Noroviruses are the most common cause of acute viral gastroenteritis in humans, with epidemics commonly occurring in hospitals and on ocean liners (Clarke & Lambden, 2005
). The virus, which is transmitted through contaminated food and water, can infect and replicate in enterocytes of the epithelial cell lining of the small and large intestine (Green, 2007
). Currently, there is neither vaccine nor antiviral therapy available.
The norovirus genome consists of a molecule of single-stranded positive-sense RNA (7.7 kb) comprising three open reading frames ORF 1, ORF 2 and ORF 3 (Lambden et al.
). ORF 1, which is located at the 5′-terminus of the genome, encodes a large nonstructural 200 kDa polyprotein. ORF 2 encodes the major capsid protein VP1 and ORF 3 codes for a small basic protein VP2 that is thought to assist in the viral assembly process (Bertolotti-Ciarlet et al.
). In vitro
translation and mutagenesis studies indicated that the 200 kDa ORF 1 polyprotein is cleaved by the action of the viral protease to generate initially three separate functional protein products (Liu et al.
). Full processing of the precursor polyprotein generates an N-terminal protein (p48), an NTPase (p41), a 3A-like protein (p22), a Vpg protein (p16), a 3C-like protease (p19) and an RNA polymerase (p57) (Liu et al.
). The protease also inhibits cellular translation by cleavage of the poly(A)-binding protein, thereby allowing preferential viral protein expression compared with host proteins (Kuyumcu-Martinez et al.
). Since processing of the 200 kDa precursor polyprotein is essential to yield functional viral proteins, the viral protease presents itself as an attractive target for antiviral strategies.
Enzymes in this family are cysteine proteases that display a trypsin-like or chymotrypsin-like serine protease fold, a property which distinguishes them from other viral proteases (Matthews et al.
). The Southampton norovirus protease has a preference for cleavage at LQ–GP and LQ–GK sequences, but it can also cleave at ME–GK, FE–AP and LE–GG (where ‘–’ indicates the scissile bond). In the nomenclature of Schechter & Berger (1967
), the substrate residues each side of the scissile bond are labelled P1 and P1′ and the remainder are labelled according to the scheme …P3, P2, P1, P1′, P2′, P3′…. The corresponding subsites in the enzyme are labelled S3, S2 etc
. It appears that the Southampton norovirus protease preferentially accommodates a glutamine or glutamate residue at the P1 position, a small amino acid at P1′ and a hydrophobic residue at P2. Modified peptide inhibitors that include the preferred amino-acid recognition sequence but possess a C-terminal moiety capable of reacting with the active-site cysteine residue have been developed for other viral cysteine proteases and in vitro
studies have shown that these completely inhibit the catalytic activity and have antiviral properties in vivo
(Dragovich et al.
). One such modified peptide inhibitor includes a Michael acceptor group at its C-terminus, which undergoes nucleophilic attack by the active-site thiol, resulting in the inhibitor becoming irreversibly bound to the enzyme (Fig. 1; Dragovich et al.
Structure of the Michael acceptor peptide inhibitor (MAPI) designed for the Southampton virus protease.
A number of noroviral proteases have been analysed by X-ray diffraction, e.g.
those from the Chiba and Norwalk viruses (Nakamura et al.
; Zeitler et al.
). In this paper, we describe the crystallization of the Southampton norovirus protease, initially in a form that diffracted to medium resolution. A marked improvement in crystal quality was achieved by cocrystallization of the enzyme with the Michael acceptor peptide inhibitor (MAPI) acetyl-Glu-Phe-Gln-Leu-Gln-X
, in which a peptide mimicking part of the natural substrate consensus sequence is coupled to a propenyl ethyl ester moiety (X
) in order to modify the active-site cysteine. The resulting cocrystals belonged to space group P
and diffracted synchrotron radiation to 1.7 Å resolution.