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.
Subject(s)
Classical Swine Fever Virus/genetics , Classical Swine Fever Virus/pathogenicity , Endopeptidases/genetics , Endopeptidases/metabolism , Interferon Regulatory Factor-3/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism , Amino Acid Substitution/genetics , Animals , Cell Line , Mutagenesis, Site-Directed , Mutation, Missense , Swine , VirulenceABSTRACT
Classical swine fever virus (CSFV) protects cells from double-stranded (ds) RNA-mediated apoptosis and IFN-alpha/beta induction. This phenotype is lost when CSFV lacks N(pro) (DeltaN(pro) CSFV). In the present study, we demonstrate that N(pro) counteracts dsRNA-mediated apoptosis and IFN-alpha/beta induction independently of other CSFV elements. For this purpose, we generated porcine SK-6 and PK-15 cell lines constitutively expressing N(pro) fused to the enhanced green fluorescent protein (EGFP). The survival of the SK6-EGFP-N(pro) cell line after polyinosinic polycytidylic acid [poly(IC)] treatment was comparable to that of CSFV-infected SK-6 cells and was significantly higher than the survival of the parent cell line. In PK-15 cells, the presence of EGFP-N(pro) prevented the DeltaN(pro) CSFV- and poly(IC)-mediated IFN-alpha/beta production. Importantly, N(pro) also inhibited IFN-alpha and IFN-beta promoter-driven luciferase expression in human cells and blocked IFN-alpha/beta induction mediated by Newcastle disease virus. This establishes a novel function for N(pro) in counteraction of the IFN-alpha/beta induction pathway.
