Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral
response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-
infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs),
cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral
components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic
protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus
and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their
transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-
infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al.,
2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010;
Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by
viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent
cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA
virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was
confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-
8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of
caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-
viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage
of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is
a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and
detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the
specific role of caspase-8 in this process.