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Journal of Virology, December 2003, p. 13361-13375, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13361-13375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The {delta} Region of Outer-Capsid Protein µ1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

Kartik Chandran,1,{dagger} John S. L. Parker,1,{ddagger} Marcelo Ehrlich,2,3 Tomas Kirchhausen,2,3 and Max L. Nibert1*

Departments of Microbiology and Molecular Genetics,1 Cell Biology,2 Center for Blood Research, Harvard Medical School, Boston, Massachusetts 021153

Received 6 June 2003/ Accepted 5 September 2003

Cell entry by reoviruses requires a large, transcriptionally active subvirion particle to gain access to the cytoplasm. The features of this particle have been the subject of debate, but three primary candidates—the infectious subvirion particle (ISVP), ISVP*, and core particle forms—that differ in whether putative membrane penetration protein µ1 and adhesin {sigma}1 remain particle bound have been identified. Experiments with antibody reagents in this study yielded new information about the steps in particle disassembly during cell entry. Monoclonal antibodies specific for the {delta} region of µ1 provided evidence for a conformational change in µ1 and for release of the {delta} proteolytic fragment from entering particles. Antiserum raised against cores provided evidence for entry-related changes in particle structure and identified entering particles that largely lack the {delta} fragment inside cells. Antibodies specific for {sigma}1 showed that it is also largely shed from entering particles. Limited coimmunostaining with markers for late endosomes and lysosomes indicated the particles lacking {delta} and {sigma}1 did not localize to those subcellular compartments, and other observations suggested that both the particles and free {delta} were released into the cytoplasm. Essentially equivalent findings were obtained with native ISVPs and highly infectious recoated particles containing wild-type proteins. Poorly infectious recoated particles containing a hyperstable mutant form of µ1, however, showed no evidence for the in vitro and intracellular changes in particle structure normally detected by antibodies, and these particles instead accumulated in late endosomes or lysosomes. Recoated particles with hyperstable µ1 were also ineffective at mediating erythrocyte lysis in vitro and promoting {alpha}-sarcin coentry and intoxication of cells in cultures. Based on these and other findings, we propose that ISVP* is a transient intermediate in cell entry which mediates membrane penetration and is then further uncoated in the cytoplasm to yield particles, resembling cores, that largely lack the {delta} fragment of µ1.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 645-3680. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu.

{dagger} Present address: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.

{ddagger} Present address: James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

Journal of Virology, December 2003, p. 13361-13375, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13361-13375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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