Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site.
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