Npro lysine 66 regulates ubiquitination-dependent degradation and innate immune evasion of classical swine fever virus.

Classical swine fever virus (CSFV) remains a significant threat to the global swine industry. The viral non-structural protein N-terminal protease (Npro) has been identified as a critical protein of CSFV evasion of host innate immune responses. Although Npro is known as an intrinsically unstable protein that undergoes degradation, the specific molecular determinants regulating its stability remain unclear. Here, we report that the 66th lysine (K66) within the N-terminal region of Npro functions as a major ubiquitination site that is essential for its efficient proteasomal degradation that is linked to downregulation of IRF3 and specific protein 1 (Sp1)/histone deacetylase 1 (HDAC1). The K66A mutation markedly reduced Npro ubiquitination and enhanced its stability together with upregulation of IRF3 and Sp1. Functionally, the K66A mutant virus (CSFV-NproK66A) did not antagonize effectively the transcriptional regulators Sp1/HDAC1 and IRF3, leading to increased expression of critical downstream antiviral effectors, including IFN-β, IFN-λ3, and ISG15, as compared with its wildtype strain. Consistent with these in vitro findings, the recombinant virus CSFV-NproK66A exhibits attenuated replication under antiviral immune pressure and marked impairment in suppressing host immune responses in response to poly(I:C) stimulation. Taken together, this study has defined K66 as an important regulatory residue that links Npro ubiquitination-associated degradation of IRF3 and Sp1 with CSFV immune evasion and provides novel insight into how CSFV fine-tunes the activity of Npro as a pestivirus-specific immune antagonist.