Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-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-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-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-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-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-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.     

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.     

Inhibition of the NF-kappaB pathway by varicella-zoster virus in vitro and in human epidermal cells in vivo

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Using human cellular DNA microarrays, we found that many nuclear factor kappa B (NF-kappaB)-responsive genes were down-regulated in VZV-infected fibroblasts, suggesting that VZV infection inhibited the NF-kappaB pathway. The activation of this pathway causes a cellular antiviral response, including the production of alpha/beta interferon, cytokines, and other proteins that restrict viral infection. In these experiments, we demonstrated that VZV interferes with NF-kappaB activation in cultured fibroblasts and in differentiated epidermal cells in skin xenografts of SCIDhu mice infected in vivo. VZV infection of fibroblasts caused a transient nuclear translocation of p50 and p65, the canonical NF-kappaB family members. In a process that was dependent upon the presence of infectious VZV, these proteins rapidly became sequestered in the cytoplasm of VZV-infected cells. Exclusion of NF-kappaB proteins from nuclei was associated with the continued presence of IkappaBalpha, which binds p50 and p65 and prevents their nuclear accumulation. IkappaBalpha levels did not diminish even though the protein became phosphorylated and ubiquitinated, as determined based on detection of the characteristic high-molecular-weight form of the protein, and the 26S proteasome remained functional in VZV-infected cells. VZV infection also inhibited the characteristic degradation of IkappaBalpha that is induced by exposure of fibroblasts to tumor necrosis factor alpha. As expected, herpes simplex virus 1 caused the persistent nuclear translocation of NF-kappaB proteins, which has been shown to facilitate its replication, whereas VZV infection progressed without persistent NF-kappaB nuclear localization. We suggest that VZV has evolved a mechanism to limit host cell antiviral defenses by sequestering NF-kappaB proteins in the cytoplasm, a strategy that appears to be unique among the herpesviruses.     

Reovirus-induced apoptosis is mediated by TRAIL

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.