The key amino acids (cysteine and histidine) across BVDV Npro sequence that affect autoprotease activity and inhibition of IFN-β synthesis

Functional dissociation analyses demonstrated that mutations at His49 and Cys69 abolished autoprotease activity while retaining IFN suppression, whereas 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 strains identified naturally occurring variations at positions 40 and 130. Further analyses showed that the 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 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.