Functional expression in Escherichia coli of cloned reovirus S1 gene encoding the viral cell attachment protein sigma 1.
A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.
D000814:Aniline Compounds; D000818:Animals; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D006385:Hemagglutination Inhibition Tests; D006387:Hemagglutination, Viral; D007739:L Cells; D012089:Mammalian orthoreovirus 3 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010957:Plasmids; D011991:Receptors, Virus / Q000378:metabolism*; D012087:Reoviridae / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D000814:Aniline Compounds; D036022:Capsid Proteins; D011991:Receptors, Virus; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus; C004354:dicloran