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Journal of Virology, November 2003, p. 11357-11366, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11357-11366.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Steven L. Allison,1* Yizhi J. Tao,2,{dagger} Gabriel O'Riordain,1 Christian W. Mandl,1 Stephen C. Harrison,2,3 and Franz X. Heinz1

Institute of Virology, University of Vienna, A-1095 Vienna, Austria,1 Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138,2 Children's Hospital and Harvard Medical School, Boston, Massachusetts 021153

Received 28 April 2003/ Accepted 29 July 2003

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

* Corresponding author. Mailing address: Institute of Virology, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79505. Fax: 43-1-40490-9795. E-mail: steven.allison{at}univie.ac.at.

{dagger} Present address: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas.

Journal of Virology, November 2003, p. 11357-11366, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11357-11366.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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