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Journal of Virology, January 2005, p. 428-440, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.428-440.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Importance of Arginine 20 of the Swine Vesicular Disease Virus 2A Protease for Activity and Virulence

Toru Inoue,1,2,{dagger} Soren Alexandersen,3,{dagger},{ddagger} Angela T. Clark,2 Ciara Murphy,3 Melvyn Quan,3 Scott M. Reid,3 Yoshihiro Sakoda,1,§ Helen L. Johns,2 and Graham J. Belsham2*

Department of Exotic Disease, National Institute of Animal Health, Kodaira, Tokyo, Japan,1 Molecular Biology Division,2 Epidemiology Division, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom3

Received 2 April 2004/ Accepted 30 August 2004

A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.

* Corresponding author. Mailing address: BBCRC Institute for Animal Health, Pirbright Lab, Ash Rd., Pirbright, Woking, Surrey GU24 0NF, United Kingdom. Phone: 44 (0) 1483 232441. Fax: 44 (0) 1483 232448. E-mail: graham.belsham{at}bbsrc.ac.uk.

{dagger} T.I. and S.A. contributed equally to this work.

{ddagger} Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark.

§ Present address: Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.

Journal of Virology, January 2005, p. 428-440, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.428-440.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Inoue, T., Zhang, Z., Wang, L., West, L., Bashiruddin, J. B., Belsham, G. J. (2007). Significance of arginine 20 in the 2A protease for swine vesicular disease virus pathogenicity. J. Gen. Virol. 88: 2275-2279 [Abstract] [Full Text]  
  • Shaw, A. E., Reid, S. M., Knowles, N. J., Hutchings, G. H., Wilsden, G., Brocchi, E., Paton, D., King, D. P. (2005). Sequence analysis of the 5' untranslated region of swine vesicular disease virus reveals block deletions between the end of the internal ribosomal entry site and the initiation codon. J. Gen. Virol. 86: 2753-2761 [Abstract] [Full Text]  


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