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Journal of Virology, November 2009, p. 11275-11282, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01263-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner{triangledown}

Stewart Goodwin,1 Tobias J. Tuthill,1 Armando Arias,2 Richard A. Killington,1 and David J. Rowlands1*

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom,1 Centro de Biologia Molecular (CSIC-UAM), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain2

Received 19 June 2009/ Accepted 18 August 2009

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin {alpha}vβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

* Corresponding author. Mailing address: Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44 (0)113 343 5641. Fax: 44 (0)113 343 5638. E-mail: d.j.rowlands{at}leeds.ac.uk

{triangledown} Published ahead of print on 26 August 2009.

Journal of Virology, November 2009, p. 11275-11282, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01263-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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