The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme

Abstract

NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn 2؉ (and, to a lesser degree, by Cd 2؉ , Pb 2؉ , and Co 2؉ ) and inhibited by Cu 2؉ and Hg 2؉ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related.

The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis

“…As in HRV2-2A pro , the zinc site connects the interbarrel loop (ligands Cys97S γ and Cys99S γ ) with the turn between the β strands dII and eII (ligands Cys145S γ and a water molecule hydrogen bonded to His148). Nevertheless, the binding sites are not identical, as the zinc ion can be removed from the NS3 proteinase simply by dialysis against EDTA (Stempniak et al, 1997). In contrast, removal of the zinc ion from HRV2-2A pro requires prior denaturation of the enzyme with 8 M urea (Sommergruber et al, 1994b;Voss et al, 1995).…”

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays

“…Residues present in CTC motif (Cys481 and Cys483) and CHC motif (Cys457 and Cys 459) may be involved in zinc metal ion coordination and catalysis (Figure 9A). It is reported that the Zn binding site present on the opposite side of active site (Herold et al, 1999), which even validated by crystal structure of hepatitis C virus NS3 proteases (Stempniak et al, 1997;Barbato et al, 1999;Arasappan et al, 2005) picornavirus 2A (Yu and Lloyd, 1992) PL1pro of HCoV-229E (Ziebuhr et al, 2007), p150 of RUBV (Zhou et al, 2007), and Lpro of FMDV (Guarne et al, 2000). The diagrammatic representation indicates presence of CHC motif, opposite to the predicted active site, could be zinc binding site of HEV-protease (Figure 9).…”

Interaction of viral proteins with metal ions: role in maintaining the structure and functions of viruses

“…The coordination of the structural zinc in the HCV NS3 proteinase is mediated by Cys-97, Cys-99, Cys-145, and His-149. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that they are structurally related [55].…”

“…HCV polyprotein processing is activated by Zn 2+ and, to a lesser degree, by Cd 2+ , Pb 2+ , and Co 2+ and is inhibited by Cu 2+ and Hg 2+ ions [54]. Zn 2+ is not directly involved in catalysis but may have a structural role [55]. It is suggested that the three cysteines, and the histidines coordinate the structural zinc in the HCV NS3 proteinase at a Zn 2+binding site while dengue 2 virus NS3 protease, though lot of homology, does not contain a Zn 2+ -binding site [56].…”