Site-directed mutagenesis of the C-terminal portion of reovirus protein σ1: Evidence for a conformation-dependent receptor binding domain

Oligonucleotide site-directed mutagenesis was used to modify the type 3 (T3) reovirus cell attachment protein σl at residues located in the three regions (designated C, D, and E in the C-terminal one-third of at) 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 of forms were synthesized in an in vitro transcription/translation system and subjected to structural and functional analysis. None of the mutations affected the ability of al to form trimers. However, mutation (all representing drastic changes) in any of the five triply conserved residues (Tyr₃₂₆, Asn₃₆₉, Phe₃₇₀ Tyr_45o, and Pros₄₅₁) caused a complete or partial abrogation of al 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-σ1 antibody. In all cases, the loss of cell binding function was associated with a drastic conformational change in the C-terminal globular head of σ1. These results suggest that conserved residues in the three highly conserved regions in the C-terminal portion of σl play important structural and functional roles and are involved in proper head folding and generation of a conformation-dependent receptor binding domain.