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Journal of Virology, July 2007, p. 7517-7528, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00605-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Ca²⁺-Binding Domain in the Rubella Virus Nonstructural Protease

Department of Chemistry,¹ Department of Biology, Georgia State University, Atlanta, Georgia 30303²

Received 21 March 2007/ Accepted 25 April 2007

The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca²⁺-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca²⁺-coordinating loop into a non-Ca²⁺-binding scaffold protein, CD2. The grafted EF-loop bound to Ca²⁺ and its trivalent analogs Tb³⁺ and La³⁺ with K_ds of 214, 47, and 14 µM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca²⁺-coordinating ligands in the EF-loop led to the elimination of Tb³⁺ binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca²⁺ ([Ca²⁺]/[protein] = 0.7 ± 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca²⁺ binding induced local conformational changes and increased thermal stability (T_m = 4.1°C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by 20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca²⁺-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.

Published ahead of print on 2 May 2007.