Analysis of functional domains on reovirus cell attachment protein sigma 1 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 sigma 1 fusion protein (F-sigma 1) 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 sigma 1 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 sigma 1 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-sigma 1 antibodies. It was found that mutant sigma 1 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-sigma 1, but none of the mutants, could bind immobilized glycophorin. These data clearly suggest that the host cell binding domain of sigma 1 is distinct from its hemagglutination domain. Also, the five neutralizing anti-sigma 1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of sigma 1.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D036022:Capsid Proteins; D004247:DNA / Q000235:genetics; D006021:Glycophorins / Q000378:metabolism; D006389:Hemagglutinins, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007739:L Cells / Q000378:metabolism; D012089:Mammalian orthoreovirus 3 / Q000235:genetics*; D051379:Mice; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D006021:Glycophorins; D006389:Hemagglutinins, Viral; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C046176:reovirus type 3 receptor; C031728:sigma 1 protein, reovirus; D004247:DNA