A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication

摘要

pThe protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Znsup2+/sup-binding ligands. This minidomain bound to Znsup2+/sup with a stoichiometry of ≈0.7 and an apparent dissociation constant of &500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Znsup2+/sup binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cyssup1175/sup, Cyssup1178/sup, Cyssup1225/sup and Cyssup1227/sup were required for the binding of Znsup2+/sup. Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)sub48/subCXC Znsup2+/sup-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication./p