The Pestivirus N Terminal Protease Npro Redistributes to Mitochondria and Peroxisomes Suggesting New Sites for Regulation of IRF3 by Npro

The N-terminal protease of pestiviruses, N-pro is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N-pro in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N-pro, but not by mutant protein N-pro C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N-pro inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N-pro and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N-pro and we show that, in common with many other viral proteins, N-pro targets mitochondria to inhibit apoptosis in response to cell stress. N-pro itself not only relocated to mitochondria but in addition, both N-pro and IRF3 associated with peroxisomes, with over 85% of N-pro puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N-pro and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N-pro accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N-pro, and highlight the role of these organelles in the anti-viral pathway.