Reovirus induces apoptosis in cultured cells and in vivo.In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-κB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-κB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirusinduced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-β mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-β substantially diminished reovirus replication and apoptosis, which suggests that IFN-β induction by NF-κB protects against reovirus-induced myocarditis. These findings reveal an organspecific role for NF-κB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis and myocarditis in infected animals. Differences in apoptosis efficiency displayed by reovirus strains are linked to the viral 1-encoding M2 gene segment. Studies using pharmacologic inhibitors of reovirus replication demonstrate that apoptosis induction by reovirus requires viral disassembly in cellular endosomes but not RNA synthesis. Since the 1 protein functions to pierce endosomal membranes during this temporal window, these findings point to an important role for 1 in activating signaling pathways that lead to apoptosis. To understand mechanisms used by 1 to induce apoptosis, a panel of 1 mutant viruses generated by reverse genetics was analyzed for the capacities to penetrate host cell membranes, activate proapoptotic signaling pathways, evoke cell death, and produce encephalitis in newborn mice. We found that single amino acid changes within the ␦ region of 1 reduce the efficiency of membrane penetration. These mutations also diminish the capacities of reovirus to activate proapoptotic transcription factors NF-B and IRF-3 and elicit apoptosis. Additionally, we observed that following intracranial inoculation, an apoptosis-deficient 1 mutant is less virulent in newborn mice in comparison to the wild-type virus. These results indicate a critical function for the membrane penetration activity of 1 in evoking prodeath signaling pathways that regulate reovirus pathogenesis.

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein 1 and membrane penetration protein 1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirusinduced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a 1-independent manner. Apoptosis following this uptake mechanism requires aciddependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L ؋ T3D reassortant viruses revealed that the 1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of 1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the 1 protein plays an essential role in stimulating proapoptotic signaling.