Analysis of functional domains on reovirus cell attachment protein σ1 using cloned s1 gene deletion mutants

Previously a reovirus (serotype 3) S1 gene cDNA was inserted into the lac cloning site of pUC13 and expressed in Escherichia coli to yield a σ1 fusion protein (F-σ1) capable of binding to mouse L fibroblasts and of agglutinating human red blood cells (S. A. Masri, L. Nagata, D. C. W. Mah, and P. W. K. Lee, 1986, Virology 149, 83-90). To probe the functional domains on the σ1 protein, restriction enzymes which divide the S1 gene into four segments (5′-I-II-III-IV-3′) of similar size were used to generate five in-frame deletion mutants (D1-D5). Corresponding mutant forms of σ1 were expressed in E. coli and were assayed for (i) host cell (mouse L fibroblasts) binding activity; (ii) glycophorin (reovirus erythrocyte receptor) binding activity (R. W. Paul and P. W. K. Lee, 1987, Virology 159, 94-101 and (iii) recognizability by a library of neutralizing monoclonal anti-σ1 antibodies. It was found that mutant σ1 forms with segment III or segment IV deleted did not exhibit any detectable L-cell binding activity, whereas mutants with these two segments intact (but lacking segment II or segments I and II) were capable of attaching to L-cell receptors, albeit with reduced efficiencies. On the other hand, only F-σ1, but none of the mutants, could bind immobilized glycophorin. These data clearly suggest that the host cell binding domain of σl is distinct from its hemagglutination domain. Also, the five neutralizing anti-σ1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of σ1.