Characterization of a torovirus main proteinase.

Abstract

Viruses of the order Nidovirales encode huge replicase polyproteins. These are processed primarily by the chymotrypsin-like main proteinases (M(pro)s). So far, M(pro)s have been studied only for corona-, arteri-, and roniviruses. Here, we report the characterization of the M(pro) of toroviruses, the fourth main Nidovirus branch. Comparative sequence analysis of polyprotein 1a of equine torovirus (EToV) strain Berne, identified a serine proteinase domain, flanked by hydrophobic regions. Heterologous expression of this domain resulted in autoprocessing at flanking cleavage sites. N-terminal sequence analysis of cleavage products tentatively identified FxxQ downward arrow(S, A) as the substrate consensus sequence. EToV M(pro) combines several traits of its closest relatives. It has a predicted three-domain structure, with two catalytic beta-barrel domains and an additional C-terminal domain of unknown function. With respect to substrate specificity, the EToV M(pro) resembles its coronavirus homologue in its preference for P1-Gln, but its substrate-binding subsite, S1, more closely resembles that of arteri- and ronivirus M(pro)s, which prefer P1-Glu. Surprisingly, in contrast to the M(pro)s of corona- and roniviruses, but like that of arterivirus, the torovirus M(pro) uses serine instead of cysteine as its principal nucleophile. Under the premise that the M(pro)s of corona- and toroviruses are more closely related to each other than to those of arteri- and roniviruses, the transition from serine- to cysteine-based proteolytic catalysis (or vice versa) must have happened more than once in the course of nidovirus evolution. In this respect, it is of interest that a mutant EToV M(pro) with a Ser165-->Cys substitution retained partial enzymatic activity.