Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.     

Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Characterization of an in vitro model of alphavirus infection of immature and mature neurons

Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.     

Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide

Sindbis virus (SV) causes acute encephalomyelitis by infecting and inducing the death of neurons. Induction of apoptosis occurs during virus entry and involves acid-induced conformational changes in the viral surface glycoproteins and sphingomyelin (SM)-dependent fusion of the virus envelope with the endosomal membrane. We have studied neuroblastoma cells to determine how this entry process triggers cell death. Acidic sphingomyelinase was activated during entry followed by activation of neutral sphingomyelinase, SM degradation, and a sustained increase in ceramide. Ceramide-induced apoptosis and SV-induced apoptosis could be inhibited by treatment with Z-VAD-fmk, a caspase inhibitor, and by overexpression of Bcl-2, an antiapoptotic cellular protein. Acid ceramidase, expressed in a recombinant SV, decreased intracellular ceramide and protected cells from apoptosis. The data suggest that acid-induced SM-dependent virus fusion initiates the apoptotic cascade by inducing SM degradation and ceramide release.     

BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

Acid sphingomyelinase deficiency increases susceptibility to fatal alphavirus encephalomyelitis

Sindbis virus (SV), an enveloped virus with a single-stranded, plus-sense RNA genome, is the prototype alphavirus in the Togaviridae family. In mice, SV infects neurons and can cause apoptosis of immature neurons. Sphingomyelin (SM) is the most prevalent cellular sphingolipid, is particularly abundant in the nervous systems of mammals, and is required for alphavirus fusion and entry. The level of SM is tightly regulated by sphingomyelinases. A defect in acid sphingomyelinase (ASMase) results in SM storage and subsequent intracellular accumulation of SM. To better understand the role of the SM pathway in SV pathogenesis, we have characterized SV infection of transgenic mice deficient in the ASMase gene. ASMase knockout (ASM-KO) mice were more susceptible to SV infection than wild-type (WT) or heterozygous (Het) animals. Titers of SV were higher in the brains of ASM-KO mice than in the brains of WT mice. More SV RNA was detected by in situ hybridization, more SV protein was detected by immunohistochemistry, and more terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were present in the cortex and hippocampus of ASM-KO mice than in those of WT or Het mice. Interleukin-6 (IL-6), but not IL-1beta or tumor necrosis factor alpha, was elevated in infected ASM-KO mice compared to levels in WT or Het mice, but studies with IL-6-KO mice and recombinant SV expressing IL-6 showed no role for IL-6 in fatal disease. Together these data indicate that the increase in susceptibility of ASM-KO mice to SV infection was the result of more-rapid replication and spread of SV in the nervous system and increased neuronal death.     

Temporal changes in chromatin, intracellular calcium, and poly(ADP-ribose) polymerase during Sindbis virus-induced apoptosis of neuroblastoma cells.

Sindbis virus (SV) induces apoptosis in many vertebrate cells, but the mechanism is unknown. To gain insight into this mechanism, the nature and time course of intracellular changes related to programmed cell death were studied in SV-infected mouse neuroblastoma cells. New virus production began at 5 h after infection and reach a peak at 12 h. Hoechst 33342 staining of DNA analyzed by flow cytometry demonstrated changes in chromatin beginning 6 h after infection. These chromatin changes were cell cycle dependent, affecting cells in G0/G1 but not S phase. Apoptosis was not dependent on increases in intracellular Ca2+ and occurred more rapidly in the absence of extracellular Ca2+. Nuclear changes were accompanied by activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), resulting in increased consumption of NAD which was apparent by 10 h after infection. SV-induced apoptosis also involved the proteolytic cleavage of PARP. This cleavage was detectable at 16 h after infection approximately the same time that DNA fragmentation was apparent by agarose gel electrophoresis. We conclude that SV-induced apoptosis of neuroblastoma cells is dependent on viral replication, is not dependent on a rise in intracellular Ca2+, and is accompanied by activation of PARP and of a protease that cleaves PARP.     

Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.     

Spermine-induced toxicity in cerebellar granule neurons is independent of its actions at NMDA receptors

The neurotoxic actions of polyamines such as spermine have been linked to their modulation of NMDA receptors, resulting in an excitotoxic cell death. Here, we demonstrate that chronic exposure to the polyamine spermine and acute exposure to the combination of spermine and glutamate result in significant toxicity to primary cultures of cerebellar granule neurons (CGNs). However, in both cases this cell death (a) lacks the characteristic cell swelling associated with the necrotic cell death induced by glutamate and (b) is characterized by the widespread formation of apoptotic nuclei. Whereas dizocilpine (MK-801) blocks the synergistic cell death resulting from acute exposure to spermine plus glutamate, neither MK-801 nor the calcium chelator EGTA appreciably attenuates CGN death resulting from chronic exposure to spermine. Consistent with previous reports, glutamate, both acute and chronic, causes CGN death that is characterized by cell swelling, sensitivity to MK-801 and EGTA, and only small numbers of apoptotic nuclei. Spermine-induced toxicity is not blocked by either the protein synthesis inhibitor cycloheximide or the pancaspase inhibitor tert-butoxycarbonyl-Asp-(O-methyl) fluoromethyl ketone. However, the antioxidant butylated hydroxyanisole is an effective blocker of spermine-induced CGN death, suggesting a free-radical component to this cell death. The intact spermine molecule, rather than a catabolic by-product, is required for cell death because the amine oxidase inhibitors N1,N2-bis(2,3-butadienyl)-1,4-butanediamine and aminoguanidine fail to block this toxicity. Thus, in CGNs, spermine-induced toxicity does not occur by its modulation of NMDA receptors, although, under some circumstances, NMDA receptor activation can modulate spermine-induced toxicity.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

vFLIP protects PC-12 cells from apoptosis induced by Sindbis virus: implications for the role of TNF-alpha.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor alpha (TNF-alpha), and pre-treatment of the cells with an anti-TNF-alpha blocking antibody or with soluble TNF-alpha receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-alpha R1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-alpha plays an important role in the apoptotic response.     

Sendai virus envelopes can mediate Epstein-Barr virus binding to and penetration into Epstein-Barr virus receptor-negative cells

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition, Clostridium perfringens neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with C. perfringens neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.     

Promoted cell death of cells expressing human MxA by influenza virus infection

Interferon-inducible MxA protein plays a crucial role in cellular protection from RNA virus infection, although the protection mechanism is not completely clarified. Here, we examined effects of MxA on either uninfected or influenza virus A/PR/8/34-infected cells. Viral protein synthesis was reduced in cells expressing MxA. Under serum-starved conditions, not only viral but also cellular protein synthesis was reduced by expression of MxA. Of interest is that MxA promoted cell death induced by apoptotic stimuli as well as influenza virus infection. These results lead to a possibility that MxA suppresses multiplication of influenza virus by affecting cellular functions including the apoptotic pathway.     

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.     

Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent

Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, virus infections that result in neuronal depletion, either by virus-mediated cell death or by induction of the cytolytic immune response, could cause permanent neurological impairment of the host. In a transgenic mouse model of measles virus (MV) infection of neurons, we have previously shown that the host T-cell response was required for resolution of infection in susceptible adult mice. In this report, we show that this protective response did not result in neuronal death, even during the peak of T-cell infiltration into the brain parenchyma. When susceptible mice were intercrossed with specific immune knockout mice, a critical role for gamma interferon (IFN-gamma) was identified in protection against MV infection and CNS disease. Moreover, the addition of previously activated splenocytes or recombinant murine IFN-gamma to MV-infected primary neurons resulted in the inhibition of viral replication in the absence of neuronal death. Together, these data support the hypothesis that the host immune response can promote viral clearance without concomitant neuronal loss, a process that appears to be mediated by cytokines.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.