Classical swine fever virus can remain virulent after specific elimination of the interferon regulatory factor 3-degrading function of Npro

J Virol. 2009 Jan;83(2):817-29. doi: 10.1128/JVI.01509-08. Epub 2008 Nov 5.

Abstract

Pestiviruses prevent alpha/beta interferon (IFN-alpha/beta) 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-alpha/beta 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-alpha/beta induction is not a prerequisite for CSFV virulence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Animals
  • Cell Line
  • Classical Swine Fever Virus / genetics*
  • Classical Swine Fever Virus / pathogenicity*
  • Endopeptidases / genetics*
  • Endopeptidases / metabolism*
  • Interferon Regulatory Factor-3 / metabolism*
  • Mutagenesis, Site-Directed
  • Mutation, Missense
  • Swine
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism*
  • Virulence
  • Virulence Factors / genetics*
  • Virulence Factors / metabolism*

Substances

  • Interferon Regulatory Factor-3
  • Viral Proteins
  • Virulence Factors
  • Endopeptidases
  • N(pro) protein, swine fever virus