The N-terminal protease (Npro) of bovine viral diarrhea virus (BVDV) possesses two major biological functions, namely autoprotease activity and antagonism of interferon (IFN) synthesis, both of which are important for viral replication and immune evasion. Given the high mutation rate of the BVDV genome, whether mutations in Npro affect these functions remains largely unknown. Because cysteine and histidine residues are critical determinants of Npro activity, we systematically investigated the roles of all cysteine and histidine residues in regulating autoprotease activity and IFN antagonism. Synonymous codon substitutions involving cysteine or histidine residues did not affect the autoprotease activity of Npro. In contrast, analyses of nonsynonymous substitutions revealed that, in addition to the previously identified catalytic residues His49 and Cys69, His40, His99, and the zinc-binding residues Cys112, Cys134, and Cys138 were also essential for efficient autoprotease activity. By comparison, substitution of His148 had no detectable effect on either autoprotease activity or IFN antagonism. Functional dissociation analyses demonstrated that mutations at Glu22 and His49 enhanced IFN-β antagonism while abolishing autoprotease activity, whereas the Cys69 mutation abolished autocleavage but retained IFN suppression. In contrast, certain substitutions at His130 and Cys161 selectively impaired IFN antagonism without affecting autocleavage, indicating that these two functions are partially separable. Notably, mutations at His40, His99, Cys112, Cys134 and Cys138 markedly impaired both autoprotease activity and IFN antagonism, suggesting that these residues represent common functional determinants of the two activities. Sequence alignment of Npro proteins from different BVDV-1 strains identified naturally occurring non-conservative variations at positions 40, 130 and 148. Further analyses showed that the naturally occurring His40Arg and His130Pro substitutions significantly affected both autoprotease activity and IFN antagonism, supporting a functional association between Npro autocleavage and IFN suppression. In contrast, His148Arg, His148Asn, and His148Gln substitutions had no detectable effect on either activity. Collectively, these findings demonstrate that cysteine and histidine residues play critical roles in regulating both autoprotease activity and IFN antagonism of BVDV-1 Npro. Mutations at specific residues differentially affect these functions, highlighting the importance of conserved cysteine and histidine residues in maintaining the multifunctional properties of Npro.
