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Journal of Virology, August 2004, p. 8732-8745, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8732-8745.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Putative Autocleavage of Outer Capsid Protein µ1, Allowing Release of Myristoylated Peptide µ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

Amy L. Odegard,1,2 Kartik Chandran,1,{dagger} Xing Zhang,3 John S. L. Parker,1,{ddagger} Timothy S. Baker,3 and Max L. Nibert1*

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

Received 24 January 2004/ Accepted 8 April 2004

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein µ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments µ1N (4 kDa; myristoylated) and µ1C (72 kDa). In this study, we found that µ1 cleavage to yield µ1N and µ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective µ1 (asparagine -> alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including µ1 conformational change and {sigma}1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin {alpha}-sarcin). In addition, after these particles were allowed to enter cells, the {delta} region of µ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective µ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of µ1N/µ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal µ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

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

{dagger} Present address: Hematology Division, Department of Medicine, Brigham and Women's Hospital, 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, August 2004, p. 8732-8745, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8732-8745.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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