ABSTRACT
Viruses
of the order Nidovirales encode huge replicase polyproteins.
These are processed primarily by the chymotrypsin-like main proteinases
(Mpros). So far, Mpros have been studied only for
corona-, arteri-, and roniviruses. Here, we report the characterization
of the Mpro 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
Mpro 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
Mpro resembles its coronavirus homologue in its preference
for P1-Gln, but its substrate-binding subsite, S1, more closely
resembles that of arteri- and ronivirus Mpros, which prefer
P1-Glu. Surprisingly, in contrast to the Mpros of corona-
and roniviruses, but like that of arterivirus, the torovirus
Mpro uses serine instead of cysteine as its principal
nucleophile. Under the premise that the Mpros 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 Mpro with a
Ser165→Cys substitution retained partial enzymatic
activity.