Expression of the rubella virus nonstructural protein ORF and demonstration of proteolytic processing.
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.
Marr
L D
LD
Department of Biology, Georgia State University, Atlanta 30302-4010.
Wang
C Y
CY
Frey
T K
TK
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D012412:Rubella virus / Q000201:enzymology / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics
D004279:DNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D003546:Cysteine Endopeptidases