirus Replication Complex , 2006, Journal of Virology.

[15]  T. Wileman.  Aggresomes and Autophagy Generate Sites for Virus Replication , 2006, Science.

[16]  P. Chan,et al.  Specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in SARS patients , 2006, Journal of Clinical Pathology.

[17]  Tsung-Han Hsieh,et al.  Severe acute respiratory syndrome coronavirus 3C‐like protease‐induced apoptosis , 2006, FEMS immunology and medical microbiology.

[18]  J. Lippincott-Schwartz,et al.  Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities. , 2005, Molecular biology of the cell.

[19]  R. Baric,et al.  Severe Acute Respiratory Syndrome Coronavirus Group-Specific Open Reading Frames Encode Nonessential Functions for Replication in Cell Cultures and Mice , 2005, Journal of Virology.

[20]  S. Weiss,et al.  Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus , 2005, Microbiology and Molecular Biology Reviews.

[21]  S. Perlman,et al.  A Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Protein Enhances Virulence of an Attenuated Murine Coronavirus , 2005, Journal of Virology.

[22]  Jiapei Chen,et al.  G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells , 2005, Virology Journal.

[23]  L. Ren,et al.  Apoptosis induced by the SARS-associated coronavirus in Vero cells is replication-dependent and involves caspase. , 2005, DNA and cell biology.

[24]  J. Donaldson,et al.  Localization and function of Arf family GTPases. , 2005, Biochemical Society transactions.

[25]  K. Kirkegaard,et al.  Topology of Double-Membraned Vesicles and the Opportunity for Non-Lytic Release of Cytoplasm , 2005, Autophagy.

[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]  W. Leung,et al.  Coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (SARS) , 2005, Modern Pathology.

[28]  K. Kirkegaard,et al.  Subversion of Cellular Autophagosomal Machinery by RNA Viruses , 2005, PLoS biology.

[29]  Runsheng Chen,et al.  Antibody responses to individual proteins of SARS coronavirus and their neutralization activities , 2005, Microbes and Infection.

[30]  J. Lippincott-Schwartz,et al.  ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells , 2005, The Journal of cell biology.

[31]  R. Baric,et al.  Single-Amino-Acid Substitutions in Open Reading Frame (ORF) 1b-nsp14 and ORF 2a Proteins of the Coronavirus Mouse Hepatitis Virus Are Attenuating in Mice , 2005, Journal of Virology.

[32]  Michelle M. Packard,et al.  Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan , 2005, Human Pathology.

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

[34]  Andrew Pekosz,et al.  Structure and Intracellular Targeting of the SARS-Coronavirus Orf7a Accessory Protein , 2005, Structure.

[35]  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.

[36]  T. Mizutani,et al.  Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells , 2004, Virology.

[37]  W. Hong,et al.  Characterization of a Unique Group-Specific Protein (U122) of the Severe Acute Respiratory Syndrome Coronavirus , 2004, Journal of Virology.

[38]  K. Chan,et al.  Evaluation and Validation of an Enzyme-Linked Immunosorbent Assay and an Immunochromatographic Test for Serological Diagnosis of Severe Acute Respiratory Syndrome , 2004, Clinical Diagnostic Laboratory Immunology.

[39]  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.

[40]  P. Mcgeer,et al.  SARS corona virus peptides recognized by antibodies in the sera of convalescent cases , 2004, Virology.

[41]  G. Gao,et al.  SARS coronavirus induces apoptosis in Vero E6 Cells , 2004, Journal of medical virology.

[42]  P. Rottier,et al.  Live, Attenuated Coronavirus Vaccines through the Directed Deletion of Group-Specific Genes Provide Protection against Feline Infectious Peritonitis , 2004, Journal of Virology.

[43]  N. Mizushima,et al.  Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy* , 2004, Journal of Biological Chemistry.

[44]  Y. Leo,et al.  Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers , 2004, Clinical Diagnostic Laboratory Immunology.

[45]  Yee-Joo Tan,et al.  Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay and Immunochromatographic Tests for Detection of Immunoglobulin G Antibodies to Severe Acute Respiratory Syndrome (SARS) Coronavirus in SARS Patients , 2004, Clinical Diagnostic Laboratory Immunology.

[46]  J. A. Comer,et al.  Ultrastructural Characterization of SARS Coronavirus , 2004, Emerging infectious diseases.

[47]  J. Lippincott-Schwartz,et al.  A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[48]  T. Hyypiä,et al.  Replication Complex of Human Parechovirus 1 , 2003, Journal of Virology.

[49]  Christian Drosten,et al.  Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome , 2003, Science.

[50]  I. Sola,et al.  Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence , 2003, Virology.

[51]  P. Daszak,et al.  Elucidation of Nipah virus morphogenesis and replication using ultrastructural and molecular approaches. , 2003, Virus research.

[52]  K. Kirkegaard,et al.  Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles , 2000, Journal of Virology.

[53]  B. Antonny,et al.  Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[54]  J. Acker,et al.  Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. , 1999, Molecular cell.

[55]  G. Banting,et al.  Efficient trafficking of TGN38 from the endosome to the trans-Golgi network requires a free hydroxyl group at position 331 in the cytosolic domain. , 1998, Molecular biology of the cell.

[56]  Luis Carrasco,et al.  Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179 , 1997, Journal of virology.

[57]  G. Banting,et al.  TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology. , 1997, Biochimica et biophysica acta.

[58]  N. Gonatas,et al.  Syncytia Formation Induced by Coronavirus Infection Is Associated with Fragmentation and Rearrangement of the Golgi Apparatus , 1996, Virology.

[59]  M. Yaspo,et al.  Primate homologues of rat TGN38: primary structure, expression and functional implications. , 1996, Journal of cell science.

[60]  N. Gonatas,et al.  Fragmentation and rearrangement of the Golgi apparatus during MHV infection of L-2 cells. , 1995, Advances in experimental medicine and biology.

[61]  B. Roizman,et al.  The herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus , 1994, Journal of virology.

[62]  H. Geuze,et al.  Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding , 1994, Journal of virology.

[63]  R. Klausner,et al.  ARF: a key regulatory switch in membrane traffic and organelle structure. , 1994, Current opinion in cell biology.

[64]  K. Howell,et al.  A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network , 1993, The Journal of cell biology.

[65]  J. Lippincott-Schwartz,et al.  Brefeldin A: insights into the control of membrane traffic and organelle structure , 1992, The Journal of cell biology.

[66]  D. Botstein,et al.  ADP-ribosylation factor is functionally and physically associated with the Golgi complex. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[67]  I. Pastan,et al.  Identification of two lysosomal membrane glycoproteins , 1985, The Journal of cell biology.