PROTEOLYTIC PROCESSING OF RUBELLA-VIRUS NONSTRUCTURAL PROTEINS

The genomic RNA of rubella virus contains two long open reading frames (ORF), a 5'-proximal ORF that codes for the nonstructural proteins an d a 3'-proximal ORF that encodes the structural proteins. The cDNA enc oding the nonstructural protein ORF of the wild-type M33 strain of rub ella virus has been obtained and sequenced. Comparison between the non structural proteins of the M33 and Therien strains of rubella virus re vealed a 98% homology in nucleotide sequence and 98.1% in deduced amin o acid sequence. To examine the processing of rubella virus nonstructu ral protein, the complete nonstructural protein ORF was expressed in B HK cells using a pSFV expression vector. Three nonstructural protein p roducts (p200, p150, and p90) with molecular weights of 200, 150, and 90 kDa were identified using antisera raised against synthetic peptide s corresponding to regions of the nonstructural proteins. p200 is the polyprotein precursor, while p150 and p90 are the cleavage products. S ite-directed mutagenesis of the Cys-1151 residue (one of the catalytic dyad residues of the viral protease) and of the Gly-1300 residue (the viral protease cleavage site) abrogated protease activity and p200 pr ecursor cleavage, respectively. Coexpression of mutant constructs in B HK cells indicated that rubella virus protease can function both in ci s and in trans. (C) 1998 Academic Press.