Characterization of a Torovirus Main Proteinase

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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↓(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 catalyticβ -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 Ser 165 →Cys substitution retained partial enzymatic activity.