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 and a 3'-proximal ORF that encodes the structural proteins. The cDNA encoding the nonstructural protein ORF of the wild-type M33 strain of rubella virus has been obtained and sequenced. Comparison between the nonstructural proteins of the M33 and Therien strains of rubella virus revealed a 98% homology in nucleotide sequence and 98.1% in deduced amino acid sequence. To examine the processing of rubella virus nonstructural protein, the complete nonstructural protein ORF was expressed in BHK cells using a pSFV expression vector. Three nonstructural protein products (p200, p150, and p90) with molecular weights of 200, 150, and 90 kDa were identified using antisera raised against synthetic peptides corresponding to regions of the nonstructural proteins. p200 is the polyprotein precursor, while p150 and p90 are the cleavage products. Site-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 precursor cleavage, respectively. Coexpression of mutant constructs in BHK cells indicated that rubella virus protease can function both in cis and in trans.
Yao
J
J
Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Yang
D
D
Chong
P
P
Hwang
D
D
Liang
Y
Y
Gillam
S
S
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral / Q000235:genetics; D008970:Molecular Weight; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012412:Rubella virus / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; D010450:Endopeptidases