A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold

A cysteine proteinase, papain-like proteinase (PL1pro), of the human corona virus 229E (HCoV) regulates the expression of the replicase polyproteins, p p1a and ppa1ab, by cleavage between Gly(111) and Asn(112), far upstream of its own catalytic residue Cys(1054). In this report, using bioinformatics t ools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connectin g the left and right hand domains of a papain-like fold. Optical emission s pectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments usin g the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation, T he reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanth roline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ af ter renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger i ncluding replacements of any of four conserved Cys residues predicted to co -ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a po tential framework for regulatory processes and may be indicative of a nonpr oteolytic activity of this enzyme.