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Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo.

ABSTRACT: Dengue virus (DENV) utilizes the endoplasmic reticulum (ER) for replication and assembling. Accumulation of unfolded proteins in the ER lumen leads to ER stress and unfolded protein response (UPR). Three branches of UPRs temporally modulated DENV infection. Moreover, ER stress can also induce autophagy. DENV infection induces autophagy which plays a promotive role in viral replication has been reported. However, the role of ER stress in DENV-induced autophagy, viral titer, and pathogenesis remain unclear. Here, we reveal that ER stress and its downstream UPRs are indispensable for DENV-induced autophagy in various human cells. We demonstrate that PERK-eIF2? and IRE1?-JNK signaling pathways increased autophagy and viral load after DENV infection. However, ATF6-related pathway showed no effect on autophagy and viral replication. IRE1?-JNK downstream molecule Bcl-2 was phosphorylated by activated JNK and dissociated from Beclin 1, which playing a critical role in autophagy activation. These findings were confirmed as decreased viral titer, attenuated disease symptoms, and prolonged survival rate in the presence of JNK inhibitor in vivo. In summary, we are the first to reveal that DENV2-induced ER stress increases autophagy activity, DENV replication, and pathogenesis through two UPR signaling pathways both in vitro and in vivo.

SUBMITTER: Lee YR

PROVIDER: S-EPMC5765116 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo.

Lee Ying-Ray YR Kuo Szu-Han SH Lin Ching-Yen CY Fu Po-Jung PJ Lin Yee-Shin YS Yeh Trai-Ming TM Liu Hsiao-Sheng HS

Scientific reports 20180111 1

Dengue virus (DENV) utilizes the endoplasmic reticulum (ER) for replication and assembling. Accumulation of unfolded proteins in the ER lumen leads to ER stress and unfolded protein response (UPR). Three branches of UPRs temporally modulated DENV infection. Moreover, ER stress can also induce autophagy. DENV infection induces autophagy which plays a promotive role in viral replication has been reported. However, the role of ER stress in DENV-induced autophagy, viral titer, and pathogenesis remai ...[more]

PMID: 29323257

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Project description:BackgroundWe previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.ResultsWe demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.ConclusionsIn this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.

Project description:Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.

Dataset Information

Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo.

Publications

Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo.

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