Abstract
The N-terminal protease of pestiviruses, Npro 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 Npro 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 Npro, but not by mutant protein Npro C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that Npro inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of Npro 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 Npro and we show that, in common with many other viral proteins, Npro targets mitochondria to inhibit apoptosis in response to cell stress. Npro itself not only relocated to mitochondria but in addition, both Npro and IRF3 associated with peroxisomes, with over 85% of Npro puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing Npro and IRF3 associated with ubiquitin. IRF3 was degraded, whereas Npro accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by Npro, and highlight the role of these organelles in the anti-viral pathway.
Highlights
Viruses employ many strategies to establish persistent infection of their host
Sodium arsenate is an inducer of the cell stress pathway and we chose this chemical to look at the role of Npro in inhibition of apoptosis, both because mitochondria play a central role in IRF3 signalling and because virus infection activates the cell stress pathway [19]
Various apoptotic signals induced by staurosporine, poly I:C, interferon, sodium arsenate and hydrogen peroxide were inhibited by wild type Npro, but not by the mutant protein, suggesting that the mechanism of apoptosis inhibition is through IRF3 and that levels of IRF3 in cells expressing mutant Npro were sufficient to promote apoptosis
Summary
Viruses employ many strategies to establish persistent infection of their host. Most involve inhibition of innate immune responses, including inhibition of Type I interferon and inhibition of apoptosis [1,2]. Our previous work has shown that this single viral protein expressed alone in cells can block Type 1 interferon production by promoting the loss of IRF3 [5], to which it binds directly through a zinc binding TRASH motif [6], leading to IRF3 degradation in proteasomes [7] Both these properties of Npro have generated useful biological tools; the autoprotease activity has been used to enhance refolding and expression of fused peptides and proteins during bacterial protein expression [8] and the direct antagonism of IRF3 has been used to generate cell lines deficient in IRF3 to block antiviral responses to study viral replication [9]
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