title: Site-directed mutagenesis of the C-terminal portion of reovirus protein sigma 1: evidence for a conformation-dependent receptor binding domain.
abstract: Oligonucleotide site-directed mutagenesis was used to modify the type 3 (T3) reovirus cell attachment protein sigma 1 at residues located in the three regions (designated C, D, and E in the C-terminal one-third of sigma 1) that are highly conserved between the three reovirus serotypes. Of the eight residues targeted for mutagenesis, five (one in region C, and two each in regions D, and E) are conserved among all three proteins. Wild-type (wt) and mutant sigma 1 forms were synthesized in an vitro transcription/translation system and subjected to structural and functional analysis. None of the mutations affected the ability of sigma 1 to form trimers. However, mutation (all representing drastic changes) in any of the five triply conserved residues (Tyr326, Asn369, Phe370, Tyr450, and Pro451) caused a complete or partial abrogation of sigma 1 cell binding function, whereas mutation in any of the other three residues (Ser325, Ser327, and Asp365) had no adverse effect. The structural integrity of the mutant proteins was then probed using trypsin, chymotrypsin, and a neutralizing monoclonal anti-sigma 1 antibody. In all cases, the loss of cell binding function was associated with a drastic conformational change in the C-terminal globular head of sigma 1. These results suggest that conserved residues in the three highly conserved regions in the C-terminal portion of sigma 1 play important structural and functional roles and are involved in proper head folding and generation of a conformation-dependent receptor binding domain.
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
authors.forename: D L
authors.initials: DL
authors.lastname: Turner
authors.forename: R
authors.initials: R
authors.lastname: Duncan
authors.forename: P W
authors.initials: PW
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins; D002918:Chymotrypsin / Q000494:pharmacology; D004252:DNA Mutational Analysis; D007739:L Cells / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011991:Receptors, Virus / Q000378:metabolism*; D012087:Reoviridae / Q000235:genetics*; D016415:Sequence Alignment; D014357:Trypsin / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure
chemical_list: D036022:Capsid Proteins; D008024:Ligands; D011991:Receptors, Virus; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus; D002918:Chymotrypsin; D014357:Trypsin