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Journal of Virology, January 2009, p. 817-829, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01509-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of N^pro

Nicolas Ruggli,^1* Artur Summerfield,¹ Ana R. Fiebach,¹ Laurence Guzylack-Piriou,¹ Oliver Bauhofer,^1, Catherine G. Lamm,^1, Sandro Waltersperger,¹ Keita Matsuno,² Luzia Liu,¹ Markus Gerber,¹ Kyung H. Choi,³ Martin A. Hofmann,¹ Yoshihiro Sakoda,² and Jon-Duri Tratschin¹

Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland,¹ Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan,² Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0647³

Received 17 July 2008/ Accepted 21 October 2008

Pestiviruses prevent alpha/beta interferon (IFN-/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral N^pro nonstructural protein. N^pro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire N^pro gene resulted in attenuation in pigs. In order to elaborate on the role of the N^pro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in N^pro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of N^pro. We describe two mutations in N^pro that eliminate N^pro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for N^pro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-/β induction is not a prerequisite for CSFV virulence.

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* Corresponding author. Mailing address: Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland. Phone: 41 31 848 9211. Fax: 41 31 848 9222. E-mail: nicolas.ruggli{at}ivi.admin.ch

Published ahead of print on 5 November 2008.

Present address: Department of Molecular Virology, University of Heidelberg, D-69120 Heidelberg, Germany.

Present address: Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK.

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Journal of Virology, January 2009, p. 817-829, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01509-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.