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Journal of Virology, July 2004, p. 6723-6734, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.6723-6734.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, Is Transported to the Cell Surface and Undergoes Endocytosis

Yee-Joo Tan,1* Eileen Teng,1 Shuo Shen,1 Timothy H. P. Tan,1 Phuay-Yee Goh,1 Burtram C. Fielding,1 Eng-Eong Ooi,2 Hwee-Cheng Tan,2 Seng Gee Lim,1 and Wanjin Hong1

Institute of Molecular and Cell Biology, Singapore 117609,1 Environmental Health Institute, National Environmental Agency, Singapore 117610,Singapore2

Received 16 December 2003/ Accepted 23 February 2004

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx{phi} and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.

* Corresponding author. Mailing address: Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Phone: 65-68743780. Fax: 65-67791117. E-mail: mcbtanyj{at}imcb.nus.edu.sg.

Journal of Virology, July 2004, p. 6723-6734, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.6723-6734.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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