CONFORMATIONAL AND FUNCTIONAL-ANALYSIS OF THE C-TERMINAL GLOBULAR HEAD OF THE REOVIRUS CELL ATTACHMENT PROTEIN

We have been investigating structure-function relationships in the reovirus cell attachment protein σ using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) σ1 protein. The full-length and truncated σ1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the v1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was σ1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of σ1. In an attempt to identify smaller, functional peptides, full-length σ1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of σ1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of σ exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain. © 1991.