The rubella virus nonstructural protease requires divalent cations for activity and functions in trans.
The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn2+, Cd2+, and Co2+, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707-4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.
Liu
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Department of Biology, Georgia State University, Atlanta 30303, USA.
Ropp
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Jackson
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Frey
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D002104:Cadmium; D002413:Cations, Divalent; D003035:Cobalt; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012412:Rubella virus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D015032:Zinc
D002413:Cations, Divalent; D017361:Viral Nonstructural Proteins; D002104:Cadmium; D003035:Cobalt; D003546:Cysteine Endopeptidases; D015032:Zinc