title: Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.
abstract: We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 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) sigma 1 protein. The full-length and truncated sigma 1 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 sigma 1 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 sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 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 sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 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.
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Duncan
authors.forename: D
authors.initials: D
authors.lastname: Horne
authors.forename: J E
authors.initials: JE
authors.lastname: Strong
authors.forename: G
authors.initials: G
authors.lastname: Leone
authors.forename: R T
authors.initials: RT
authors.lastname: Pon
authors.forename: M C
authors.initials: MC
authors.lastname: Yeung
authors.forename: P W
authors.initials: PW
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D036022:Capsid Proteins; D002918:Chymotrypsin / Q000494:pharmacology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D012087:Reoviridae / Q000235:genetics / Q000648:ultrastructure*; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000648:ultrastructure*
chemical_list: D036022:Capsid Proteins; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus; D002918:Chymotrypsin; D014357:Trypsin