Characterization of the zinc binding activity of the rubella virus nonstructural protease

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a prot ease that cleaves the NSP precursor (240 kDa) at a single site to produce t wo products, A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP p recursor is necessary for RUB replication. Based on computer alignments, th e RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X, Liu, S. L, Ropp, R, J, Jackson, and T, K, Frey, J, Virol, 72:4463-1466, 1998), To analyze the function of metal cation bin ding in protease activity, Zn binding studies were performed using the mini mal NS protease domain within the NSP ORF, When expressed as a maltose bind ing protein (MBP) fusion protein by bacteria, the NS protease exhibited act ivity both in the bacteria and in vitro following purification when denatur ed and refolded in the presence of Zn, Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn-65 binding assay revealed two Zn binding domains: one located at a predi cted metal binding motif beginning at Cys1175 and the other one close to th e cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic s ite, in the second domain are essential for zinc binding, All of these resi dues are also necessary for the protease activity, as were several other Cy s residues not involved in Zn binding. Far-UV circular dichroism (CD) analy sis of the MBP-NS protease fusion protein showed that the protease domain c ontained a large amount of alpha-helical structure, which is consistent wit h the results of secondary-structural prediction. Both far-UV-CD and fluore scence studies suggested that Zn did not exert a major effect on the overal l structure of the fusion protein, Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril, In conjunction with the finding th at the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus m etalloprotease rather than a PCP.