JEV-induced Apoptosis Research Articles

Japanese encephalitis virus infection induces mitochondrial-mediated apoptosis through the proapoptotic protein BAX

The Japanese encephalitis virus (JEV), a zoonotic flavivirus, is Asia’s primary cause of viral encephalitis. JEV induces apoptosis in a variety of cells; however, the precise mechanisms underlying this apoptosis resulting from JEV infection remain to be elucidated. Our previous studies showed that the proapoptosis gene BAX may have a role in JEV proliferation. In this study, we constructed a PK-15 cell line (BAX.KO) with a knockout of the BAX gene using CRISPR/Cas9. The knockout of the BAX gene effectively inhibited the proliferation of JEV, resulting in a 39.9% decrease in viral protein levels, while BAX overexpression produced the opposite effect. We confirmed that JEV induces apoptosis of PK-15 using 4′,6-diamidino-2-phenylindole (DAPI) staining and Annexin V-FITC/PI staining. Furthermore, we found that the phosphorylation of P53 and the expression levels of BAX, NOXA, PUMA, and cleaved-caspase-3/9 were significantly upregulated after JEV infection. Moreover, we found that JEV infection not only caused mitochondrial damage, the release of mitochondrial cytochrome C (Cyt C), and the downregulation of the apoptosis-inhibiting protein BCL-2 but also reduced the mitochondrial membrane potential (MOMP) and the accumulation of intracellular reactive oxygen species (ROS). These factors collectively encourage the activation of the mitochondrial apoptosis pathway. In contrast, BAX gene knockout significantly reduces the apoptotic changes caused by JEV infection. Treatment with the caspase3 inhibitor attenuated JEV-induced viral proliferation and release, leading to a decrease in viral protein levels of 46% in PK-15 cells and 30% in BAX.KO cells. In conclusion, this study clarified the molecular mechanisms of JEV-induced apoptosis and provided a theoretical basis for revealing the pathogenic mechanisms of JEV infection.

Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway.

Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

Japanese encephalitis virus induces apoptosis by inhibiting Foxo signaling pathway

Japanese encephalitis virus (JEV) infection induces brain tissue disease characterized by neuron death. however, little is known about the underlying mechanism. Using RNA sequencing, we profiled global mRNA expression changes in response to in vitro and in vivo JEV infection. Integration analysis of in vitro and in vivo mRNA transcriptome revealed that JEV infection regulated apoptosis-related Foxo signaling pathway. Foxo expression was reduced by JEV infection in vitro and in vivo. Knockdown of Foxo promoted apoptosis, while its overexpression reduced apoptosis in JEV-infected Neuro-2a cells. JEV infection in Neuro-2a cells decreased the expression of Foxo downstream genes including pro-apoptotic protein Bim, anti-apoptotic protein Bcl-6 and p21. Overexpression of anti-apoptotic proteins Bcl-6 and p21 repressed JEV-induced apoptosis. These findings suggest that Foxo primarily exerts an anti-apoptotic function via Bcl-6 and p21 in JEV-infected Neuro-2a cells. A STAT3 binding site was identified in the promoter region of Foxo by TFBIND software and confirmed by ChIP and reporter assays. JEV infection reduced STAT3 expression as well as its binding at the Foxo promoter compared to mock infection in Neuro-2a cells. Moreover, STAT3 knockdown reduced Foxo promoter activity and Foxo expression. Therefore, JEV reduced Foxo expression, at least in part, by downregulating STAT3. Taken together, we found that JEV induced cell apoptosis by inhibiting STAT3-Foxo-Bcl-6/p21 pathway, which provides a novel insight into JEV-caused encephalitis.

MiR-370 mimic inhibits replication of Japanese encephalitis virus in glioblastoma cells.

Japanese encephalitis (JE) is one of the most severe viral infections of the central nervous system. No effective treatment for JE currently exists, because its pathogenesis remains largely unknown. The present study was designed to screen the potential microRNAs (miRNAs) involved in JE. Glioblastoma cells were collected, after being infected with the Japanese encephalitis virus (JEV). Total miRNAs were extracted and analyzed using an miRNA chip. One of the most severely affected miRNAs was selected, and the role of miR-370 in JEV infection was investigated. Cell viability and apoptosis of the host cells were evaluated. JEV replication was detected via analysis of gene E expression. Real-time polymerase chain reaction was used to determine the levels of endogenous miR-370 and expression of innate immunity-related genes. Following JEV infection, 114 miRNAs were affected, as evidenced by the miRNA chip. Among them, 30 miRNAs were upregulated and 84 were downregulated. The changes observed in five miRNAs were confirmed by real-time polymerase chain reaction. One of the significantly downregulated miRNAs was miR-370. Therefore, miR-370 mimic was transfected into the cells, following which the levels of endogenous miR-370 were significantly elevated. Concurrently, JEV replication was significantly reduced 24 hours after transfection of miR-370 mimic. Functionally, miR-370 mimic mitigated both JEV-induced apoptosis and the inhibition of host cell proliferation. Following JEV infection, interferon-β and nuclear factor-kappa B were upregulated, whereas miR-370 mimic prevented the upregulation of the genes induced by JEV infection. The present study demonstrated that miR-370 expression in host cells is downregulated following JEV infection, which further mediates innate immunity-related gene expression. Taken together, miR-370 mimic might be useful to prevent viral replication and infection-induced host cell injury.

Transcriptional regulation and activation of the mitogen-activated protein kinase pathway after Japanese encephalitis virus infection in neuroblastoma cells

Japanese encephalitis virus (JEV), the most frequent and the single most important cause of encephalitis worldwide, has spread throughout most of Asia. For the development of appropriate and effective therapy, there is an immediate requirement to understand the role of host factors in JEV-induced neuropathogenesis. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in JEV infection of mouse neuroblastoma (N2A) cells. The MAPK pathway was studied at the transcriptional level to access the gene expression profile at different time points after JEV infection. The effector MAPK genes were also analyzed for protein expression and activation. Gene expression analysis showed a significant regulation of extracellular signal-regulated kinases (ERK)1, ERK2 and c-Jun N-terminal kinase (JNK)3 genes along with their downstream transcription factors such as Mef2c, c-Jun and Sfn. Experiments with the JNK inhibitor, SP600125, and the ERK inhibitor, PD98059, showed the involvement of JNK in JEV-induced caspase-3 activation and apoptosis, but ERK1/2 had no effect. Overall, our results show the transcriptional regulation of the MAPK pathway and the essential role of JNK in JEV-induced apoptosis in neuroblastoma cells. These findings provide a new insight into the role of the mitogen- and stress-activated kinases in JEV pathogenesis and opens up new avenues of therapeutics.

Japanese encephalitis virus down-regulates thioredoxin and induces ROS-mediated ASK1-ERK/p38 MAPK activation in human promonocyte cells

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes severe neurological disease with high mortality. Molecular mechanisms of JEV pathogenesis such as upstream apoptotic processes and pathways are not yet completely resolved or understood. In this study, JEV replication in human promonocyte cells induced time-dependent apoptosis and activated virus dose-dependent caspases 3, 8 and 9. Proteomic analysis demonstrated up- and down-regulated (more or less than 1.5-fold) proteins in JEV-infected promonocyte cells. Biological process categorization showed processes of antioxidation, free radical removal, and sulfur redox metabolism entailed many identified up- and down-regulated proteins. Down-regulation of thioredoxin, confirmed by using Western blotting, was involved in the apoptosis process of the oxidative stress response pathway. JEV infection caused increased intracellular ROS production and activation of ASK1-ERK/p38 MAPK signaling. ERK/p38 MAPK inhibitor PD98059 treatment definitely suppressed this apoptosis. Down-regulation of thioredoxin, increased intracellular ROS, and activation of ASK1-ERK/p38 MAPK signaling all were associated with JEV-induced apoptosis. These results are suggestive of an oxidative stress-pathway as a key element of JE pathogenesis.

Japanese encephalitis virus infection activates caspase-8 and -9 in a FADD-independent and mitochondrion-dependent manner

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, replicates primarily at the endoplasmic reticulum and thereby triggers apoptosis of infected cells. This study investigated the hierarchical activation of the caspase network induced by JEV infection. It was found that JEV activated the initiators caspase-8 and -9, as well as effector caspase-3, in infected baby hamster kidney and mouse neuroblastoma (N18) cells. In neuronal N18 cells, JEV infection triggered cytochrome c release from mitochondria, which in turn activated caspase-9 and -3. Treatment of JEV-infected N18 cells with cyclosporin A or ruthenium red, which attenuate mitochondrial injuries, blocked activation of caspase-9 or -3, typifying that, in neuronal cells, this apoptosis involves the mitochondrial pathway. Alternatively, in caspase-3-deficient MCF-7 cells, JEV persisted and readily triggered a typical apoptotic response, including cytochrome c release and full activation of caspase-9 and -8 along with caspase-6, indicating that JEV did not require caspase-3 to manifest caspase-8 activation and apoptosis. Interestingly, a Fas-associated death-domain-containing protein (FADD) dominant-negative mutant, which interfered with transmission of the extracellular death signals into cells through the Fas/tumour necrosis factor (TNF) receptor, failed to block JEV-induced apoptosis and caspase-8 activation, implying that receptor oligomerization of the Fas/TNF pathway might not participate in JEV-induced apoptosis. Taken together, these results illustrate that JEV infection triggers caspase cascades involving the initiators caspase-8 and -9, probably through FADD-independent but mitochondrion-dependent pathways.

Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response.

The malfunctioning of the endoplasmic reticulum (ER) of cells in hosts ranging from yeast to mammals can trigger an unfolded protein response (UPR). Such malfunctioning can result from a variety of ER stresses, including the inhibition of protein glycosylation and calcium imbalance. To cope with ER stresses, cells may rely on the UPR to send a signal(s) from the ER to the nucleus to stimulate appropriate cellular responses, including induction of chaperone expression. During Japanese encephalitis virus (JEV) infection, the lumen of the ER rapidly accumulates substantial amounts of viral proteins for virus progeny production. In the present study, we demonstrate that as evidenced by certain chaperone inductions, JEV infection triggers the UPR in fibroblast BHK-21 cells and in neuronal N18 and NT-2 cells, in which JEV results in apoptotic cell death. By contrast, no UPR was observed in apoptosis-resistant K562 cells infected by JEV. JEV infection also activates expression of CHOP/GADD153, a distinctive transcription factor often induced by the UPR, and appears to trigger activation of p38 mitogen-activated protein kinase, a posttranslational activator of CHOP. Ectopic enforcement of CHOP expression enhanced JEV-induced apoptosis, whereas treatment with a p38-specific inhibitor, SB203580, partially blocked JEV-induced apoptosis. Interestingly, bcl-2 overexpression and treatment with a pancaspase inhibitor, z-VAD-fmk, inhibited CHOP induction and diminished JEV-induced apoptosis, suggesting that Bcl-2 and caspases could be the upstream regulators of CHOP. Our results thus suggest that virus-induced ER stress may participate, via p38-dependent and CHOP-mediated pathways, in the apoptotic process triggered by JEV infection.

Antiapoptotic but not antiviral function of human bcl-2 assists establishment of Japanese encephalitis virus persistence in cultured cells.

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human bcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog, bcl-XL, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced bcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 which plays a role in the establishment of JEV persistence.

Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells.

Infection by Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans and induces severe cytopathic effects in different types of cultured cells. This study attempted to determine whether apoptosis contributes to virus-induced cell death in a culture system by characterizing JEV lytic infection in baby hamster kidney BHK-21 cells, murine neuroblastoma N18 cells, and human neuronal progenitor NT2 cells. According to our results, the replication of JEV, and not the UV-inactivated virions per se, triggered apoptosis in these cell lines, as evidenced by nuclear condensation, DNA fragmentation ladder, and in situ end labeling of DNA strand breaks with terminal transferase (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). Different strains of JEV, regardless of whether they are neurovirulent to mice, could induce apoptosis of the infected cells. In addition, enforced expression of the human protooncogene bcl-2 in BHK-21 cells, which did not influence virus production, appeared to delay the process of JEV-induced apoptosis, despite the fact that most infected cells were inevitably killed after prolonged cultures. However, Bcl-2 proteins expressed in N18 cells failed to block JEV-induced apoptosis, although they did prevent Sindbis virus-induced apoptosis from occurring in the same cells. This finding suggests that these two viruses may utilize similar but not identical mechanisms to kill their infected cells. The results presented here thus demonstrate that apoptosis can be a general mechanism for JEV-induced cell death and that enforced bcl-2 expression may be inadequate in protecting all cell types from JEV-induced apoptosis in cell cultures.