Identification of an NF-κB-Dependent Gene Network in Cells Infected by Mammalian Reovirus

Abstract

Reovirus infection activates NF-B, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-B elicits this cellular response. To identify host genes activated by NF-B following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IB␣ (mIB␣) under the control of an inducible promoter. Induction of mIB␣ inhibited the activation of NF-B and blocked the expression of NF-B-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-B signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIB␣. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-B-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-B control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-B-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-␣, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-B-directed expression of cellular genes that activate death signaling pathways in infected cells.

Genome-Wide Analysis of Gene Expression in T Cells to Identify Targets of the NF-κB Transcription Factor c-Rel

“…Significant functional compensation between members of the NF-B/Rel family has previously been shown for a small group of genes in response to TNF-␣ in fibroblasts lacking various NF-B family members (34). In addition, there is significant overlap both in the functional groupings and in the specific genes affected by c-Rel deletion and those affected in other expression profiling studies where NF-B activation was globally inhibited (35)(36)(37). Small scale studies of genes affected by RelA deletion in RelA Ϫ/Ϫ mouse embryonic fibroblasts (34) or RelA RNA interference treatment in HeLa cells (38) also identified genes such as IP-10, Bcl2l1, and IB␣ as RelA targets, suggesting a common gene expression program executed by the NF-B/Rel family of transcription factors.…”

Apoptosis Induced by Mammalian Reovirus Is Beta Interferon (IFN) Independent and Enhanced by IFN Regulatory Factor 3- and NF-κB-Dependent Expression of Noxa

“…Efficient apoptosis induction following reovirus infection is dependent on the transcription factors IRF-3 and NF-B (20,34,35). Despite this association, microarray analysis of NF-Bdependent genes induced following reovirus infection did not identify a readily apparent mechanism for the initiation of apoptosis (58). However, a number of transcriptional networks that couple to apoptotic signaling pathways were identified.…”

“…Microarray studies of gene expression following reovirus infection revealed that several ISGs are rapidly upregulated following infection in an NF-B-dependent manner (58). However, those studies did not identify a mediator of proapoptotic signaling that could readily explain the cell death pathways initiated by reovirus infection.…”

“…IPS-1 subsequently engages the kinases inhibitor of B kinase alpha (IKK-␣), IKK-␤, IKK-, and Tank-binding kinase 1 (TBK1), which activate transcription factors, including activating transcription factor 2 (ATF-2)/c-Jun, IFN regulatory factor 3 (IRF-3), and NF-B (53,71,90). Once in the nucleus, these transcription factors induce the expression of a variety of inflammatory and antiviral genes, including IFN-␤ (58,68).…”

RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

“…High-density microarray analyses of cells expressing a regulated dominant-negative NF-B inhibitor have elucidated the genes under control of the canonical NF-B activation pathway in epithelial cells subjected to viral infections (40,55) and cytokine stimulation (56) and those controlled by its distinct activation modes (54). These studies have shown that in response to TNF stimulation, NF-B binding controls the expression of a noncontiguous group of genes whose products control a variety of biological processes, including leukocyte activation/chemotaxis, negative regulators of the TNF-IKK pathway, cellular metabolism, antigen processing, and others (54).…”