Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation

Classical Swine Fever Virus NproLimits Type I Interferon Induction in Plasmacytoid Dendritic Cells by Interacting with Interferon Regulatory Factor 7

“…The plasmids pCI-Ub-Core, pFLAG-IRF3, and pFLAG-IRF7 were described elsewhere (6,42). For the mammalian two-hybrid assays (Promega), plasmid pFN10A(ACT)-IRF3 expressing the porcine IRF3 fused to the VP16 transactivator domain and pFN11A(BIND)-derived plasmids expressing GAL4-N pro and the mutants GAL4-N (C 161 A) were used; these are described elsewhere (43,53). Mutants with deletions in the IRF3 and IRF7 genes were obtained by PCR-based site-directed mutagenesis using standard techniques.…”

“…N pro harbors an autoprotease domain in the N-terminal half of the protein, resulting in cotranslational release of N pro from the nascent polyprotein. In the C-terminal half of N pro , a metal-binding TRASH motif consisting of C 112 -X21-C 134 -X3-C 138 mediates the coordination of one zinc (Zn) atom per molecule (53). This domain is essential for N pro to interact with IRF3 and to mediate the degradation of IRF3 by the proteasome.…”

“…A recent report described a novel Zn-binding TRASH motif in N pro , involving the residues C 112 , C 134 , D 136 , and C 138 . Zn binding by N pro is a prerequisite for the interaction of N pro with IRF3, leading to the proteasomal degradation of IRF3 and to the inhibition of type I IFN induction (53). Considering the structural similarity of IRF3 and IRF7, we asked whether N pro also relays on the same Zn-binding domain to interact with IRF7.…”

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

“…Previous studies demonstrated that the amino acid residues C112, C134, D136 and C138 of CSFV N pro form a TRASH domain and are essential for the suppression of IFN-a/b induction Szymanski et al, 2009). Four CSFV isolates in Thailand were classified into genotype 1.1 based on the E2 gene sequence (Fig.…”

“…N pro is not essential for viral replication (Tratschin et al, 1998), but is involved in pathogenicity by suppressing IFN-a/b induction through IRF-3 degradation in host cells (Mayer et al, 2004;Hilton et al, 2006;Bauhofer et al, 2007;Ruggli et al, 2009;Tamura et al, 2014). A TRASH zinc-binding domain located in the C-terminal half of N pro , and involving the amino acid residues at positions 112, 134, 136 and 138, is required for mediating IRF-3 degradation Szymanski et al, 2009;Tamura et al, 2014).…”