The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus 1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that 1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic ␣-helices within a carboxyl-terminal portion of 1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by 1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated when 1 was coexpressed with 3, with which it is known to coassemble. We propose that 1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of 1 to associate with intracellular membranes. Moreover, during reovirus infection, association with 3 may regulate apoptosis induction by 1.

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease.

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 1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed 1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of 1 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 1-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 1 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 1, indicating that caspases were not required. Additionally, 1-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.

Mammalian orthoreoviruses replicate and assemble in the cytosol of infected cells. A viral nonstructural protein, NS, forms large inclusion-like structures called viral factories (VFs) in which assembling viral particles can be identified. Here we examined the localization of the cellular chaperone Hsc70 and found that it colocalizes with VFs in infected cells and also with viral factory-like structures (VFLs) formed by ectopically expressed NS. Small interfering RNA (siRNA)-mediated knockdown of Hsc70 did not affect the formation or maintenance of VFLs. We further showed that dominant negative mutants of Hsc70 were also recruited to VFLs, indicating that Hsc70 recruitment to VFLs is independent of the chaperone function. In support of this finding, NS was immunoprecipitated with wild-type Hsc70, with a dominant negative mutant of Hsc70, and with the minimal substrate-binding site of Hsc70 (amino acids 395 to 540). We identified a minimal region of NS between amino acids 222 and 271 that was sufficient for the interaction with Hsc70. This region of NS has not been assigned any function previously. However, neither point mutants with alterations in this region nor the complete deletion of this domain abrogated the NS-Hsc70 interaction, indicating that a second portion of NS also interacts with Hsc70. Taken together, these findings suggest a specific chaperone function for Hsc70 within viral factories, the sites of reovirus replication and assembly in cells. Mammalian orthoreoviruses have a genome of 10 doublestranded RNA (dsRNA) segments that are encased in a double-layered, nonenveloped capsid. The replication and assembly of reoviruses are thought to take place in distinct cytoplasmic inclusion bodies called viral factories (VFs) (33). The matrix of these structures is formed by the nonstructural viral protein NS (5). The factories are not static elements but can fuse with other viral factories in the same infected cell (J. S. L. Parker, unpublished findings). During the course of infection, other viral proteins are recruited to the viral factories at distinct times (3,8,28). By thinsection electron microscopy, the matrix of viral factories appears to consist of fibrils that have a distinct kink (9, 10, 35). However, no atomic resolution structure of NS is available. If expressed alone, without other viral proteins, the 80-kDa NS protein forms viral factory-like structures (VFLs) that resemble VFs in infected cells (5). The carboxyl-terminal (C-terminal) one-third of NS, comprising amino acids (aa) 471 to 721, is sufficient for VFL formation (2). This minimal factory-forming region has two predicted coiled-coil domains linked by a putative zinc hook and followed by a short C-terminal tail (26). The first one-third of NS (aa 1 to 221) has been identified as a scaffold for the recruitment of the viral proteins 1, 2, 2, 2, and NS; in contrast, the RNA-dependent RNA polymerase (RdRp), 3, interacts with the C-terminal minimal factory-forming region (5, 27, 28). So far, no function has been elucidated for the middle ...

Xanthomonas campestris pv. pelargonii (Xcp) and Ralstonia solanacearum (Rs) are the two most important bacterial pathogens of commercially cultivated geraniums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition, we designed a new primer pair specific for Rs that amplifies a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process, a negative PCR could result from either an accurate indication that the plant was pathogen‐free or from a false negative assay. We therefore designed `amplification competence' primers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494‐bp amplification product that confirms amplification competence and validates a negative assay result. Thus, the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample.