IκB Kinase Subunits α and γ Are Required for Activation of NF-κB and Induction of Apoptosis by Mammalian Reovirus

Abstract

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-B and downstream cellular genes. To better understand the mechanism of NF-B activation by reovirus, NF-B signaling intermediates under reovirus control were investigated at the level of Rel, IB, and IB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This Mammalian reoviruses are nonenveloped viruses that contain a genome of 10 segments of double-stranded RNA (52). Following infection of newborn mice, reovirus disseminates systemically, causing injury to the central nervous system (CNS), heart, and liver (76). Apoptosis induced by reovirus appears to be the primary mechanism for virus-induced encephalitis (53, 54, 59) and myocarditis (22,23,54). Disassembly of internalized virus in the endocytic pathway provides the initial viral trigger for stimulating the signaling pathways that elicit an apoptotic response (19,21).Transcription factor NF-B plays an important regulatory role in apoptosis evoked by reovirus in cultured cells (20) and in vivo (54). Inducible members of the NF-B family are sequestered in the cytoplasm by inhibitory IB proteins, including IB␣, IB␤, IBε, and p100/NF-B2 (3,31,68,79,82). In response to a wide variety of NF-B inducers, IB proteins are phosphorylated at specific serine residues, earmarking these molecules for destruction by the ubiquitin-proteasome pathway (7,12,31,56,75,82). Phosphorylation of IB proteins is mediated by cytokine-inducible IB kinases (IKKs) IKK␣ and IKK␤ (47,78), which can form higher-order complexes containing a regulatory subunit called IKK␥/Nemo (26,50,62,88,91). A primary function of IKK␤ is to modulate the inhibitory interaction of IB␣ with the prototypical form of NF-B containing p50/RelA dimers (25,26,50,58,69). This regulatory circuit, termed the classical pathway of NF-B activation, is strictly dependent on the presence of IKK␥/Nemo (64,65,88). In contrast, IKK␣ functions in an alternative IKK␥-independent pathway of NF-B activation that leads to the proteolytic processing of p100 and the production of a functional p52 Rel subunit (16,66,71). Unlike the classical IKK␤-directed pathway of NF-B activation, the alternative pathway involving IKK␣ is dependent on its prior phosphorylation by NF-Binducing kinase (NIK) (43,66,85). In addition to the cytoplasmic function of IKK␣, a nuclear role for IKK␣ in the transcriptional activation of NF-B-responsive genes has been suggested by in vitro studies (1,32,33,41,48,69,87).To better understand the mechanism of NF-B activation by reovirus, we conducted experiments to define the NF-B/Rel, IB, and IKK proteins that are under reovirus control. These studies revealed that NF-B/Rel proteins are mobilized to the nuclear compartment with biphasic kinetics follo...

Retinoic Acid-inducible Gene-I and Interferon-β Promoter Stimulator-1 Augment Proapoptotic Responses Following Mammalian Reovirus Infection via Interferon Regulatory Factor-3

“…1C). The kinetics of reovirus-induced activation of IRF-3 parallel kinetics of NF-B activation by reovirus (20,33).…”

“…NF-B complexes containing p50 and RelA (p65) are activated following reovirus infection over a time course similar to the activation of IRF-3 (20,33). NF-B complexes activated by reovirus also contain c-Rel at 8 h post-infection and p52 at 20 -24 h post-infection (33).…”

“…NF-B complexes activated by reovirus also contain c-Rel at 8 h post-infection and p52 at 20 -24 h post-infection (33). Although other NF-B family members are activated during reovirus infection, p50 and RelA are clearly required for reovirus-induced apoptosis (20), suggesting that the initial activation of NF-B is essential for cell death signaling.…”

“…Although other NF-B family members are activated during reovirus infection, p50 and RelA are clearly required for reovirus-induced apoptosis (20), suggesting that the initial activation of NF-B is essential for cell death signaling. Reovirus-induced NF-B activation requires the ␣ and ␥ subunits of the multi-component IKK complex (33), in contrast to the canonical activation of p50/Rel-A heterodimers, which requires IKK␤ and IKK␥ (60). The signal transducers that couple reovirus to the IKK complex are not known.…”

“…Nuclear extracts were prepared as described previously (33). Extracts (10 g of total protein) were resolved by electrophoresis in 4 -20% polyacrylamide gels and transferred to nitrocellulose membranes.…”