SITE-DIRECTED MUTAGENIC ANALYSIS OF REOVIRUS SIGMA-3 PROTEIN-BINDING TO DSRNA

The S4 gene of reovirus encodes a double-stranded RNA-binding protein, sigma 3, that can inhibit activation of the interferon-induced dsRNA- dependent protein kinase, PKR. In this study, we attempted to localize the region of sigma 3 involved in dsRNA-binding by constructing delet ion and point mutations, expressing the mutated proteins in COS cells, and testing the ability of the native mutated proteins to bind dsRNA- agarose. Transfection of S4 into COS cells resulted in expression of t wo forms of sigma 3, a full-length protein, and a protein containing a small truncation at the amino-terminal end. The truncation is likely due to a proteolytic event. Deletions of as few as 10 amino acids from the amino-terminal end of the protein or 10 amino acids from the carb oxyl-terminal end of the protein resulted in loss of dsRNA-binding act ivity. A putative dsRNA-binding domain has previously been localized t o an 85 amino acid region located between amino acids 234 and 297 (Mil ler, J. E., and Samuel, C. E., J. Virol. 66, 5347-5356 1992). Mutagene sis of basic residues located within two distinct motifs of this regio n showed that some basic residues are absolutely required for binding to dsRNA while others can be changed with little effect. (C) 1994 Acad emic Press, Inc.