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

ABSTRACTAccumulated 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 bothin vitroandin 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.IMPORTANCEJapanese 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.

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Japanese Encephalitis Virus Infection Initiates Endoplasmic Reticulum Stress and an Unfolded Protein Response

Journal of Virology ◽

10.1128/jvi.76.9.4162-4171.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4162-4171 ◽

Cited By ~ 236

Author(s):

Hong-Lin Su ◽

Ching-Len Liao ◽

Yi-Ling Lin

Keyword(s):

Endoplasmic Reticulum ◽

Japanese Encephalitis Virus ◽

Unfolded Protein Response ◽

Japanese Encephalitis ◽

Specific Inhibitor ◽

Encephalitis Virus ◽

Protein Glycosylation ◽

Unfolded Protein ◽

Induced Apoptosis ◽

Protein Response

ABSTRACT 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.

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Activation of endoplasmic reticulum stress mediates oxidative stress–induced apoptosis of granulosa cells in ovaries affected by endometrioma

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz066 ◽

2019 ◽

Vol 26 (1) ◽

pp. 40-52 ◽

Cited By ~ 2

Author(s):

Chisato Kunitomi ◽

Miyuki Harada ◽

Nozomi Takahashi ◽

Jerilee M K Azhary ◽

Akari Kusamoto ◽

...

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Granulosa Cells ◽

Ovarian Function ◽

Stress Sensor ◽

Local Factor ◽

High Oxidative Stress ◽

Induced Apoptosis ◽

Sensor Proteins

Abstract Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress–induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

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Endoplasmic Reticulum Stress Sensor Protein Kinase R–Like Endoplasmic Reticulum Kinase (PERK) Protects Against Pressure Overload–Induced Heart Failure and Lung Remodeling

Hypertension ◽

10.1161/hypertensionaha.114.03811 ◽

2014 ◽

Vol 64 (4) ◽

pp. 738-744 ◽

Cited By ~ 50

Author(s):

Xiaoyu Liu ◽

Dongmin Kwak ◽

Zhongbing Lu ◽

Xin Xu ◽

John Fassett ◽

...

Keyword(s):

Heart Failure ◽

Endoplasmic Reticulum ◽

Protein Kinase ◽

Endoplasmic Reticulum Stress ◽

Pressure Overload ◽

Protein Kinase R ◽

Stress Sensor ◽

Lung Remodeling ◽

Sensor Protein

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Japanese encephalitis virus induces apoptosis by the IRE1/JNK pathway of ER stress response in BHK-21 cells

Archives of Virology ◽

10.1007/s00705-015-2715-5 ◽

2015 ◽

Vol 161 (3) ◽

pp. 699-703 ◽

Cited By ~ 25

Author(s):

Mingjie Huang ◽

Ahui Xu ◽

Xiaoyu Wu ◽

Yanni Zhang ◽

Yunli Guo ◽

...

Keyword(s):

Stress Response ◽

Er Stress ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Jnk Pathway ◽

Er Stress Response

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

Journal of General Virology ◽

10.1099/vir.0.2008/000182-0 ◽

2008 ◽

Vol 89 (8) ◽

pp. 1930-1941 ◽

Cited By ~ 22

Author(s):

Chang-Huei Tsao ◽

Hong-Lin Su ◽

Yi-Ling Lin ◽

Han-Pang Yu ◽

Shu-Ming Kuo ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Caspase 3 ◽

Encephalitis Virus ◽

Dominant Negative Mutant ◽

Caspase 8 ◽

Dependent Manner ◽

Caspase 9 ◽

Effector Caspase ◽

Induced Apoptosis

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.

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Japanese encephalitis virus activates autophagy through XBP1 and ATF6 ER stress sensors in neuronal cells

Journal of General Virology ◽

10.1099/jgv.0.000792 ◽

2017 ◽

Vol 98 (5) ◽

pp. 1027-1039 ◽

Cited By ~ 30

Author(s):

Manish Sharma ◽

Sankar Bhattacharyya ◽

Kiran Bala Sharma ◽

Shailendra Chauhan ◽

Suramya Asthana ◽

...

Keyword(s):

Er Stress ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Neuronal Cells ◽

Encephalitis Virus ◽

Stress Sensors

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Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection

Virology Journal ◽

10.1186/s12985-015-0269-5 ◽

2015 ◽

Vol 12 (1) ◽

pp. 32 ◽

Cited By ~ 6

Author(s):

Hey Lyoo ◽

Soo Park ◽

Ji Kim ◽

Yong Jeong

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Persistent Infection ◽

Encephalitis Virus ◽

Induced Apoptosis

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NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

The Journal of Cell Biology ◽

10.1083/jcb.201006121 ◽

2010 ◽

Vol 191 (6) ◽

pp. 1113-1125 ◽

Cited By ~ 187

Author(s):

Gang Li ◽

Christopher Scull ◽

Lale Ozcan ◽

Ira Tabas

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Cellular Mechanisms ◽

Apoptosis Pathway ◽

Stress Oxidative ◽

Induced Apoptosis ◽

And Oxidative Stress

Endoplasmic reticulum (ER)–induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress–apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA–dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.

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