Reovirus-induced G(2)/M cell cycle arrest requires sigma1s and occurs in the absence of apoptosis

Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G(2)/M cell cycle arrest. Using T1L x T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G(2)/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein sigma1 and the nonstructural protein sigma1s. A sigma1s-deficient reovirus strain, T3C84-MA, fails to induce G(2)/M arrest, yet retains the capacity to induce apoptosis, indicating that sigma1s is required for reovirus-induced G(2)/M arrest. Expression of sigma1s in C127 cells increases the percentage of cells in the G(2)/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G(2)/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G(2)/M arrest, indicating that G(2)/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: role of the S1 and M2 genes.

The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle.     

Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.     

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis

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 σ1 and membrane penetration protein μ1, 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 reovirus-induced 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 σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent 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 μ1-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 σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.     

Activation of Polo-like kinase 3 by hypoxic stresses

Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl(2). Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells.     

Genetic mapping of reovirus virulence and organ tropism in severe combined immunodeficient mice: organ-specific virulence genes.

We used reovirus reassortant genetics and severe combined immunodeficient (SCID) mice to define viral genes important for organ tropism and virulence in the absence of antigen-specific immunity. Adult SCID mice infected with reovirus serotype 1 strain Lang (T1L) died after 20 +/- 6 days, while infection with serotype 3 strain Dearing (T3D) was lethal after 77 +/- 22 days. One hundred forty-five adult SCID mice were infected with T1L, T3D, and 25 different T1L x T3D reassortant reoviruses, and gene segments associated with the increased virulence of T1L were identified. Gene segments S1, L2, M1, and L1 accounted for > 90% of the genetically determined increase in T1L virulence. Gene segment M1 was independently important for virulence, with S1, L2, and L1 alone or in combination also playing a role. T1L grew to higher titers in multiple organs and caused more severe hepatitis than T3D. Seventy adult SCID mice, T1L, T3D, and 15 T1L x T3D reassortant viruses were used to map genetic determinants of viral titers in the brain, intestines, and liver, as well as the severity of hepatitis. Different sets of gene segments were important for determining viral titers in different organs. Gene segments L1 (encoding a core protein) and L2 (encoding the core spike of the virion) were important in all of the organs analyzed. The M1 gene segment (encoding a core protein), but not the S1 gene segment, was a critical determinant of reovirus titer in the liver and severity of hepatitis. The S1 gene segment (encoding the viral cell attachment protein and a nonstructural protein), but not the M1 gene segment, was a critical determinant of titers in intestines and brains. These studies demonstrate that viral growth in different organs is dependent on different subsets of the genes important for virulence. The virion-associated protein products of the four gene segments (L1, L2, M1, and S1) important for virulence and organ tropism in SCID mice likely form a structural unit, the reovirus vertex. Organs (the brain and intestines versus the liver) differ in properties that determine which virulence genes, and thus which parts of this structural unit, are important.     

Activation of the JNK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on CXCR4 and CD74

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.     

Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by reovirus T3D

Reovirus T3D is an oncolytic agent that preferentially targets tumor cells expressing an activated Ras oncogene. Ras signaling interferes with the cellular stress response that inhibits translation of reovirus RNAs. Murine C26 colorectal carcinoma cells express a mutant KrasD12 gene. Reovirus T3D efficiently kills C26 cells, but not C26 cells in which the KrasD12 mRNA is stably repressed by expression of KrasD12-directed short-hairpin RNAs. Surprisingly, neither reovirus T3D protein synthesis nor T3D virus yields were suppressed by deletion of KrasD12. Rather, reovirus-induced tumor cell apoptosis was completely abrogated as a result of Kras knockdown. We conclude that sensitization of C26 tumor cells to reovirus-induced apoptosis underlies the Ras dependency of reovirus T3D oncolysis.     

Daxx Upregulation within the Cytoplasm of Reovirus-Infected Cells Is Mediated by Interferon and Contributes to Apoptosis

Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm.     

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.     

Hyaluronidase activation of c-Jun N-terminal kinase is necessary for protection of L929 fibrosarcoma cells from staurosporine-mediated cell death

Hyaluronidase counteracts the growth inhibitory function of transforming growth factor beta (TGF-beta), whereas secretion of autocrine TGF-beta and hyaluronidase is necessary for progression and metastasis of various cancers. Whether hyaluronidase and TGF-beta1 induce resistance to staurosporine in L929 fibrosarcoma cells was investigated. When pretreated with TGF-beta1 for 1-2 h, L929 cells resisted staurosporine apoptosis. In contrast, without pretreatment, hyaluronidase protected L929 cells fromstaurosporine apoptosis. Hyaluronidase rapidly activated p42/44 MAPK (or ERK) in L929 cells and TGF-beta1 retarded the activation. Nonetheless, TGF-beta1 synergistically increased hyaluronidase-mediated inhibition of staurosporine apoptosis. Hyaluronidase rapidly activated c-Jun N-terminal kinase (JNK1 and JNK2) in L929 cells in 20 min. Dominant negative JNK1, JNK2, and JNK3 abolished the hyaluronidase inhibition of staurosporine apoptosis, but not the TGF-beta1 protective effect. Unlike the resistance to staurosporine, pretreatment of L929 cells with hyaluronidase is necessary to generate resistance to other anticancer drugs, including doxorubicin, daunorubicin, actinomycin D, and camptothecin, and the induced resistance was also blocked by dominant-negative JNKs. Together, hyaluronidase-mediated JNK activation is necessary to generate resistance to various anticancer drugs in L929 cells.     

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.     

Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors

The cellular pathways of apoptosis have not been fully characterized; however, calpain, a cytosolic calcium-activated cysteine protease, has been implicated in several forms of programmed cell death. Reoviruses induce apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of calpain in reovirus-induced apoptosis in vitro by measuring calpain activity as well as evaluating the effects of calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and calpain activity, measured as cleavage of the fluorogenic calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of apoptosis by calpain inhibitors is independent of effects on viral replication. We conclude that calpain activation is a component of the regulatory cascade in reovirus-induced apoptosis.