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Journal of Virology, May 2002, p. 5184-5197, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5184-5197.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sites and Determinants of Early Cleavages in the Proteolytic Processing Pathway of Reovirus Surface Protein 3

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 Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455³

Received 13 July 2001/ Accepted 13 February 2002

Entry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein 3. In the virion, 3 mostly covers the membrane-penetration protein µ1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which 3 maintains µ1 in this inactive state and the structural changes that accompany 3 processing and µ1 activation, however, are not well understood. In this study we characterized the early steps in 3 processing and determined their effects on µ1 function and particle infectivity. We identified two regions of high protease sensitivity, "hypersensitive" regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved 3 as an early step in processing. Further processing of 3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L x T3D reassortant virions differed in the sites of early 3 cleavage, with T1L 3 being cleaved mainly at residues 238 to 244 and T3D 3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L 3 occurring faster than cleavage of T3D 3. Analyses using chimeric and site-directed mutants of recombinant 3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the 3 crystal structure. The results indicate that proteolytic processing of 3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.

Dedicated to the memory of Carles Martí Henneberg.

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