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Journal of Virology, April 2006, p. 4157-4167, Vol. 80, No. 8
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.8.4157-4167.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Characterization of a Torovirus Main Proteinase
Saskia L. Smits,1 Eric J. Snijder,2 and Raoul J. de Groot1*Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands,1 Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands2
Received 12 October 2005/ Accepted 2 February 2006
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 catalytic
ß-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.
* Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands. Phone: 31 30 2531463/2485. Fax: 31 30 2536723. E-mail: R.Groot{at}vet.uu.nl.
Journal of Virology, April 2006, p. 4157-4167, Vol. 80, No. 8
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.8.4157-4167.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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