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Journal of Virology, December 2002, p. 12211-12222, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12211-12222.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Hydrophilic Amino-Terminal Arm of Reovirus Core Shell Protein {lambda}1 Is Dispensable for Particle Assembly

Jonghwa Kim,1,2 Xing Zhang,3 Victoria E. Centonze,4,{dagger} Valorie D. Bowman,3 Simon Noble,2,{ddagger} Timothy S. Baker,4 and Max L. Nibert1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry,2 Integrated Microscopy Resource, University of Wisconsin—Madison, Madison, Wisconsin 53706,4 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 479073

Received 10 April 2002/ Accepted 6 August 2002

The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridae family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins {lambda}1, {lambda}2, and {sigma}2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, µ1 and {sigma}3, to produce virion-like particles. Coexpression of core shell protein {lambda}1 and core nodule protein {sigma}2 was sufficient to yield CLPs that could withstand purification, whereas expression of {lambda}1 alone was not, indicating a required role for {sigma}2 as a previous study also suggested. In addition, CLPs that lacked {lambda}2 (formed from {lambda}1 and {sigma}2 only) could not be coated with µ1 and {sigma}3, indicating a required role for {lambda}2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of {lambda}1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of {lambda}1 deletion mutants, we showed that the amino-terminal 230 residues of {lambda}1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of {lambda}1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of {lambda}1 with the threefold and/or fivefold copies of {sigma}2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.

* Corresponding author. Mailing 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 Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900.

{ddagger} Present address: Nature Medicine, New York, NY 10010-1707.

Journal of Virology, December 2002, p. 12211-12222, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12211-12222.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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