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Journal of Virology, April 1999, p. 2963-2973, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Reovirus Virion-Like Particles Obtained by Recoating Infectious Subvirion Particles with Baculovirus-Expressed sigma 3 Protein: an Approach for Analyzing sigma 3 Functions during Virus Entry

Judit Jané-Valbuena,1,2 Max L. Nibert,1,2,* Stephan M. Spencer,1,2 Stephen B. Walker,3 Timothy S. Baker,3 Ya Chen,4 Victoria E. Centonze,4 and Leslie A. Schiff5

Department of Biochemistry, College of Agricultural and Life Sciences,1 and Institute for Molecular Virology2 and Integrated Microscopy Resource,4 The Graduate School, University of Wisconsin---Madison, Madison, Wisconsin 53706; Department of Biological Sciences, Purdue University, West Lafayette, Indiana 479073; and Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 554555

Received 20 August 1998/Accepted 8 December 1998

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein sigma 3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed sigma 3 protein to infectious subvirion particles (ISVPs) that lack sigma 3. A level of sigma 3 binding approaching 100% of that in native virions was routinely achieved. The sigma 3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study sigma 3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of sigma 3 but contain outer-capsid protein µ1/µ1C mostly cleaved at the delta -phi junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that sigma 3, and not the delta -phi cleavage of µ1/µ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric sigma 3 proteins to localize the primary determinants of a strain-dependent difference in sigma 3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

* Corresponding author. Mailing address: Institute for Molecular Virology, University of Wisconsin---Madison, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 262-4536. Fax: (608) 262-7414. E-mail: mlnibert{at}facstaff.wisc.edu.

Journal of Virology, April 1999, p. 2963-2973, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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