Sites and Determinants of Early Cleavages in the Proteolytic Processing Pathway of Reovirus Surface Protein s3

ABSTRACTEntry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein s3. In the virion, s3 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 s3 maintains µ1 in this inactive state and the structural changes that accompany s3 processing and µ1 activation, however, are not well understood. In this study we characterized the early steps in s3 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 s3 as an early step in processing. Further processing of s3 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 × T3D reassortant virions differed in the sites of early s3 cleavage, with T1L s3 being cleaved mainly at residues 238 to 244 and T3D s3 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 s3 occurring faster than cleavage of T3D s3. Analyses using chimeric and site-directed mutants of recombinant s3 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 s3 crystal structure. The results indicate that proteolytic processing of s3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.