To analyze the proteins produced from the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF), a DNA containing the RUB NSP-ORF was introduced into the expression vector pTM3 in which the sequences to be expressed are downstream from a T7 RNA polymerase promoter. In cells infected with a vaccinia virus recombinant which expresses T7 RNA polymerase and transfected with this plasmid, three RUB-specific products with electrophoretic mobilities of 200, 150, and 97 kDa were clearly visible. By computer alignment, the presence of a cysteine protease was predicted within the NSP-ORF (A. E. Gorbalenya et al., FEBS Lett. 288, 201-205, 1991). When the Cys proposed as the catalytic residue of this protease (Cys1151) was mutated to a Gly, only the 200-kDa product was produced, demonstrating that the Cys is important in the activity of the protease responsible for the processing of the RUB NSPs and that the 150- and 97-kDa species are processing products. Transfections with deletion mutants revealed that the 150-kDa processing product is derived from the amino-terminal two-thirds of the ORF and that both the protease and the cleavage site on the COOH-terminal side of the 150-kDa product are between amino acids 1005 and 1507 of the ORF.