Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase

Abstract

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.

Expression, purification, and in vitro activity of an arterivirus main proteinase

“…For optimization of this cleavage assay, nsp4His was preferred over MBP-nsp4 because the purification of the latter was a more elaborate process. Although it was reported that the addition of six His residues strongly inhibited the enzymatic activity of the human coronavirus 229E 3CL pro proteinase (Ziebuhr et al, 1997), in both our assays the catalytic activity of nsp4His was very comparable to that of partially purified uncleaved or cleaved MBP-nsp4. Also, the in vivo trans-cleavage activity of nsp4His, as determined using the recombinant vaccinia virus/T7 expression system (data not shown), was comparable to that of untagged nsp4, suggesting that the presence of the (His) 6 tag did not affect nsp4 proteolytic activity.…”

“…Although the purified EAV nsp4His proteinase showed catalytic activity on all peptides, its activity was very weak compared to that of other 3C-like proteinases in similar experiments (Cordingley et al, 1989;Hammerle et al, 1991;Hegyi et al, 2002;Jewell et al, 1992;Ziebuhr et al, 1997). Therefore, to investigate whether a differently purified nsp4 proteinase might perform better, the catalytic activity of nsp4His proteinase was compared to that of a partially purified MBP-nsp4 proteinase.…”

“…Consequently, an alternative assay based on the use of synthetic peptides as substrates was developed. Using this approach other 3C-(like) proteinases, like those of the human coronavirus 229E, the feline infectious peritonitis virus, and several picornaviruses (Cordingley et al, 1989;Hammerle et al, 1991;Hegyi et al, 2002;Jewell et al, 1992;Ziebuhr et al, 1997), have been successfully characterized.…”

Nidovirales: Evolving the largest RNA virus genome

“…The 3CL pro s of the four main nidovirus branches are profoundly diverged and (according to conventional criteria) do not group together, although they have become significantly separated from their viral and cellular homologs. Even the catalytic center, which is uniformly conserved in cellular homologs, has evolved into quite different forms, including a Cys-His catalytic dyad in roni-and coronaviruses, a Ser-His-Asp catalytic triad in arteriviruses, and a Ser-His-based catalytic center in toroviruses (Anand et al, 2002Barrette-Ng et al, 2002;Draker et al, 2006;Ziebuhr et al, 1997Ziebuhr et al, , 2003. The crystal structures of coronavirus and arterivirus 3CL pro s revealed that both groups of enzymes have a three-domain structure, with domains I and II forming a (chymotrypsin-like) two-ß-barrel fold consisting of 12 or 13 ß-strands (Anand et al, 2002Barrette-Ng et al, 2002;Yang et al, 2003).…”

Characterization of the norovirus 3C-like protease

“…These studies allowed us to identify the active-site amino acid residues with a combination of site-directed mutagenesis studies (Boniotti et al, 1994;Cheah et al, 1990;Gosert et al, 1997;Hammerle et al, 1991). Several 3C and 3C-like proteases were purified and their biochemical properties were well characterized (Baum et al, 1991;Chisholm et al, 2001;Davis et al, 1997;Ziebuhr et al, 1997). They are characterized by their proteolytic activities being inhibited by both cysteine and serine protease inhibitors.…”