title: Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.
abstract: The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein.
authors.affiliation: *Molecular Microbiology Group, University of Southampton, Southampton SO16 6YD, U.K.
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Herod
authors.affiliation: *Molecular Microbiology Group, University of Southampton, Southampton SO16 6YD, U.K.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Prince
authors.affiliation: *Molecular Microbiology Group, University of Southampton, Southampton SO16 6YD, U.K.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Skilton
authors.affiliation: †Otago School of Medical Sciences, Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Vernon K
authors.initials: VK
authors.lastname: Ward
authors.affiliation: ‡Laboratory for Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW3 2PF, U.K.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cooper
authors.affiliation: *Molecular Microbiology Group, University of Southampton, Southampton SO16 6YD, U.K.
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D029322:Norovirus / Q000201:enzymology*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases