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Journal of Virology, June 2002, p. 6257-6267, Vol. 76, No. 12
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.12.6257-6267.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Membrane Association and Dimerization of a Cysteine-Rich, 16-Kilodalton Polypeptide Released from the C-Terminal Region of the Coronavirus Infectious Bronchitis Virus 1a Polyprotein

Lisa F. P. Ng1 and D. X. Liu1,2*

Institute of Molecular Agrobiology, The National University of Singapore, Singapore 117604,1 School of Biological Sciences, Nanyang Technological University, Singapore 6376162

Received 6 December 2001/ Accepted 20 March 2002

More than 10 mature proteins processed from coronavirus gene 1-encoded polyproteins have been identified in virus-infected cells. Here, we report the identification of the most C-terminal cleavage product of the 1a polyprotein as a 16-kDa protein in infectious bronchitis virus-infected Vero cells. Indirect immunofluorescence demonstrated that the protein exhibits a distinct perinuclear punctate staining pattern, suggesting that it is associated with cellular membranes. Positive staining observed on nonpermeabilized cells indicates that the protein may get transported to the cell surface, but no secretion of the protein out of the cells was observed. Treatment of the membrane fraction prepared from cells expressing the 16-kDa protein with Triton X-100, a high pH, and a high concentration of salts showed that the protein may be tightly associated with intracellular membranes. Dual-labeling experiments demonstrated that the 16-kDa protein colocalized with the 5'-bromouridine 5'-triphosphate-labeled viral RNA, suggesting that it may be associated with the viral replication machinery. Sequence comparison of the 16-kDa protein with the equivalent products of other coronaviruses showed multiple conserved cysteine residues, and site-directed mutagenesis studies revealed that these conserved residues may contribute to dimerization of the 16-kDa protein. Furthermore, increased accumulation of the 16-kDa protein upon stimulation with epidermal growth factor was observed, providing preliminary evidence that the protein might be involved in the growth factor signaling pathway.

* Corresponding author. Mailing address: School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Block 5, Level 3, Singapore 637616. Phone: 65-872-7000. Fax: 65-872-7007. E-mail: dxliu{at}ntu.edu.sg.

Journal of Virology, June 2002, p. 6257-6267, Vol. 76, No. 12
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.12.6257-6267.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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