Japanese Encephalitis Virus, Coronavirus, Autophagy, and the ER stress response

The accumulation of misfolded proteins in the ER lumen induces a stress response commonly known as the Unfolded Protein Response (UPR) or ER stress response, an adaptive signalling pathway increasing the expression of ER chaperones, inhibiting mRNA translation, and stimulating ER associated degradation (ERAD) of accumulated proteins. The degradation via the ERAD pathway in particular requires the formation of double membrane vesicles -more commonly referred to as autophagosomes - which subsequently fuse with lysosomes to form the autolysosome.  The ERAD pathway can be induced by all three branches of the ER stress response -PERK, ATF6, and IRE1- which increase the expression of ER degradation enhancer, mannosidase alpha-like-1/-2 (EDEM1/2) proteins in addition to other components of the ERAD pathway either by ATF4 (in conjunction with sXBP1) by cleaved ATF6α. Binding of cytosolic misfolded proteins to components of the ERAD pathway allows the retrotranslocation of these protein into the ER lumen where ER chaperones may assist these proteins to be folded correctly and/or be glycosylated in a process which involves binding to EDEM1/2/3.  Alternatively proteins might however be targeted for degradation; in this case the EDEM/protein complex induces the formation of specific autophagosomes, the EDEMosome. In addition to components of the ERAD pathway, ATF4 also induces the expression of autophagy related genes, including but not limited to LC3, ATG16L, p62/SQSTM1, NBR, and ATG7, thus linking the induction of ER stress to the induction of autophagy. As an alternative to ERAD/EDEMosome mediated degradation, proteins which are ubiquitylated are targeted for the proteasome or autophagy by binding to p62/SQSTM1 or NBR; degradation via the EDEMosome in contrast is exclusively mediated by the formation of autophagosomes. 

The accumulation of protein aggregates in the ER lumen can induce autophagy dependent
and independent pathways of degradation

In the case of viral infected cells, several viruses have been shown to induce the ER stress response signaling pathway and as a result autophagy rather than apoptosis, either during viral replication or following binding of the virus to its receptor. In these cases, the UPR leads to the induction of the cellular Interferon response and viral induced autophagy can lead to the degradation of viral components in the lysosome as well as being processed in multivesicular bodies and subsequent MHC class I/II presentation of viral antigens. On the other hand, prolonged ER stress induced by viral proteins in the absence of autophagy can and would induce apoptosis or necrosis, both which can be considered as an antiviral response as well.

Crosstalk between UPR and induction of autophagy

In the case of JEV (and other viruses) premature apoptosis and the induction of autophagy induced by the localisation of both the non-structural and structural proteins to the ER would decrease viral replication due to cell death or degradation of viral proteins. Both apoptosis and autophagy can be induced by UPR. In the case of autophagy, both the PERK and ATF6 induced pathways induce the formation of autophagosomes whereas the IRE1 pathway inhibits autophagy via CHOP  and promotes DR5 dependent apoptotic pathways. It should be noted however that CHOP has a dual role since at least during short term ER stress CHOP increases the expression of autophagy related genes such as ATG5 and ATG7 required for  the formation of autophagic vesicles whereas only during prolonged ER stress autophagy is inhibited. to further complicate the matter, CHOP also releases Beclin1 from cytoplasmic Beclin1-Bcl2 complexes and thus induces the formation of autophagic vesicles as well as facilitating nuclear translocation of Bcl2, the latter forming a complex with ASPP2 and inducing a process referred to as autophagic apoptosis in hepatocellular carcinoma cells via induction CHOP expression.

Since both the PERK and ATF6 dependent pathways are activated prior activation of IRE1, these pathways might benefit JEV especially early in infection by preventing apoptosis. Indeed, following the infection of neuronal cells with a neurotrophic strain of JEV, an induction of autophagy as evidenced by the accumulation of LC3-II positive autophagosomes can be observed as early as 16 hrs p.i., coinciding with the detection of NS5, suggesting that newly synthesised proteins rather than incoming structural proteins are responsible for the induction of autophagy. Since these vesicles are not only positive for LC3-II but also for EDEM1, they represent EDEMosomes and can potentially be degraded by the autolysosomal pathway. Furthermore, the formation of EDEMosomes or autophagosomes is necessary for preventing apoptosis as infected ATG5 or ATG7 deficient cells are highly susceptible to virus induced apoptosis. On the other hand, maturation of the autophagosome also enhances the degradation of viral components via the autolysosome; indeed levels of viral RNA,  as measured by qRT-PCR, are increased in ATG5 and ATG7 deficient cells compared to wt cells. 

In the case of Coronavirus infected cells, the expression of the nsp-6 protein not only induces the formation of omegasomes but also inhibits the formation of mature autolysosomes via inhibition of the mTORC1 complex. It seems possible therefore, that JEV also inhibits the formation of mature autophagosomes and indeed, late in infection the presence of LC3-II positive autophagosomes decreases. If however this is due to the expression of a viral protein is unclear; to the author of this post it seems most likely that the prolonged ER stress induced by the viral NS and structural proteins activates the IRE1 dependent pathway late in infection and thus inhibits autophagy via CHOP induction. It remains to be seen however, if in cells deficient for CHOP and other components of the IRE1 pathway, viral induced autophagy is affected or not and if these cells are undergoing apoptosis in a PERK/ATF6 dependent manner only. As mentioned before, CHOP plays a dual role so it might be possible that in JEV infected cells CHOP is actually required for the induction of autophagy.

As for the signaling pathway leading to the formation of autophagic vesicles, the Core protein of JEV has been shown to induce p38 MAPK, thus activating PERK and ATF6 dependent signaling pathways and since in JEV infected cells autophagy genes are not unregulated it might be possible that JEV selectively activates ATF6α. In addition to upregulating the expression of DR5, both PERK and ATF6α also induce the expression of autophagy- and ERAD- related genes as mentioned above. It might therefore be possible that prior the induction of IRE1, JEV infected cells predominantly induce autophagy via ATF6α and only the prolonged induction of ER stress induces IRE1 dependent activation of apoptosis via CHOP and autophagy inhibition. In other words, the induction of EDEMosomes following the localisation of JEV proteins may represent not only a scaffold for viral RNA synthesis but also an antiviral response, which is reflected by the increase of viral RNA in ATG5/7 deficient cell lines. If however other proteins that the Core protein contribute to the formation of EDEMosomes remains to be investigated and I am curious to see the results.

                   Coronavirus and the ER stress response

As discussed in previous posts, nsps’ derived from both Arteri- and Coronavirus’ induce the formation of EDEMosomes and both nsp-7 from PRRSV and nsp--6 induce the formation of omegasomes; furthermore, both proteins (in addition to others) are localised at the ER and part of the viral RTC. As we have seen, the co-localisation of (viral) proteins with EDEM-1 suggests that the protein underwent a retrotranslocation to the ER thus allowing ER localisation in the absence of a classical localisation sequence. The coronaviral nsp-3/-4/-6 proteins are also N-glycosylated, suggesting that once inside the ER they undergo glycosylation. So far however, none of these proteins has been shown to induce a prolonged ER stress response, raising the question what are the unique properties of JEV that induce a stress response? MHV does induce p38 MAPK, IBV infection does induce the ER stress response, and both SARS-CoV and MHV infected cells exhibit increased mRNA levels of homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1 (HERPUD1).  The induction of the UPR following Coronavirus infection has indeed been proposed to induce apoptosis as well as the induction of chemokines (which can be inhibited by various coronaviral proteins. In contrast to JEV however, where the Core protein has been shown to induce UPR (and as the author hypothesizes that the viral NS2A, NS2B, M, and E proteins do so likewise), in the case of Coronavirus’ it is not known which one of the nsp proteins involved -if any- do indeed cause short term or prolonged ER stress. On the other hand, in the case of CoV we know which proteins are sufficient to induce EDEMosomes/omegasomes - if however the structural proteins play a role in the formation of the RTC remains to be seen. 

Coronavirus (top) and Japanese Encephalitis Virus (bottom) induce the formation of EDEMosomes and
inhibit the formation of autolysosomes

Further reading

Matsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, Nishimura K, & Matsuo S (2013). Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology open, 2 (10), 1084-90 PMID: 24167719

Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, Ogawa S, Kaufman RJ, Kominami E, & Momoi T (2007). ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell death and differentiation, 14 (2), 230-9 PMID: 16794605 

Shin YJ, Han SH, Kim DS, Lee GH, Yoo WH, Kang YM, Choi JY, Lee YC, Park SJ, Jeong SK, Kim HT, Chae SW, Jeong HJ, Kim HR, & Chae HJ (2010). Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress. Arthritis research & therapy, 12 (1) PMID: 20122151 

Liu K, Shi Y, Guo X, Wang S, Ouyang Y, Hao M, Liu D, Qiao L, Li N, Zheng J, & Chen D (2014). CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2. Cell death & disease, 5 PMID: 25032846

Armstrong, J., Flockhart, R., Veal, G., Lovat, P., & Redfern, C. (2009). Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells Journal of Biological Chemistry, 285 (9), 6091-6100 DOI: 10.1074/jbc.M109.014092

Shoulders MD, Ryno LM, Genereux JC, Moresco JJ, Tu PG, Wu C, Yates JR 3rd, Su AI, Kelly JW, & Wiseman RL (2013). Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell reports, 3 (4), 1279-92 PMID: 23583182

Rzymski T, Milani M, Pike L, Buffa F, Mellor HR, Winchester L, Pires I, Hammond E, Ragoussis I, & Harris AL (2010). Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene, 29 (31), 4424-35 PMID: 20514020


B'chir W, Chaveroux C, Carraro V, Averous J, Maurin AC, Jousse C, Muranishi Y, Parry L, Fafournoux P, & Bruhat A (2014). Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation. Cellular signalling, 26 (7), 1385-91 PMID: 24657471 

Yu Z, Wang AM, Adachi H, Katsuno M, Sobue G, Yue Z, Robins DM, & Lieberman AP (2011). Macroautophagy is regulated by the UPR-mediator CHOP and accentuates the phenotype of SBMA mice. PLoS genetics, 7 (10) PMID: 22022281

Sharma M, Bhattacharyya S, Nain M, Kaur M, Sood V, Gupta V, Khasa R, Abdin MZ, Vrati S, & Kalia M (2014). Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes. Autophagy, 10 (9) PMID: 25046112

Li JK, Liang JJ, Liao CL, & Lin YL (2012). Autophagy is involved in the early step of Japanese encephalitis virus infection. Microbes and infection / Institut Pasteur, 14 (2), 159-68 PMID: 21946213

Banerjee S, Narayanan K, Mizutani T, & Makino S (2002). Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells. Journal of virology, 76 (12), 5937-48 PMID: 12021326

Fung TS, & Liu DX (2014). Coronavirus infection, ER stress, apoptosis and innate immunity. Frontiers in microbiology, 5 PMID: 24987391

Cottam EM, Whelband MC, & Wileman T (2014). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10 (8) PMID: 24991833

Japanese Encephalitis Virus (JEV), ER stress, and Apoptosis

Japanese Encephalitis Virus (JEV) is a causative agent of acute encephalitis in humans, and being an arthropod borne virus transmitted predominately by mosquitoes (Culex tritaeniorhynchus, Culex gelidus, Culex fuscocephala and Culex vishnui) that primarily target domestic animals and humans, with an estimated mortality up to 50000 deaths reported per annum, as well as bats.

The JEV virion

Since JEV is a enveloped virus belonging to the Flaviviridae it contains a positive sense ssRNA genome of approx. 11 kb in size with a 5’cap but no modification on the 3’ end. Following receptor mediated endocytosis, which is partly mediated by the viral envelope  (E) glycoprotein via binding to highly sulfated cellular heparansulfate, the genome is released into the cytoplasm of the cell and translated into a single polyprotein. Similar to the orf1ab polyprotein encoded by Coronaviruses, the JEV polyprotein is subsequently cleaved co- and posttranslationally into several non-structural (NS) and structural proteins by cellular and viral proteases. 

The JEV polyprotein is cleaved into non-structural and structural proteins

As it the case with Coronavirus and other positive strand RNA viruses, JEV replicates in the cytoplasm of infected cells, including the formation of replication centers that contain enzymes necessary for viral replication as well as viral ssRNA and dsRNA. Indeed, in both rat PC-12 and (hamster) BHK-21 cells viral particles have been shown to mature at and bud from the membranes of the intrinsic secretory system, namely the ER and the Golgi. As it the case in other Flavivirus infected cells, JEV induces the proliferation and hypertrophy of the rough ER, suggesting that the formation of JEV replication centers and/or the maturation and subsequent release of viral particles induces ER stress and subsequently not only an antiviral response by inducing the expression of cytokines and chemokines but also inducing apoptosis.

                            Unfolded protein response: general outline

The induction of ER stress induces a protective response collectively known as the unfolded protein response (UPR), mediated by three ER transmembrane receptors, pancreatic ER kinase (PKR)‐like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol‐requiring enzyme 1 (IRE1). Under normal conditions, these are inactivated by being associated with an ER chaperone, GRP78, which dissociates upon the accumulation of un-or misfolded proteins, triggering UPR by sequentially activating first PERK, second ATF6, and lastly IRE1. Generally, activated PERK phosphorylates eukaryotic initiation factor 2α (eIF2α), thus inhibiting general -but not IRES initiated- translation and increasing the eIF2α independent translation of ATF4; ATF4 in turn translocates to the nucleus where it activates the expression of genes encoding for ER chaperones. The activation and nuclear translocation of ATF6 regulates the expression of ER chaperones and X box‐binding protein 1 (XBP1), a transcription factor whose mRNA must undergo splicing by activated IRE1 in order to be translated. sXBP1 then translocates to the nucleus where it controls the expression of co-chaperones and a PERK inhibitor, p58IPK.  One of the key proteins expressed upon prolonged ER stress is GADD153 (growth arrest- and DNA damage-inducible gene 153), better known as CHOP (C/EBP homologous protein). The overexpression of CHOP induces not only cell cycle arrest but also apoptosis in a caspase-8 and DR5 dependent manner and murine embryonic fibroblasts derived from CHOP -/- animals indeed display less apoptosis. CHOP not only increases the expression of DR5 and promotes ligand-independent DR5 engagement but also downregulates the expression of Bcl2, thus promoting the accumulation of Bax/Bak heterodimers at the ER as well at Mitochondria. In addition, IRE1 forms a complex with Bax, Bak, TRAF2, and ASK1 thus phosphorylating Bcl2 in a JNK dependent manner. CHOP can also be activated by extracellular stimuli such as pro-inflammatory cytokines via activation of a stress-inducible p38 mitogen-activated protein kinase (MAPK).

                           JEV replication complexes and UPR

In contrast to the RTCs of Corona- or Arterivirus, JEV does not induce the formation of double membrane vesicles but utilizes the ER cisternae for viral replication, which is reflected by the viral RNA dependent RNA Polymerase (NS5) being localized in the ER lumen rather than the cytoplasm side of the ER as in Coronaviruses. Another difference is that there are indications that both the viral NS5 and NS3 proteins are localized to the nuclear matrix. In addition, the viral Core protein can be found in the nucleus as well and nucleolar localization has been postulated to be beneficial for viral replication maybe by recruiting B23 to sites of viral assembly. Although it has not proven, B23 -or another nuclear protein might be act as a chaperone for JEV proteins and/or be required for JEV core mediated inhibition of stress granule formation.  Indeed, the JEV core protein has been shown to inhibit JEV induced ER stress response in particular pathways induced by PERK, PKR, and eIF2α phosphorylation following treatment with Arsenite by binding a cellular protein, Caprin-1 (cytoplasmic activation/proliferation-associated protein-1 ), which is an initiation factor for the formation of stress granules. Additionally, the author of this post postulates that the JEV Core protein might relocalises Stress Granule and Nucleolar Protein (SGNP) to sites of viral replication akin to the relocalisation of B23.

The JEV replication complex is anchored in the ER membrane

                    JEV induced ER stress and the induction apoptosis

Since JEV replicates and assembles at the ER, not only the accumulation of viral proteins but also the depletion of lipids induces UPR. Indeed, the infection of bot neuronal and non-neuronal cells activates the expression of CHOP, triggers the expression of pro-inflammatory cytokines, subsequently inducing apoptosis in  activation of p38 MAPK in a Caspase-8 and (potentially) DR5 dependent manner. Indeed, the treatment of infected cells with either a MAPK inhibitor (SB203580), CsA, the expression of baculovirus p35 protein, or poxvirus CrmA each inhibit JEV induced apoptosis. It remains however to be seen how the virus evades the induction of apoptosis in the early stages of viral replication since it would be disadvantageous for viral replication. In the opinion of the author of this post,  JEV might be able to inhibit or delay PERK and ATF6 mediated induction of the UPR. One candidate might be the Core protein, which -as described above- associates with and relocalises B23 and SGNP from the nucleolus to the site of viral replication in addition to forming complex with Caprin-1 and hnRNP A2. This is contrast to the viral NS2B, NS3 and E proteins that induce apoptosis via the activation of the intrinsic- caspase-9 and -3 dependent- pathway by activation of p38 MAPK and ASK1but without activation of caspase-8. Although it is not clear how NS2B and NS3 induce ER stress, in the case of JEV E protein binding to GRP78 might activate PERK, ATF6, and/or IRE1.

JEV NS2B and NS3 might induce translocation of Bax/Bak complexes and induction of Caspase-3/-9/-12 via JNK by IRE1 mediated formation of Bax/Bak/ASK1/TRAF2 complexes

The activation of Caspase-8 might be dependent on induction of DR5 expression and whether the activation of caspase-8 involves the TRAIL pathway or if the expression and intracellular accumulation of DR5 drives ligand independent activation of DR5 is not clear. If so, then JEV mediated activation of Caspase-8 would differ from Dengue Virus that activates caspase-8 via TRAIL, Fas, and TNF-α receptor mediated pathways. Since the JEV Core protein has been shown to increase the expression of pro-inflammatory cytokines, Core might induce an autocrine loop wherein the expression of Core increases the expression of both the cytokines as well as their respective receptors. Alternatively -or in addition- intracellular DR5 might accumulate and initiate Caspase-8 mediated apoptosis.

JEV Core, NS2B, NS3, and E protein might activate DR5 via PERK and ATF6 by inducing p38 MAPK in later stages of the infection

Finally it should be noted that following induction of PERK, it takes 24 hrs until Caspase-3 and PARP cleavage can be detected and that ATF4 -and not CHOP- might be be sufficient for the induction of apoptosis in cells transfected with ATF4 and CHOP respectively. If therefore JEV NS2B, NS3 and E induced apoptosis is dependent on ATF4 but not CHOP remains to be seen. How then is apoptosis induced by pro-inflammatory cytokines prevented in the early stages of infection? One possibility might be that that non coding RNA derived from the viral 5' and 3' non coding region inhibits IRF3 mediated signalling which together with the delayed activation of caspase cleavage might prevent the induction of apoptosis during the early stages of infection. Also, does JEV induce apoptosis in arthropod cell lines and if not why? The activation of CHOP is further complicated by results obtained in cells infected with JEV and treated with a caspase inhibitor or with SB203580indicating that caspase activation is required for activation. This would suggest that ER stress response pathways activated early in the infection activate caspase-3 via activation of PERK. Moreover,  NS1' of neurotrophic strains of JEV is cleaved probably by Caspase-12 or -7 in neuronal cells, which is required for replication of neurotrophic JEV in neuronal cells. 

Summary of the ER stress pathways induced upon infection JEV

In summary, the expression and localisation of JEV non-structural and structural proteins is sufficient to induce apoptosis by inducing ER stress. This in contrast to the nsp’s from Corona- and Arterivirus’ that so far have not been shown to induce a ER stress response. If the induction of the formation of autophagy like vesicles may play a role will be discussed in another post.








Further reading

Shailendra K. Saxena (2013). Japanese Encephalitis Virus: The Complex Biology of an Emerging Pathogen Encephalitis DOI: 10.5772/54111 

Unni SK, Růžek D, Chhatbar C, Mishra R, Johri MK, & Singh SK (2011). Japanese encephalitis virus: from genome to infectome. Microbes and infection / Institut Pasteur, 13 (4), 312-21 PMID: 21238600 

Cui J, Counor D, Shen D, Sun G, He H, Deubel V, & Zhang S (2008). Detection of Japanese encephalitis virus antibodies in bats in Southern China. The American journal of tropical medicine and hygiene, 78 (6), 1007-11 PMID: 18541785

Mori Y, Okabayashi T, Yamashita T, Zhao Z, Wakita T, Yasui K, Hasebe F, Tadano M, Konishi E, Moriishi K, & Matsuura Y (2005). Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. Journal of virology, 79 (6), 3448-58 PMID: 15731239 

Tsuda Y, Mori Y, Abe T, Yamashita T, Okamoto T, Ichimura T, Moriishi K, & Matsuura Y (2006). Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication. Microbiology and immunology, 50 (3), 225-34 PMID: 16547420

Szebeni, A., & Olson, M. (2008). Nucleolar protein B23 has molecular chaperone activities Protein Science, 8 (4), 905-912 DOI: 10.1110/ps.8.4.905 

Zhu CH, Kim J, Shay JW, & Wright WE (2008). SGNP: an essential Stress Granule/Nucleolar Protein potentially involved in 5.8s rRNA processing/transport. PloS one, 3 (11) PMID: 19005571

Uchil PD, Kumar AV, & Satchidanandam V (2006). Nuclear localization of flavivirus RNA synthesis in infected cells. Journal of virology, 80 (11), 5451-64 PMID: 16699025

Katoh H, Okamoto T, Fukuhara T, Kambara H, Morita E, Mori Y, Kamitani W, & Matsuura Y (2013). Japanese encephalitis virus core protein inhibits stress granule formation through an interaction with Caprin-1 and facilitates viral propagation. Journal of virology, 87 (1), 489-502 PMID: 23097442 

Liao CL, Lin YL, Shen SC, Shen JY, Su HL, Huang YL, Ma SH, Sun YC, Chen KP, & Chen LK (1998). Antiapoptotic but not antiviral function of human bcl-2 assists establishment of Japanese encephalitis virus persistence in cultured cells. Journal of virology, 72 (12), 9844-54 PMID: 9811720

Ghosh Roy S, Sadigh B, Datan E, Lockshin RA, & Zakeri Z (2014). Regulation of cell survival and death during Flavivirus infections. World journal of biological chemistry, 5 (2), 93-105 PMID: 24921001 

Tsao, C., Su, H., Lin, Y., Yu, H., Kuo, S., Shen, C., Chen, C., & Liao, C. (2008). Japanese encephalitis virus infection activates caspase-8 and -9 in a FADD-independent and mitochondrion-dependent manner Journal of General Virology, 89 (8), 1930-1941 DOI: 10.1099/vir.0.2008/000182-0 

Yiang GT, Chen YH, Chou PL, Chang WJ, Wei CW, & Yu YL (2013). The NS3 protease and helicase domains of Japanese encephalitis virus trigger cell death via caspase‑dependent and ‑independent pathways. Molecular medicine reports, 7 (3), 826-30 PMID: 23291778 

Yang TC, Shiu SL, Chuang PH, Lin YJ, Wan L, Lan YC, & Lin CW (2009). Japanese encephalitis virus NS2B-NS3 protease induces caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells. Virus research, 143 (1), 77-85 PMID: 19463724 

Wu, Y., Chang, C., Hung, C., Tsai, M., Schuyler, S., & Wang, R. (2011). Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-128 

Chen SO, Fang SH, Shih DY, Chang TJ, & Liu JJ (2009). Recombinant core proteins of Japanese encephalitis virus as activators of the innate immune response. Virus genes, 38 (1), 10-8 PMID: 19009340 

Yamaguchi H, & Wang HG (2004). CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells. The Journal of biological chemistry, 279 (44), 45495-502 PMID: 15322075

Lu, M., Lawrence, D., Marsters, S., Acosta-Alvear, D., Kimmig, P., Mendez, A., Paton, A., Paton, J., Walter, P., & Ashkenazi, A. (2014). Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis Science, 345 (6192), 98-101 DOI: 10.1126/science.1254312 

Han J, Back SH, Hur J, Lin YH, Gildersleeve R, Shan J, Yuan CL, Krokowski D, Wang S, Hatzoglou M, Kilberg MS, Sartor MA, & Kaufman RJ (2013). ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nature cell biology, 15 (5), 481-90 PMID: 23624402  

Chang RY, Hsu TW, Chen YL, Liu SF, Tsai YJ, Lin YT, Chen YS, & Fan YH (2013). Japanese encephalitis virus non-coding RNA inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3. Veterinary microbiology, 166 (1-2), 11-21 PMID: 23755934

Sun J, Yu Y, & Deubel V (2012). Japanese encephalitis virus NS1' protein depends on pseudoknot secondary structure and is cleaved by caspase during virus infection and cell apoptosis. Microbes and infection / Institut Pasteur, 14 (11), 930-40 PMID: 22504173