scholarly journals Calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis

2013 ◽  
Vol 94 (7) ◽  
pp. 1477-1485 ◽  
Author(s):  
Jia-Rong Lu ◽  
Wen-Wen Lu ◽  
Jian-Zhong Lai ◽  
Fu-Lian Tsai ◽  
Szu-Hsien Wu ◽  
...  
Keyword(s):  
Enterovirus 71 ◽  
Confocal Imaging ◽  
Ev71 Infection ◽  
Calcium Flux ◽  
Apoptotic Pathway ◽  
Fractionation Analysis ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Factor ◽  
The Impact

Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca2+)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca2+-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca2+ dynamics in the infected cells and found elevated levels of mitochondrial Ca2+. Treatment with ruthenium red, a mitochondrial Ca2+ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca2+ flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca2+ homeostasis on EV71 infection.

scholarly journals Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

2016 ◽  
Vol 90 (21) ◽  
pp. 9862-9877 ◽  
Author(s):  
Haolong Cong ◽  
Ning Du ◽  
Yang Yang ◽  
Lei Song ◽  
Wenliang Zhang ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Enterovirus 71 ◽  
Terminal Region ◽  
Ev71 Infection ◽  
Apoptotic Pathway ◽  
Hydrophobic Region ◽  
Mitochondrial Apoptotic Pathway ◽  
Proapoptotic Protein ◽  
2B Protein ◽  
Induced Apoptosis

ABSTRACTTo survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochromec(Cytc) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XLabrogates 2B-induced release of Cytcand caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax.IMPORTANCEEV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cytcrelease and mitochondrial permeabilization during EV71 infection.

VGLL4 Promotes EV71 Virus Proliferation and Accelerates the Apoptosis of Infected RD Cells

2021 ◽  
Author(s):  
Shengyu Wang ◽  
Xin Sun ◽  
Junhua Qiao ◽  
Cancan He ◽  
Langfei Tian ◽  
...  
Keyword(s):  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Eukaryotic Expression ◽  
Host Cells ◽  
Ev71 Infection ◽  
Western Blot Assay ◽  
Multifunctional Protein ◽  
Rd Cells ◽  
Infected Cells ◽  
Induced Apoptosis

Abstract Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot and mouth disease (HFMD) which affects public health increasingly. Apoptosis plays an important role in EV71 infection, but the molecular mechanism involved in EV71 induced apoptosis is not completely clear. VGLL4 is a multifunctional protein in host cells, which has been studied in tumor and cell apoptosis, but has not been reported in pathogen. In this study, the mammalian eukaryotic expression plasmid of VGLL4 fused with HA tag (HA-VGLL4) and the model of overexpression VGLL4 RD cells were successfully constructed. The effect of VGLL4 on the proliferation of EV71 was detected by western-blot assay, fluorescence quantitative PCR and cytotoxicity assay (CCK assay), and the mechanism of its effect on the proliferation of the virus was researched. The experimental results showed that VGLL4 may promote the replication of EV71 by promoting the apoptosis of infected cells. VGLL4 can be an important target for prevention and treatment of EV71 infection.

Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1566-1566
Author(s):  
Tiffany Khong ◽  
Janelle Sharkey ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Murine Model ◽  
Dna Methyltransferase ◽  
Myeloma Cell ◽  
Apoptotic Pathway ◽  
P16 Gene ◽  
Induced Apoptosis ◽  
The Impact

Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

scholarly journals Biphasic Modulation of Apoptotic Pathways in Cryptosporidium parvum-Infected Human Intestinal Epithelial Cells

2008 ◽  
Vol 77 (2) ◽  
pp. 837-849 ◽  
Author(s):  
Jin Liu ◽  
Mingqi Deng ◽  
Cheryl A. Lancto ◽  
Mitchell S. Abrahamsen ◽  
Mark S. Rutherford ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Expression Patterns ◽  
Nuclear Condensation ◽  
Successful Infection ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
The Impact

ABSTRACT The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

scholarly journals The Major Apoptotic Pathway Activated and Suppressed by Poliovirus

2003 ◽  
Vol 77 (1) ◽  
pp. 45-56 ◽  
Author(s):  
George A. Belov ◽  
Lyudmila I. Romanova ◽  
Elena A. Tolskaya ◽  
Marina S. Kolesnikova ◽  
Yuri A. Lazebnik ◽  
...  
Keyword(s):  
Program Implementation ◽  
Caspase 3 ◽  
Chromatin Condensation ◽  
Productive Infection ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Poliovirus Infection ◽  
Proapoptotic Protein ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACT Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.

scholarly journals Reovirus-Induced Apoptosis Is Mediated by TRAIL

2000 ◽  
Vol 74 (17) ◽  
pp. 8135-8139 ◽  
Author(s):  
Penny Clarke ◽  
Suzanne M. Meintzer ◽  
Spencer Gibson ◽  
Christian Widmann ◽  
Timothy P. Garrington ◽  
...  
Keyword(s):  
Death Receptor ◽  
Tnf Receptor ◽  
Death Domain ◽  
Apoptotic Pathway ◽  
Cellular Receptors ◽  
Trail Receptors ◽  
Infected Cells ◽  
Soluble Trail ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

scholarly journals Streptococcus suis Serotype 2 Infection Causes Host Immunomodulation through Induction of Thymic Atrophy

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Shujie Wang ◽  
Chuang Lyu ◽  
Guixin Duan ◽  
Fandan Meng ◽  
Yongbo Yang ◽  
...  
Keyword(s):  
Interleukin 2 ◽  
Bacterial Pathogen ◽  
Streptococcus Suis ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Content Type ◽  
Thymocyte Apoptosis ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Factor ◽  
The Impact

ABSTRACT Streptococcus suis serotype 2 is an important bacterial pathogen of swine and is also an emerging zoonotic agent that may be harmful to human health. Although the virulence genes of S. suis have been extensively studied, the mechanisms by which they damage the central immune organs have rarely been studied. In the current work, we wanted to uncover more details about the impact and mechanisms of S. suis on specific populations of thymic and immune cells in infected mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed that S. suis infection induced apoptosis in CD3+, CD14+, and epithelial cells from the thymus. S. suis infection resulted in a rapid depletion of mitochondrial permeability and release of cytochrome c (CytC) and apoptosis-inducing factor (AIF) through upregulation of Bax expression and downregulation of Bcl-xl and Bcl2 expression in thymocytes. Moreover, S. suis infection increased cleavage of caspase-3, caspase-8, and caspase-9. Thus, S. suis induced thymocyte apoptosis through a p53- and caspase-dependent pathway, which led to a decrease of CD3+ cells in the thymus, subsequently decreasing the numbers of CD4+ and CD8+ cells in the peripheral blood. Finally, expression dysregulation of proinflammatory cytokines in the serum, including interleukin 2 (IL-2), IL-6, IL-12 (p70), tumor necrosis factor (TNF), and IL-10, was observed in mice after S. suis type 2 infection. Taken together, these results suggest that S. suis infection can cause atrophy of the thymus and induce apoptosis of thymocytes in mice, thus likely suppressing host immunity.

scholarly journals EV71 Infection Induces IFNβ Expression in Neural Cells

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1121 ◽  
Author(s):  
Hsing-I Huang ◽  
Jhao-Yin Lin ◽  
Sheng-Hung Chen
Keyword(s):  
Enterovirus 71 ◽  
Ev71 Infection ◽  
Neural Cells ◽  
Innate Immune Responses ◽  
Toll Like Receptor ◽  
Virus Rna ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Human Neural Cells ◽  
Mitochondrial Antiviral Signaling

Enterovirus 71 (EV71) can invade the central nervous system (CNS) and cause neurological disease. Accumulating evidence indicates that EV71 can directly infect neurons in the CNS. Innate immune responses in the CNS have been known to play an essential role in limiting pathogen infections. Thus, investigating the effects of EV71 infection of neural cells is important for understanding disease pathogenesis. In this study, human neural cells were infected with EV71, and interferonβ (IFNβ) expression was examined. Our results show that IFNβ expression was upregulated in EV71-infected neural cells via pattern recognition receptors (PRRs) sensing of virus RNA. The PRRs Toll-like receptor 3 (TLR3), Toll-like receptor 8 (TLR8), and melanoma differentiation-associated gene-5 (MDA-5), but not retinoic acid-inducible gene-I (RIG-I) and Toll-like receptor 7 (TLR7), were found to be EV71-mediated IFNβ induction. Although viral proteins exhibited the ability to cleave mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF) in neural cells, levels of viral protein expression were low in these cells. Furthermore, neural cells efficiently produced IFNβ transcripts upon EV71 vRNA stimulation. Treating infected cells with anti-IFNβ antibodies resulted in increased virus replication, indicating that IFNβ release may play a role in limiting viral growth. These results indicate that EV71 infection can induce IFNβ expression in neural cells through PRR pathways.

scholarly journals Bax Is Activated during Rotavirus-Induced Apoptosis through the Mitochondrial Pathway

2007 ◽  
Vol 81 (9) ◽  
pp. 4457-4464 ◽  
Author(s):  
Sandra Martin-Latil ◽  
Laurence Mousson ◽  
Arnaud Autret ◽  
Florence Colbère-Garapin ◽  
Bruno Blondel
Keyword(s):  
Mitochondrial Pathway ◽  
Cellular Level ◽  
Viral Gastroenteritis ◽  
Small Interfering Rnas ◽  
Apoptotic Pathway ◽  
Osmotic Diarrhea ◽  
Dividing Cells ◽  
Intestinal Functions ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACT Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis, and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, which, in turn, contributes to osmotic diarrhea and rotavirus pathogenesis. Here we show that infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase; all three phenomena are features of apoptosis. RRV induced the release of cytochrome c from mitochondria to the cytosol, indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited cytochrome c release in RRV-infected cells. Thus, mitochondrial dysfunction induced by rotavirus is Bax dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improving our understanding of rotavirus pathogenesis at the cellular level.

scholarly journals Enterovirus 71 Induces INF2 Cleavage via Activated Caspase-2 in Infected RD Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Bei Wang ◽  
Chongyang Zhang ◽  
Congci Yu ◽  
Yue Zhu ◽  
Qing Tang ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Mitochondrial Fission ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Ev71 Infection ◽  
Causative Pathogen ◽  
Rd Cells ◽  
Infected Cells ◽  
Caspase 2 ◽  
Feedback Regulatory Mechanism

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease. The lack of understanding of the virus’s pathogenesis hinders the development of anti-virus drugs and the control of EV71 infection. Our previous studies have demonstrated that both mitochondria and endoplasmic reticulum (ER) were altered significantly in EV71 infected cells, but the mechanism is still unclear. In this study, we investigated the effects of EV71 infection on the expression of INF2, a key regulator factor in ER-Mitochondria communication and mitochondrial fission. We found that INF2 was cleaved in EV71 infected RD cells. The INF2 cleavage occurred at Aspartic 1,051 of INF2 and is mediated by activated caspases, predominantly by activated caspase-2. The subcellular localization of INF2 and caspase-2 was significantly altered in infected cells. We speculate that caspase-2-mediated INF2 cleavage is involved in forming viral replication organelles (ROs) and is a positive feedback regulatory mechanism of mitochondrial disorders caused by EV71 infection.

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