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J Virol, May 1998, p. 4463-4466, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

Xin Liu,1 Susan L. Ropp,1 Richard J. Jackson,2 and Teryl K. Frey1,*

Department of Biology, Georgia State University, Atlanta, Georgia 30303,1 and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom2

Received 2 September 1997/Accepted 6 February 1998

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.

* Corresponding author. Mailing address: Department of Biology, Georgia State University, University Plaza, Atlanta, GA 30303. Phone: (404) 651-3105. Fax: (404) 651-2509. E-mail: tfrey{at}panther.gsu.edu.

J Virol, May 1998, p. 4463-4466, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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