Avian reovirus S1133-induced apoptosis is associated with Bip/GRP79-mediated Bim translocation to the endoplasmic reticulum.

In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.

Viruses, apoptosis, and neuroinflammation--a double-edged sword.

Apoptosis, or programmed cell death, is a fundamental and widespread cell biological process that is distinct from cell necrosis and can be induced by a wide variety of stimuli including viral infections. Apoptosis may occur via either the intrinsic or extrinsic pathways and confers several advantages to the virally infected host including the prevention of further viral propagation and the potential inhibition and resolution of inflammatory processes. Several viruses have been shown to have the capacity to induce apoptosis in susceptible cells including herpes simplex virus, Varicella-zoster virus, rabies virus, human immunodeficiency virus, and reovirus. Apoptosis has also been observed in human African trypanosomiasis which is an infection caused by a protozoan parasite. The mechanisms leading to apoptosis may differ depending on the type of infection. Apoptosis has been reported in several neurodegenerative diseases and also psychiatric disorders but the true clinical significance of such observations is not certain, and, though interesting, it is very difficult to ascribe causation in these conditions. The presence of inflammation in the central nervous system in any neurological condition, including those associated with a viral infection, is not necessarily an absolute marker of serious disease and the notion of 'good' versus 'bad' inflammation is considered to be valid in some circumstances. The precise relationship between viruses, apoptosis, and inflammation is viewed as a complex one requiring further investigation to unravel and understand its nature.

Nuclear localization of the p17 protein of avian reovirus is correlated with autophagy induction and an increase in viral replication.

p17 is a nonstructural protein of avian reovirus (ARV) that induces autophagy in infected cells. In the present study, we investigated the effect of p17 and its nuclear localization signal (NLS) on autophagy and viral replication. When Vero cells and DF1 cells were transfected with mutant p17 in which lysine (K) at position 122 and arginine (R) at position 123 were mutated to alanine (A), the expression level of LC3 II decreased dramatically after transfection. The expression of the polypeptide encompassing the first 103 amino acids of p17, a region that did not contain the NLS, did not have a significant effect on autophagy. Moreover, when cells overexpressing mutant p17 were infected with the ARV GX2010/1 strain, the viral titer was significantly decreased compared with the expression of wild-type p17. In general, the NLS of p17 facilitates the induction of autophagy and is correlated with an increase in virus production.

Autophagy inhibitors reduce avian-reovirus-mediated apoptosis in cultured cells and in chicken embryos.

Avian reovirus (ARV)-induced apoptosis contributes to the pathogenesis of reovirus in infected chickens. However, methods for effectively reducing ARV-triggered apoptosis remain to be explored. Here, we show that pretreatment with chloroquine (CQ) or E64d plus pepstatin A decreases ARV-mediated apoptosis in chicken DF-1 cells. By acting as autophagy inhibitors, CQ and E64d plus pepstatin A increase microtubule-associated protein 1 light chain 3-II (LC3II) accumulation in ARV-infected cells, which results in decreased ARV protein synthesis and virus yield and thereby contributes to the reduction of apoptosis. Furthermore, ARV-mediated apoptosis in the bursa, heart and intestines of chicken embryos is attenuated by CQ and E64d plus pepstatin A treatment. Importantly, treatment with these autophagy inhibitors increases the survival of infected chicken embryos. Together, our data suggest that pharmacological inhibition of autophagy might represent a novel strategy for reducing ARV-mediated apoptosis.

Critical role of eukaryotic elongation factor 1 alpha 1 (EEF1A1) in avian reovirus sigma-C-induced apoptosis and inhibition of viral growth.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. It is well established that the ARV sigma-C protein induces apoptosis in host cells. However, the underlying molecular mechanism of this induction is still unclear. We report here the identification of eukaryotic elongation factor 1 alpha 1 (EEF1A1) as the interacting partner of σC. We found that σC-induced apoptosis in DF-1 cells could be completely abolished by knockdown of EEF1A1 by siRNA. Furthermore, knockdown of EEF1A1 markedly reduced ARV-induced apoptosis associated with decreased caspase-9 and -3 activation and cytochrome C release, leading to increased ARV growth in host cells. Thus, EEF1A1 plays a critical role in σC-induced apoptosis and inhibition of viral growth.

Nonstructural protein σ1s mediates reovirus-induced cell cycle arrest and apoptosis.

Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.

Apoptosis induction influences reovirus replication and virulence in newborn mice.

Apoptosis is a type of controlled cell death that is essential for development and tissue homeostasis. It also serves as a robust host response against infection by many viruses. The capacity of neurotropic viruses to induce apoptosis strongly correlates with virulence. However, the precise function of apoptosis in viral infection is not well understood. Reovirus is a neurotropic virus that induces apoptosis in a variety of cell types, including central nervous system neurons, leading to fatal encephalitis in newborn mice. To determine the effect of apoptosis on reovirus replication in the host, we generated two otherwise isogenic viruses that differ in a single amino acid in viral capsid protein µ1 that segregates with apoptotic capacity. Apoptosis-proficient and apoptosis-deficient viruses were compared for replication, dissemination, tropism, and tissue injury in newborn mice and for the capacity to spread to uninfected littermates. Our results indicate that apoptotic capacity enhances reovirus replication in the brain and consequent neurovirulence but reduces transmission efficiency. The replication advantage of the apoptosis-proficient strain is limited to the brain and correlates with enhanced infectivity of neurons. These studies reveal a new cell type-specific determinant of reovirus virulence.

Epizootic hemorrhagic disease virus induces and benefits from cell stress, autophagy, and apoptosis.

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Avian reovirus S1133-induced DNA damage signaling and subsequent apoptosis in cultured cells and in chickens.

In this study, intracellular signaling in ARV S1133-mediated apoptosis was investigated. A microarray was used to examine the gene expression profiles of cells upon ARV S1133 infection and ARV-encoded pro-apoptotic protein σC overexpression. The analysis indicated that in the set of DNA-damage-responsive genes, DDIT-3 and GADD45α were both upregulated by viral infection and σC overexpression. Further investigation demonstrated that both treatments caused DNA breaks, which increased the expression and/or phosphorylation of DNA damage response proteins. ROS and lipid peroxidation levels were increased, and ARV S1133 and σC caused apoptosis mediated by DNA damage signaling. ROS scavenger NAC, caffeine and an ATM-specific inhibitor significantly reduced ARV S1133- and σC-induced DNA breaks, DDIT-3 and GADD45α expression, H2AX phosphorylation, and apoptosis. Overexpression of DDIT-3 and GADD45α enhanced the oxidative stress and apoptosis induced by ARV S1133 and σC. In conclusion, our results demonstrate the involvement of the DNA-damage-signaling pathway in ARV S1133- and σC-induced apoptosis.

Inhibition of cellular DNA synthesis by reovirus occurs through a receptor-linked signaling pathway that is mimicked by antiidiotypic, antireceptor antibody.

Mammalian reovirus type 3 binds to a 67-kD surface glycoprotein on the membrane of neuronal cells. This interaction initiates the infective reovirus cycle. The physiological function of this virus receptor is not known, however, initial studies illustrate a striking structural and antigenic homology to the beta adrenergic receptor family. The earliest known pathologic effect of reovirus type 3 is an inhibition of host cell DNA synthesis within 8-10 h after virus attachment. The studies reported here demonstrate that binding and aggregation of reovirus receptor molecules provides the signal for this inhibitory process. Inhibition of DNA synthesis in the neuroblastoma cell line B104.G4 was unaffected by using replication-defective virus or when lysosomal processing of normal virus was blocked. Inhibition was mimicked by an antiidiotypic, antireceptor mAb. Inhibition was not observed when monovalent mAb fragments were bound to receptors, but was reconstituted when these fragments were aggregated by the addition of anti-Ig. The signal for the inhibitory effect was generated within the first 60 min after mAb binding. These observations demonstrate that reovirus and antiidiotypic pathogenicity can result from the perturbation of membrane proteins that may perform normal physiological functions.

Assessment of the productivity effects associated with epizootic hemorrhagic disease in dairy herds.

Epizootic hemorrhagic disease is caused by a Culicoides-borne Orbivirus. In cattle, the disease is characterized by reduced milk production and mortality. Recent outbreaks of epizootic hemorrhagic disease virus (EHDV) in North Africa, Israel, and Turkey increase the risk of its invasion into central and northern Europe. An outbreak of EHDV in Israel during the fall of 2006 enabled an assessment of the consequent production losses to the dairy cattle industry. Reduction in milk production and involuntary culling were modeled using a 4-yr database of monthly milk and mortality records from 48 affected and 63 unaffected herds. These indices were compared between periods of outbreak and no outbreak and assessed for various levels and exposure onset. Geospatial kriging interpolation of serological results from 127 herds was used to assess the total outbreak losses for the dairy cattle industry in Israel. Herds affected during the first, second, and third month of the outbreak (September-November) experienced an average loss of 207 (95% CI=154-261), 137 (63-211), and 52 (27-76) kg of milk/milking cow, respectively, during the outbreak period. An average excess mortality and involuntary culling of 1.47/100 cows was documented in herds affected in September. High correlation was observed between EHDV seroprevalence and milk loss; average milk loss for herds with seropositivity of 26 to 50, 51 to 75, and 76 to 100% was 84, 133, and 204 kg of milk/milking cow, respectively. A 1.42% (0.91-1.93%) increase in mortality was observed in herds with seroprevalence above 50%. Losses for the dairy cattle industry interpolated from these data were estimated at US$2,491,000 (US$1,591,000-3,391,000), an average loss of US$26.5/cow in the Israeli dairy cattle. This equals 0.55% of the average total value production of a dairy cow in Israel. This is the first study to estimate the production losses caused by EHDV or any bluetongue-like disease.

Epizootic hemorrhagic disease in cattle, Western Turkey.

In 2007, an outbreak of epizootic hemorrhagic disease (EHD) occurred in Turkey. On the basis of clinical investigation, 41 cattle were suspected to have EHD. Reverse transcription-PCR and sequence analyses indicated that the virus belonged to EHD virus serotype 6, thus confirming EHD virus infection of cattle in Turkey.

Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental reovirus-induced acute flaccid paralysis and spinal motor neuron cell death.

Acute flaccid paralysis (AFP) describes the loss of motor function in 1 or more limbs commonly associated with viral infection and destruction of motor neurons in the anterior horns of the spinal cord. Therapy is limited, and the development of effective treatments is hampered by a lack of experimental models. Reovirus infection of neonatal mice provides a model for the study of CNS viral infection pathogenesis. Injection of the Reovirus serot Type 3 strains Abney (T3A) or Dearing (T3D) into the hindlimb of 1-day-old mice resulted in the development of AFP in more than 90% of infected mice. Acute flaccid paralysis began in the ipsilateral hindlimb at 8 to 10 days postinfection and progressed to paraplegia 24 hours later. Paralysis correlated with injury, neuron loss, and spread of viral antigen first to the ipsilateral and then to the contralateral anterior horns. As demonstrated by the activation of caspase 3 and its colocalization with viral antigen in the anterior horn and concomitant cleavage of poly-(adenosine diphosphate-ribose) polymerase, AFP was associated with apoptosis. Calpain activity and inducible nitric oxide synthase expression were both elevated in the spinal cords of paralyzed animals. This study represents the first detailed characterization of a novel and highly efficient experimental model of virus-induced AFP that will facilitate evaluation of therapeutic strategies targeting virus-induced paralysis.

Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease.

Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA- and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA- developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid-binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia.

The requirement of environmental acidification for Ibaraki virus infection to host cells.

The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. IBAV outer capsid proteins, VP5 and VP2, were removed from virion when purified IBAV was exposed to low pH environment. Further experiment showed that the exposure to low pH buffer facilitated IBAV infection when the cellular endosomal pathway was impaired by bafilomycin A1. Results obtained in this study suggest that acidic environment is essential to initiate IBAV infection.

Mechanisms of reovirus-induced cell death and tissue injury: role of apoptosis and virus-induced perturbation of host-cell signaling and transcription factor activation.

Reoviruses have provided insight into the roles played by specific viral genes and the proteins they encode in virus-induced cell death and tissue injury. Apoptosis is a major mechanism of cell death induced by reoviruses. Reovirus-induced apoptosis involves both death-receptor and mitochondrial cell death pathways. Reovirus infection is associated with selective activation of mitogen activated protein kinase (MAPK) cascades including JNK/SAPK. Infection also perturbs transcription factor signaling resulting in the activation of c-Jun and initial activation followed by strain-specific inhibition of NF-kappaB. Infection results in changes in the expression of genes encoding proteins involved in cell cycle regulation, apoptosis, and DNA damage and repair processes. Apoptosis is a major mechanism of reovirus-induced injury to key target organs including the CNS and heart. Inhibition of apoptosis through the use of caspase or calpain inhibitors, minocycline, or in caspase 3(-/-) mice all reduce virus-associated tissue injury and enhance survival of infected animals. Reoviruses induce apoptotic cell death (oncolysis) in a wide variety of cancer cells and tumors. The capacity of reoviruses to grow efficiently in transformed cells is enhanced by the presence of an activated Ras signaling pathway likely through mechanisms involving inhibition of antiviral PKR signaling and activation of Ras/RalGEF/p38 pathways. The potential of reovirus-induced oncolysis in therapy of human cancers is currently being investigated in phase I/II clinical trials.

Modulation of the immune response and tumor growth by activated Ras.

As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.

Tumor necrosis factor-alpha induction by reovirus serotype 3.

We have reported previously that reovirus, when used in combination with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) chemotherapy, mediates the rejection of murine ascites tumors. Surviving animals reject a challenge with the same, but not a different, tumor, which suggests that tumor-specific immunity is induced by the treatment regimen. The present study was designed to characterize the interaction between reovirus and murine peritoneal macrophages, both in vitro and in vivo, to determine whether such a relationship may play a role in immune modulation resulting in tumor rejection. The results demonstrated that reovirus can efficiently infect peritoneal macrophages in vitro and stimulate the secretion of tumor necrosis factor-alpha (TNF-alpha). In vivo administration of reovirus, however, did not produce high levels of infection in peritoneal exudate cells, even though the cells were stimulated to express detectable levels of membrane TNF-alpha. These results suggested that infection is not necessary for TNF-alpha expression and this hypothesis was supported by the observation that this expression was also stimulated in vitro by UV-inactivated reovirus. These findings suggest that one mechanism for immune stimulation by reovirus may be through the induction of TNF-alpha.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.