JVI Accepts, published online ahead of print on 4 November 2009

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J. Virol. doi:10.1128/JVI.01662-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The ORF 3a Protein of SARS-CoV Promotes Membrane Rearrangement and Cell Death.

Eric C. Freundt, Li Yu^*, Cynthia S. Goldsmith, Sarah Welsh, Aaron Cheng, Boyd Yount, Wei Liu, Matthew B. Frieman, Ursula J. Buchholz, Gavin R. Screaton, Jennifer Lippincott-Schwartz, Sherif R. Zaki, Xiao-Ning Xu, Ralph S. Baric, Kanta Subbarao, and Michael J. Lenardo

Laboratory of Immunology, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Infectious Disease Pathology Branch, Division of Viral and Rickettsial Diseases, Center for Disease Control and Prevention, Atlanta, GA 30333, USA; Department of Epidemiology, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7435, USA; Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA; Hammersmith Hospital, Imperial College, London W12 0NN, UK; Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK

^* To whom correspondence should be addressed. Email: liyulab{at}mail.tsinghua.edu.cn.

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. Here, we have 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 following 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. Additionally, 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, over-expression 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 accumulation of intracellular vesicles.