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.

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