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Journal of Virology, June 2001, p. 5335-5342, Vol. 75, No. 11
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.11.5335-5342.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

Kartik Chandran,1,dagger Xing Zhang,2 Norman H. Olson,2 Stephen B. Walker,2 James D. Chappell,3 Terence S. Dermody,3 Timothy S. Baker,2 and Max L. Nibert1,*

Department of Biochemistry and Institute for Molecular Virology, University of Wisconsin---Madison, Madison, Wisconsin 537061; Department of Biological Sciences, Purdue University, West Lafayette, Indiana 479072; and Departments of Pediatrics and Microbiology and Immunology and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 372323

Received 15 November 2000/Accepted 7 March 2001

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins---sigma 1, µ1, and sigma 3---to enter host cells. sigma 1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma 1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma 1, µ1, and sigma 3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma 1. The recoated particles bound to and infected cultured cells in a sigma 1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma 1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma 1 protein. Additional experiments showed that recoated particles containing sigma 1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma 1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma 1 with respect to its structure, assembly into particles, and roles in entry.

* Corresponding author. Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-4829. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu.

dagger Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Mass.

Journal of Virology, June 2001, p. 5335-5342, Vol. 75, No. 11
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.11.5335-5342.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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