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

Abstract

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

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…Whether restoring the N pro function in the triple mutant GPE Ϫ virus would further enhance its pathogenicity is under current investigation. Previous data suggest that the IRF3-degrading function of N pro may support the pathogenicity of low-and moderately virulent strains but has only little influence on the pathogenicity of highly virulent viruses: mutations to knock out the N pro -mediated IRF3 degradation did not alter the pathogenicity of the highly virulent CSFV strain Eystrup and resulted in only slight attenuation of the moderately virulent vA187-1 virus (43). E rns forms disulfide-linked homodimers and possesses RNase activity (45,50).…”

“…Comparison of the genomes of the GPE Ϫ vaccine virus and the parent ALD virus revealed 225 nucleotide substitutions and 46 amino acid differences (22), which did not provide any conclusive information on the molecular basis of the attenuation. Numerous reports suggest that the viral proteins N pro , C, E rns , E1, E2, p7, and NS4B may be involved in determining the virulence of CSFV in pigs (7,8,9,10,11,12,13,32,36,37,38,39,40,43,46,54). So far, however, all conclusions on CSFV virulence determinants rely on mutations that result in downregulation of pathogenicity.…”

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

“…N pro (A187-1)/D136N showed higher stability than N pro (KPP)/S17P; N61K, although it was described to be defective in mediating IRF-3 degradation . This suggests that the lack of IRF-3 degradation by this TRASH domain mutant is indeed due to the lack of interaction with IRF-3 as described by Ruggli et al (2009) rather than to N pro instability, although a contribution by the latter cannot be excluded.…”

“…The KPP/93 strain showed low pathogenicity in pigs. In previous studies, the suppression of IFN-a/b induction by N pro was related to pathogenicity in pigs (Mayer et al, 2004;Ruggli et al, 2009;Tamura et al, 2014), suggesting that inability of KPP/93 N pro to suppress IFN-a/b induction may contribute to the low pathogenicity of the KPP/93 strain in pigs. In the present study, we identified amino acid residues of N pro responsible for the suppression of IFN-a/b induction by N pro .…”

“…The amino acid residues H49 and C69 in the N-terminal domain of N pro form a catalytic diad responsible for the autoprotease activity (Gottipati et al, 2013;Zögg et al, 2013). N pro is not essential for viral replication (Tratschin et al, 1998), but is involved in pathogenicity by suppressing IFN-a/b induction through IRF-3 degradation in host cells (Mayer et al, 2004;Hilton et al, 2006;Bauhofer et al, 2007;Ruggli et al, 2009;Tamura et al, 2014). A TRASH zinc-binding domain located in the C-terminal half of N pro , and involving the amino acid residues at positions 112, 134, 136 and 138, is required for mediating IRF-3 degradation Szymanski et al, 2009;Tamura et al, 2014).…”