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Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity

Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity
Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity
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.
murine norovirus (mnv), norovirus, protease, polyprotein, positive sense, proteolysis
1470-8728
461-472
Herod, Morgan R.
8f27d0e7-1226-44b8-92dc-234b0da4c141
Prince, Cynthia A.
77360375-60d7-4c57-88a1-a25daf65040a
Skilton, Rachel J.
b02d4f32-609c-4074-b616-ec819b018dbe
Ward, Vernon K.
3dbf2165-fec7-437f-af39-693ea61ca31f
Cooper, Jonathan B.
0e4076fa-7476-4efe-b46b-9f2d84f52314
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Herod, Morgan R.
8f27d0e7-1226-44b8-92dc-234b0da4c141
Prince, Cynthia A.
77360375-60d7-4c57-88a1-a25daf65040a
Skilton, Rachel J.
b02d4f32-609c-4074-b616-ec819b018dbe
Ward, Vernon K.
3dbf2165-fec7-437f-af39-693ea61ca31f
Cooper, Jonathan B.
0e4076fa-7476-4efe-b46b-9f2d84f52314
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b

Herod, Morgan R., Prince, Cynthia A., Skilton, Rachel J., Ward, Vernon K., Cooper, Jonathan B. and Clarke, Ian N. (2014) Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity. Biochemical Journal, 464 (3), 461-472. (doi:10.1042/BJ20140959). (PMID:25275273)

Record type: Article

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.

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More information

e-pub ahead of print date: 15 December 2014
Published date: 15 December 2014
Keywords: murine norovirus (mnv), norovirus, protease, polyprotein, positive sense, proteolysis
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 373167
URI: https://eprints.soton.ac.uk/id/eprint/373167
ISSN: 1470-8728
PURE UUID: d9635467-f3f7-44e4-ab5a-5683d3120746
ORCID for Ian N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

Catalogue record

Date deposited: 12 Jan 2015 11:13
Last modified: 06 Jun 2018 13:19

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