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Journal of Virology, April 2003, p. 4370-4382, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4370-4382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

Ivo C. Lorenz,1 Jürgen Kartenbeck,2 Anna Mezzacasa,1 Steven L. Allison,3 Franz X. Heinz,3 and Ari Helenius1*

Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland,1 Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany,2 Institute of Virology, University of Vienna, A-1095 Vienna, Austria3

Received 20 August 2002/ Accepted 6 January 2003

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

* Corresponding author. Mailing address: Institute of Biochemistry, ETH Hönggerberg, HPM E6.3, CH-8093 Zürich, Switzerland. Phone: 41-1-6326817. Fax: 41-1-6321269. E-mail: ari.helenius{at}bc.biol.ethz.ch.

Journal of Virology, April 2003, p. 4370-4382, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4370-4382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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