IDENTIFICATION OF A SHORT DOMAIN WITHIN THE NONSTRUCTURAL PROTEIN NS2OF EPIZOOTIC HEMORRHAGIC-DISEASE VIRUS THAT IS IMPORTANT FOR SINGLE-STRAND RNA-BINDING ACTIVITY

The role that a conserved amino acid motif, found in the non-structura l protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis o f the NS2 of epizootic haemorrhagic disease virus. An NS2 mutant in wh ich this motif (amino acids 75 to 83) was deleted was expressed in Spo doptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)Sepharose. The deletion mutant also differed from wildtype NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant. Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclu sion bodies in the cytoplasm of baculovirus-infected insect cells. To further characterize the role of this motif in RNA-binding, specific r esidues within the region were substituted by site-directed mutagenesi s and the mutants were expressed in Escherichia coli as fusion protein s. Analysis of the different mutant proteins indicated that in each ca se ssRNA-binding was impaired relative to that of the wild-type NS2 co ntrol. The degree of impairment corresponded to the number of amino ac id substitutions and the largest effects were associated with non-cons erved substitutions. It is suggested that the conserved motif is an im portant structural determinant in the interaction of NS2 with ssRNA.