Coronal sections of neonatal mouse brain 7 days after intracranial inoculation of 10,000 plaque forming units (PFU) of reovirus strain T3D or mock inoculation. Hematoxylin and eosin-stained tissue reveals marked destruction of brain tissue in the T3D-infected brain ( B ) as compared to the uninfected brain ( A ). By immunohis- tochemistry, serial sections of T3D-infected brain tissue were stained for viral antigen ( C ), TUNEL/apoptosis marker ( D ), and active caspase 3 ( E ). Staining for viral antigen, TUNEL, and caspase 3 were undetectable in the mock-infected brains (data not shown). (With permission from Richardson-Burns et al. 2002) 

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... (Diaz-Meco et al. 1999). Reovirus infection results in a viral strain-specific pattern activation of the c-Jun N-terminal kinase (JNK) and the JNK-associated transcription factor c-Jun (Clarke et al. 2001b). The capacity of reovirus strains to activate JNK correlates closely with their capacity to induce apoptosis (Clarke et al. 2001b). In addition, experiments using T1L T3D reassortants indicate that the same viral gene segments that determine apoptosis induction (S1 and M2) are also key determinants of JNK activation (Clarke et al. 2001b). Furthermore, our preliminary experiments indicate that reovirus-induced apoptosis is inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells and in cells treated with inhibitors of JNK activity. These results indicate that JNK is required for reovirus-induced apoptosis. Our recent experiments also indicate that JNK is required for the efficient release of smac and cytochrome c from the mitochondria of reovirus-infected cells, suggesting that JNK promotes mitochondrial pathways of apoptosis in reovirus-infected cells (Clarke et al. 2004). Both JNK-induced phosphorylation of Bcl-2 family proteins and c-Jun-in- duced expression of the BH3-only protein Bim have previously been shown to promote mitochondrial apoptotic signaling. Experiments to determine the mechanism by which JNK and c-Jun influence reovirus- induced apoptosis are currently under way. Reovirus infection induces the activation of transcription factors NF- k B and c-Jun (Clarke et al. 2001b, 2003a; Connolly et al. 2000). This suggests that activation of specific cellular genes contributes to virus-induced cellular signaling, including apoptotic signaling, in infected cells. High- density oligonucleotide microarrays used to perform a global analysis of virus-induced cellular gene expression after reovirus infection of HEK293 cells (DeBiasi et al. 2003; Poggioli et al. 2002) showed that the expression of 24 genes related to apoptosis were altered in cells infected with the apoptosis-inducing reovirus strain T3A (Table 1). These genes encode proteins with potential roles in DR, endoplasmic reticulum stress, and mitochondrial apoptotic signaling and cysteine proteases (caspases and calpains) (DeBiasi et al. 2003). Only five of these genes were also differentially expressed after T1L (weakly apoptotic) infection, emphasizing their potential importance in reovirus-induced apoptosis. To date the best-characterized example of a potentially apoptosis-inducing gene identified by microarray analysis to be differentially expressed after reovirus infection is the survival motor neuron (SMN) gene. This gene was found by polymerase chain reaction (PCR) analysis to be upregulated at the transcriptional level in reovirus-infected HEK293 cells and at the translational level in the hearts of reovirus-infected baby mice (DeBiasi et al. 2003). The SMN protein has been shown to interact with Bcl-2, conferring a synergistic protective effect against Bax-induced or Fas-mediated apoptosis that has been shown to underlie the pathogenesis of spinal muscular atrophy (Sato et al. 2000; Iwahashi et al. 1997). Changes in the expression of genes encoding proteins known to be involved in DNA repair and cell cycle regulation were also identified in this study and may also affect virus-induced pathogenesis (DeBiasi et al. 2003). In addition to inducing TRAIL-mediated apoptosis, reovirus infection also sensitizes cells to TRAIL-induced apoptosis by a mechanism that results in an increase in the activation of caspases 8 and 3 and is blocked by the caspase 8 inhibitor IETD-FMK (Clarke et al. 2000, 2001a). Reovirus infection and TRAIL treatment have synergistic rather than merely additive effects on apoptosis, and infection can confer TRAIL sensitivity to previously TRAIL-resistant cells as well as increasing the TRAIL sensitivity of partially resistant lines (Clarke et al. 2000, 2001a). This finding may increase the potential utility of TRAIL as an agent for cancer thera- py, which is currently limited by the fact that cancer cells of all types differ in sensitivity to TRAIL-induced apoptosis. The ability of reovirus to sensitize cells to TRAIL does not appear to reflect an increase in the expression of TRAIL receptors as assayed in several human cancer cell lines (Clarke et al. 2001a) and may instead be the result of inhibition of TRAIL-induced activation of NF- k B in reovirus-infected cells (Clarke et al. 2003a). The ability of reovirus to sensitize cells to TRAIL also suggests that reovirus-infected cells in vivo are also susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4 + cells that bear membrane-bound TRAIL. T3 reovirus strains infect neurons within specific regions of neonatal mouse brains, producing a lethal meningoencephalitis. Viral antigen and pathology colocalize in the brain and have a predilection for the cortex, hippocampus and thalamus (Fig. 2). T3 reovirus infection also induces apoptosis in the brains of newborn mice (Oberhaus et al. 1997; Richardson-Burns et al. 2002). Thus fragmentation of DNA into oligonu- cleosomal-length ladders can be detected in tissue samples prepared from T3D- but not mock-infected brains at 8–9 days PI, which coincides with maximal viral growth (Oberhaus et al. 1997). The presence of apoptotic cells also correlates with areas of tissue injury and viral infection in T3-infected brain sections (Fig. 2) (Oberhaus et al. 1997; Richardson- Burns et al. 2002). Most cells in infected brain regions are both TUNEL (TdT-mediated dUTP nick-end labeling)-positive (apoptotic) and reovirus antigen-positive (infected). However, there are cells in these regions that are apoptotic but antigen negative, suggesting that apoptosis occurs both as a result of direct viral infection and in uninfected “bystander” cells (Oberhaus et al. 1997). Reovirus infection in a mouse neuroblasto- ma-derived cell line (NB41a3) and in primary mouse cortical cultures (MCC) derived from embryonic (E20) mice is also associated with increased levels of caspase 3 activity and is blocked with the caspase 3 inhibitor DEVD-FMK (Richardson-Burns et al. 2002). Studies of reovirus infection in neuronal cultures also provides further evidence of bystander apoptosis. In both MCC and NB4 cells dual labeling with immunocy- tochemistry and TUNEL showed that although a great majority of infected cells were undergoing apoptosis there was also a subset of apoptotic cells that were uninfected but located in proximity to virus-infected cells (Richardson-Burns et al. 2002). Bystander apoptosis could result from the release of TRAIL, or other death ligands, from reovirus-infected cells. If this is the case, the amount of bystander apoptosis would reflect the sensitivity of the surrounding cells to the released ligand. Reovirus infection also induces increased caspase 8 activation in infected neurons, indicating that neuronal apoptosis, like that in its epi- thelial cell counterparts, involves DR activation (Richardson-Burns et al. 2002). However, the ligand-receptor trigger for this activation appears to be less specific. Thus, whereas reovirus-induced apoptosis in HEK293 cells is selectively inhibited by blocking TRAIL ligand-receptor interac- tion, reovirus-induced apoptosis in NB4 cells is inhibited by treating cells with both soluble TRAIL receptors (Fc:DR5) and soluble TNF receptors (TNFR) (FcTNFR-1), and reovirus-induced apoptosis in MCCs is inhibited by Fc:TNFR-1 and Fc:FasL. Mitochondrial apoptotic pathways are also activated after reovirus infection of neurons. Preliminary studies indicate that proapoptotic Bcl-2 family proteins, including Bid, Bax, and Bim, are activated in virus-infected neurons, resulting in the release of proapoptotic mitochondrial factors. However, there are again differences between mitochondrial signaling pathways activated after reovirus infection of neuronal and epi- thelial cells. In HEK293 cells, reovirus infection is associated with robust release of cytochrome c and smac and the subsequent activation of caspase 9 and inhibition of cellular IAPs. In neuronal cultures, however, our preliminary results indicate that reovirus infection results in the discor- dant release of smac and cytochrome c . Smac is released around 17 h PI and coincides with the cleavage of cellular IAPs. In contrast, cytochrome c release occurs only at low levels and at later times after infection, resulting in only low levels of activation of caspase 9 in these cells (Richardson-Burns et al. 2002). Consistent with these findings, the caspase 9 inhibitor Z-LEHD-FMK has little effect on reovirus-induced neuronal apoptosis, which is significantly inhibited by caspase 8 (Z-IETD-FMK), caspase 3 (Z-DEVD-FMK), or pan-caspase inhibitors. The T1L T3D reassortant virus 8B efficiently produces myocarditis in infected neonatal mice. Similar to results seen in mouse brain, DNA ex- tracted from the hearts of 8B-infected mice is fragmented into oligonu- cleosomal-length ladders, indicative of apoptosis (DeBiasi et al. 2001) and areas of TUNEL-positive cells in 8B-infected hearts correlates with areas of histological damage and reovirus antigen (DeBiasi et al. 2001). Injury to the heart following reovirus infection occurrs in the absence of an inflammatory response, also suggesting that it results from apoptotic cell death (DeBiasi et al. 2001). Treatment of mice with the calpain inhibitor CX295 [dipeptide a -ke- toamide calpain inhibitor z-Leu-aminobutyric acid-CONH(CH 2 )-3-mor- pholine] is protective against reovirus-induced myocarditis and results in a dramatic reduction in histopathologic evidence of myocardial injury (Fig. 3), a reduction in serum creatine phosphokinase (an intracellular enzyme whose release into the serum is a quantitative marker of skeletal and cardiac muscle damage), and improved weight gain (DeBiasi et al. 2001). Prevention of myocardial injury ...

Context 2

... determinants of JNK activation (Clarke et al. 2001b). Furthermore, our preliminary experiments indicate that reovirus-induced apoptosis is inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells and in cells treated with inhibitors of JNK activity. These results indicate that JNK is required for reovirus-induced apoptosis. Our recent experiments also indicate that JNK is required for the efficient release of smac and cytochrome c from the mitochondria of reovirus-infected cells, suggesting that JNK promotes mitochondrial pathways of apoptosis in reovirus-infected cells (Clarke et al. 2004). Both JNK-induced phosphorylation of Bcl-2 family proteins and c-Jun-in- duced expression of the BH3-only protein Bim have previously been shown to promote mitochondrial apoptotic signaling. Experiments to determine the mechanism by which JNK and c-Jun influence reovirus- induced apoptosis are currently under way. Reovirus infection induces the activation of transcription factors NF- k B and c-Jun (Clarke et al. 2001b, 2003a; Connolly et al. 2000). This suggests that activation of specific cellular genes contributes to virus-induced cellular signaling, including apoptotic signaling, in infected cells. High- density oligonucleotide microarrays used to perform a global analysis of virus-induced cellular gene expression after reovirus infection of HEK293 cells (DeBiasi et al. 2003; Poggioli et al. 2002) showed that the expression of 24 genes related to apoptosis were altered in cells infected with the apoptosis-inducing reovirus strain T3A (Table 1). These genes encode proteins with potential roles in DR, endoplasmic reticulum stress, and mitochondrial apoptotic signaling and cysteine proteases (caspases and calpains) (DeBiasi et al. 2003). Only five of these genes were also differentially expressed after T1L (weakly apoptotic) infection, emphasizing their potential importance in reovirus-induced apoptosis. To date the best-characterized example of a potentially apoptosis-inducing gene identified by microarray analysis to be differentially expressed after reovirus infection is the survival motor neuron (SMN) gene. This gene was found by polymerase chain reaction (PCR) analysis to be upregulated at the transcriptional level in reovirus-infected HEK293 cells and at the translational level in the hearts of reovirus-infected baby mice (DeBiasi et al. 2003). The SMN protein has been shown to interact with Bcl-2, conferring a synergistic protective effect against Bax-induced or Fas-mediated apoptosis that has been shown to underlie the pathogenesis of spinal muscular atrophy (Sato et al. 2000; Iwahashi et al. 1997). Changes in the expression of genes encoding proteins known to be involved in DNA repair and cell cycle regulation were also identified in this study and may also affect virus-induced pathogenesis (DeBiasi et al. 2003). In addition to inducing TRAIL-mediated apoptosis, reovirus infection also sensitizes cells to TRAIL-induced apoptosis by a mechanism that results in an increase in the activation of caspases 8 and 3 and is blocked by the caspase 8 inhibitor IETD-FMK (Clarke et al. 2000, 2001a). Reovirus infection and TRAIL treatment have synergistic rather than merely additive effects on apoptosis, and infection can confer TRAIL sensitivity to previously TRAIL-resistant cells as well as increasing the TRAIL sensitivity of partially resistant lines (Clarke et al. 2000, 2001a). This finding may increase the potential utility of TRAIL as an agent for cancer thera- py, which is currently limited by the fact that cancer cells of all types differ in sensitivity to TRAIL-induced apoptosis. The ability of reovirus to sensitize cells to TRAIL does not appear to reflect an increase in the expression of TRAIL receptors as assayed in several human cancer cell lines (Clarke et al. 2001a) and may instead be the result of inhibition of TRAIL-induced activation of NF- k B in reovirus-infected cells (Clarke et al. 2003a). The ability of reovirus to sensitize cells to TRAIL also suggests that reovirus-infected cells in vivo are also susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4 + cells that bear membrane-bound TRAIL. T3 reovirus strains infect neurons within specific regions of neonatal mouse brains, producing a lethal meningoencephalitis. Viral antigen and pathology colocalize in the brain and have a predilection for the cortex, hippocampus and thalamus (Fig. 2). T3 reovirus infection also induces apoptosis in the brains of newborn mice (Oberhaus et al. 1997; Richardson-Burns et al. 2002). Thus fragmentation of DNA into oligonu- cleosomal-length ladders can be detected in tissue samples prepared from T3D- but not mock-infected brains at 8–9 days PI, which coincides with maximal viral growth (Oberhaus et al. 1997). The presence of apoptotic cells also correlates with areas of tissue injury and viral infection in T3-infected brain sections (Fig. 2) (Oberhaus et al. 1997; Richardson- Burns et al. 2002). Most cells in infected brain regions are both TUNEL (TdT-mediated dUTP nick-end labeling)-positive (apoptotic) and reovirus antigen-positive (infected). However, there are cells in these regions that are apoptotic but antigen negative, suggesting that apoptosis occurs both as a result of direct viral infection and in uninfected “bystander” cells (Oberhaus et al. 1997). Reovirus infection in a mouse neuroblasto- ma-derived cell line (NB41a3) and in primary mouse cortical cultures (MCC) derived from embryonic (E20) mice is also associated with increased levels of caspase 3 activity and is blocked with the caspase 3 inhibitor DEVD-FMK (Richardson-Burns et al. 2002). Studies of reovirus infection in neuronal cultures also provides further evidence of bystander apoptosis. In both MCC and NB4 cells dual labeling with immunocy- tochemistry and TUNEL showed that although a great majority of infected cells were undergoing apoptosis there was also a subset of apoptotic cells that were uninfected but located in proximity to virus-infected cells (Richardson-Burns et al. 2002). Bystander apoptosis could result from the release of TRAIL, or other death ligands, from reovirus-infected cells. If this is the case, the amount of bystander apoptosis would reflect the sensitivity of the surrounding cells to the released ligand. Reovirus infection also induces increased caspase 8 activation in infected neurons, indicating that neuronal apoptosis, like that in its epi- thelial cell counterparts, involves DR activation (Richardson-Burns et al. 2002). However, the ligand-receptor trigger for this activation appears to be less specific. Thus, whereas reovirus-induced apoptosis in HEK293 cells is selectively inhibited by blocking TRAIL ligand-receptor interac- tion, reovirus-induced apoptosis in NB4 cells is inhibited by treating cells with both soluble TRAIL receptors (Fc:DR5) and soluble TNF receptors (TNFR) (FcTNFR-1), and reovirus-induced apoptosis in MCCs is inhibited by Fc:TNFR-1 and Fc:FasL. Mitochondrial apoptotic pathways are also activated after reovirus infection of neurons. Preliminary studies indicate that proapoptotic Bcl-2 family proteins, including Bid, Bax, and Bim, are activated in virus-infected neurons, resulting in the release of proapoptotic mitochondrial factors. However, there are again differences between mitochondrial signaling pathways activated after reovirus infection of neuronal and epi- thelial cells. In HEK293 cells, reovirus infection is associated with robust release of cytochrome c and smac and the subsequent activation of caspase 9 and inhibition of cellular IAPs. In neuronal cultures, however, our preliminary results indicate that reovirus infection results in the discor- dant release of smac and cytochrome c . Smac is released around 17 h PI and coincides with the cleavage of cellular IAPs. In contrast, cytochrome c release occurs only at low levels and at later times after infection, resulting in only low levels of activation of caspase 9 in these cells (Richardson-Burns et al. 2002). Consistent with these findings, the caspase 9 inhibitor Z-LEHD-FMK has little effect on reovirus-induced neuronal apoptosis, which is significantly inhibited by caspase 8 (Z-IETD-FMK), caspase 3 (Z-DEVD-FMK), or pan-caspase inhibitors. The T1L T3D reassortant virus 8B efficiently produces myocarditis in infected neonatal mice. Similar to results seen in mouse brain, DNA ex- tracted from the hearts of 8B-infected mice is fragmented into oligonu- cleosomal-length ladders, indicative of apoptosis (DeBiasi et al. 2001) and areas of TUNEL-positive cells in 8B-infected hearts correlates with areas of histological damage and reovirus antigen (DeBiasi et al. 2001). Injury to the heart following reovirus infection occurrs in the absence of an inflammatory response, also suggesting that it results from apoptotic cell death (DeBiasi et al. 2001). Treatment of mice with the calpain inhibitor CX295 [dipeptide a -ke- toamide calpain inhibitor z-Leu-aminobutyric acid-CONH(CH 2 )-3-mor- pholine] is protective against reovirus-induced myocarditis and results in a dramatic reduction in histopathologic evidence of myocardial injury (Fig. 3), a reduction in serum creatine phosphokinase (an intracellular enzyme whose release into the serum is a quantitative marker of skeletal and cardiac muscle damage), and improved weight gain (DeBiasi et al. 2001). Prevention of myocardial injury by apoptosis inhibitors is accompa- nied by a virtually complete inhibition of apoptotic myocardial cell death, strongly suggesting that virus-induced apoptosis is a key mechanism of cell death, tissue injury, and mortality in reovirus-infected mice and that inhibitors of apoptosis may prove useful in the treatment of virus-induced diseases (DeBiasi et al. 2001). Early studies showed that there is little correlation in continuous non- neuronal cell lines between the efficiency with which ...