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