Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways

Manish Sharma a, 1, Kiran Bala Sharma a, Shailendra Chauhan a, Sankar Bhattacharyya a, Sudhanshu Vrati a, b, **, Manjula Kalia a, *
aVaccine & Infectious Disease Research Centre, Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, India
bRegional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India

a r t i c l e i n f o

Article history: Received 15 May 2018 Accepted 20 May 2018 Available online xxx

Keywords:
Japanese encephalitis virus Diphenyleneiodonium
N-Acetyl-L- cysteine Reactive oxygen species Autophagy
ER stress
a b s t r a c t

Diphenyleneiodonium (DPI) and N-acetyl-L-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fi broblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy in- duction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of in- hibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.
© 2018 Elsevier Inc. All rights reserved.

1.Introduction

Japanese encephalitis virus (JEV) belongs to the Flaviviridae family that includes several arthropod borne medically important viruses like West Nile virus (WNV), Dengue virus (DENV), and Zika virus [1,2]. JEV is the leading cause of virus induced encephalitis in endemic regions of south-east Asia and western Pacific [3].
Virus infection results in subversion of host cellular pathways to favour virus replication, and this leads to activation of several stress responses in the infected cell. The three major effects seen in fl a- vivirus infections are induction of oxidative stress, ER stress leading to activation of the UPR and autophagy [4e8]. Autophagy induction in a virus infected cell further influences innate immune responses,

inflammation, metabolic pathways, cell adhesion and cytoskeletal organization, survival, and thus directly impacts pathogenesis [9,10].
Both oxidative stress and ER stress have been shown to be the major contributors of autophagy induction in RNA virus infections [7,11e13]. Recent studies from our laboratory have established an anti-viral role of autophagy for JEV replication, and a crucial role of the XBP1 and ATF6 ER stress sensors for autophagy activation in JEV infected neuronal cells [6,7].
Oxidative stress caused by free radical generation is a major contributor to JEV induced cell death, neuroinfl ammation and pathogenesis [14e17]. To evaluate the contribution of oxidative stress in JEV induced autophagy we employed two widely used anti-oxidants- DPI and NAC, and checked their effect on levels of ROS and autophagy induction in the context of JEV infection. DPI, an inhibitor of flavoenzymes, including NADPH oxidase [18,19], has

Abbreviations: DPI, Diphenyleneiodonium; NAC, N-acetyl-L-cysteine; JEV, Japa- nese encephalitis virus; ROS, reactive oxygen species; UPR, unfolded protein response.
* Corresponding author.
** Corresponding author. Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India.
E-mail addresses: [email protected] (S. Vrati), [email protected] (M. Kalia).
1 Department of Neuroscience, The Scripps Research Institute, Jupiter, FL, USA.
been extensively used in several studies to inhibit ROS production. NAC is a powerful scavenger of free-radicals that enhances gluta- thione biosynthesis [20,21]. We observed that while NAC reduced ROS levels in several different cell types subjected to pharmaco- logical oxidative stress or infected with JEV, DPI increased ROS levels and thus, enhanced oxidative stress. Further, though DPI

https://doi.org/10.1016/j.bbrc.2018.05.149

0006-291X/© 2018 Elsevier Inc. All rights reserved.

treatment prevented autophagy induction, as monitored by LC3-II conversion in JEV infected Neuro2a cells, and this effect was mediated by block of the ER stress pathways and not due to reduction of oxidative stress. Our data suggest that DPI sensitive enzymes are involved in the activation of ER stress sensors during JEV infection.

2.Material and methods

2.1.Cells and virus

Mouse neuroblastoma (Neuro2a) and porcine stable kidney (PS) cells were obtained from Cell Repository, National Centre for Cell Sciences, Pune, India. Mouse embryonic fi broblasts (MEF) were a gift from Prof. Mizushima and obtained through RIKEN- BioResource Cell Bank (RCB 2710). HeLa (ATCC-CCL2) cells were purchased from ATCC. For all studies JEV isolate Vellore P20778 generated in PS cells was used. JEV was titrated by plaque assay formation on PS monolayers as described earlier [22]. Plaque assay results are presented as Mean ± standard deviation (SD) of three independent experiments.

2.2.Reagents, antibodies and plasmids

Antibodies against CHOP/GADD153 (ab11419), SQSTM1 (ab56416) were from Abcam; GAPDH (2118), LC3 (3868) were from Cell Signalling Technology. Thapsigargin (T9033), DMF (242926), DPI (D2926) and NAC (A9165) were from Sigma. JEV-NS3 rabbit polyclonal antibody has been described before [6]. The plasmid- p5xATF6-GL3 (#11976) was obtained from Addgene (deposited by Ron Prywes) [23]. The plasmid pCI-Neo-hRluc was a gift from Dr. Witold Filipowicz (FMI, Basel, Switzerland).

2.3.Cell treatment and virus infection

Cells were treated with vehicle control/Thapsigargin/DMF/NAC/
DPI, mock-or JEV-infected and processed for western blotting, or RNA extraction. JEV infection was done at MOI 5 for 1 h at 37 ti C which results in an infection effi ciency of ~90%. Following infection, cells were washed twice with PBS and complete medium was added. ER stress inducer Thapsigargin was added to cells at 1 mM for 8 h. DMF and DPI were dissolved in DMSO and used at 70 mM and 1 mM respectively. NAC was dissolved in water and used at 3 mM. DPI and NAC were added to JEV infected cells at 16 h post infection (pi) and maintained till harvest (24 hpi). After infection/treatment cells were washed twice with PBS and processed. Culture super- natants was collected for plaque assays at 24 hpi.

2.4.Assay for Xbp1 splicing

The Xbp1 splicing assay was done as described previously [24]. The Xbp1 transcript was amplifi ed using the following primers Xbp1-F (5-AAACAGAGTAGCAGCGCAGACTGC-3) and Xbp1-R (5- TCCTTCTGGGTAGACCTCTGGGAG-3).

2.5.ATF6 promotor activation

Neuro2a cells were co-transfected with 1 mg of p5XATF6-GL3 and 100 ng of pCI-Neo-hRluc. After 24 h, cells were mock/JEV (5MOI) infected for 24 h. DPI/NAC was added to JEV infected cells at 16 hpi and maintained till harvest. Quantifi cation of Firefl y and Renilla luciferase activities in the samples was done using Dual- Luciferase Reporter Assay System, Promega and analyzed on Orion II microplate Luminometer (MPL4, Berthold Detection Sys- tem, Germany). Results were plotted as relative luciferase activity

(fi refl y/renilla) normalized to mock infection/treatment.

2.6.Determination of oxidative stress

The oxidative stress indicator CM-H2DCFDA (Life Technologies) was used to quantify ROS levels. Neuro2a cells were infected with JEV at 5 MOI for 24 h, washed with pre-warmed PBS and incubated with 5 mM CM-H2DCFDA in PBS for 15 min at 37 ti C. Dye was removed by two washes in PBS. DMF (70 mM, 8 h) was used as a positive control for ROS production. DPI (1 mM) and NAC (3 mM) were added to cells at 16 hpi and maintained till harvest. Fluores- cence intensity was quantified by flow cytometry (BD FACS-Canto II) and data was analyzed by FlowJo software.

2.7.Statistical analysis

Student t-test was used for statistical analysis. Differences were considered signifi cant at values of *P < 0.05; **P < 0.01.

3.Results

3.1.DPI enhances oxidative stress in JEV infected cells

Production of ROS is the leading cause of oxidative stress in RNA virus infections. To assay oxidative stress in JEV-infected cells we measured the oxidation of H2DCFDA, an indicator of production of intracellular ROS. This was tested in three cell lines- Neuro2a (mouse neuronal), MEF (mouse embryonic fi broblasts) and HeLa (human epithelial). The pharmacological reagent DMF (ROS inducer) was used as a positive control along with JEV infection, and, the anti-oxidants NAC and DPI were tested for their effect on levels of ROS generation. JEV infection, and DMF treatment lead to an increase in ROS levels in all the three cell types as compared to mock-infected cells (Fig. 1). NAC treatment was effective in reducing ROS levels comparable to those of control cells or even lower. DPI treatment however, further increased ROS levels in both mock- and JEV- infected cells. This was seen in all the three cell types suggesting that DPI treatment does not inhibit but rather enhances ROS levels during JEV infection (Fig. 1).

3.2.DPI blocks activation of autophagy and ER stress pathways in JEV infected Neuro2a cells

Since autophagy has been linked to levels of oxidative stress in cells we tested the effect of both DPI and NAC on the induction of autophagy and activation of ER stress pathways during JEV infec- tion. Levels of LC3-II and SQSTM1, which are the markers of auto- phagy induction and autophagosome substrate respectively were estimated by western blotting [25,26]. We observed that DPI completely and NAC partially blocked JEV induced autophagy as seen by decreased levels of the autophagy marker LC3-II and a corresponding increase in levels of SQSTM1 (Fig. 2A). However since DPI treatment was enhancing oxidative stress in JEV- infected Neuro2a cells, its effect on autophagy inhibition was not due to any effect on ROS levels.
Recent studies from our laboratory have demonstrated a crucial role of the ER stress responses for induction of autophagy during JEV infection [7]. Therefore, we tested the effect of DPI and NAC treatment on the activation of ER stress pathways in JEV- infected cells. As a readout of PKR-like ER kinase (PERK) activation we looked at expression levels of the transcription factor CHOP (GADD153), which is a well characterized readout of persistent ER stress [27]. As expected, expression of CHOP was observed in JEV- infected cells [7,28], however, in DPI- treated JEV- infected cells expression of CHOP was significantly suppressed (Fig. 2A).

M. Sharma et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6 3

Fig. 1. Effect of DPI and NAC on ROS levels in JEV infection. Mock/JEV (5MOI) infected-Neuro2a/MEF/HeLa cells were treated with vehicle control/DPI/NAC at 16 hpi. In the DMF treated group cells were treated with DMF alone/DMP þ DPI/DMF þ NAC. After 8 h (24 hpi), levels of ROS were estimated by treating cells with CM-H2DCFDA and measuring DCF fluorescence intensity. The experiment was performed using biological triplicates, and similar trends were observed in three independent experiments. (*, P < 0.05; **, P < 0.01).

Fig. 2. Effect of DPI and NAC on activation of autophagy and ER stress in JEV infection. (A) Neuro2a cells were mock/JEV infected at 5MOI and treated with DMF/DPI/NAC as described in Materials and Methods. At 24 hpi cell lysates were blotted with the indicated antibodies (upper panel), and total RNA was amplified using Xbp1 primers to check for Xbp1 splicing (lower panel). Blots shown are from one representative experiment and similar results were observed in three independent experiments. (B) Neuro2a cells were co- transfected with plasmids p5xATF6-GL3 and pCI-Neo-hRluc at 24 h post-transfection were mock/JEV infected (5MOI, 24 h) and treated with vehicle control/DPI/NAC. Firefly and renilla luciferase activities were quantified in the cell lysates. Graph shows mean relative luciferase activity (firefly/renilla) normalized to mock infection from three independent experiments. (*, P < 0.05; **, P < 0.01).

We next characterized the effect of DPI on the second ER stress pathway mediated by IRE1-XBP1. Activation of IRE1 leads to an unconventional post-transcriptional splicing of Xbp1 mRNA (uXbp1) to produce a spliced form of Xbp1 (sXbp1) mRNA that leads to translation of the XBP1s protein, which functions as a tran- scription factor leading to activation of several ER stress elements genes [29,30]. In an RT-PCR reaction uXbp1 is observed as a 442 bp fragment and sXbp1 as 416 bp fragment [24]. While NAC treatment had no effect on Xbp1 splicing in JEV-infected cells, DPI inhibited this pathway also in JEV infection (Fig. 2A).
ATF6 is the third sensor of ER stress, which is targeted to the Golgi, where it is proteolytically cleaved, and its 50 kDa DNA- binding domain translocates to the nucleus to activate gene expression [31]. Activation of ATF6 in JEV infection can be seen
through an ATF6 promoter activation assay [7] (Fig. 2B). Neuro2a cells were co-transfected with plasmids p5xATF6-GL3 and pCI- Neo-hRluc. In response to JEV infection signifi cant activation of the ATF6 promoter was observed as measured by relative increase in luciferase activity (Fig. 2B), which was partially inhibited by NAC, but reduced below basal levels by DPI treatment. Collectively these data demonstrate that DPI inhibits ER stress induced during JEV infection, indicating the potential role of DPI sensitive enzymes in the activation of ER stress pathways.
We further tested if DPI- mediated inhibition of ER stress pathways would also be observed in Thapsigargin-treated cells, a widely used ER stress inducer that depletes the ER calcium stores, and also inhibits autophagy fl ux by blocking fusion of autophgo- somes to lysosomes [32,33]. Both DPI and NAC had no effect on

Thapsigargin-induced ER stress-as seen by CHOP production, Xbp1 splicing and ATF6 promoter activation (Fig. 3). This is not unex- pected since the mechanism of UPR induction by Thapsigargin vs JEV infection is likely to be different.

3.3.Effect of DPI on virus replication

We further tested the effect of DPI and NAC treatment on pro- duction of infectious virions assayed by virus titres in the super- natant. DPI treatment significantly reduced virus titres while NAC treatment did not have any effect (Fig. 4). Since DPI treatment in- hibits virus-induced ER stress pathways, it is likely that this has an overall dampening effect on virus replication and egress, which occurs in close association with ER derived membranes. The inhi- bition mediated by DPI on JEV infectious virus particle release has also been shown in an earlier study [16].

4.Discussion

JEV, a highly pathogenic fl avivirus, crosses the blood-brain barrier and induces massive neuroinfl ammation and neuronal cell death. Generation of ROS following JEV infection has been reported in several studies [14,16,34], and is implicated to be one of the prime causes of JEV- induced inflammation and pathogenesis [15e17,35]. JEV infection also results in autophagy upregulation in neuronal and non-neuronal cell types, which primarily exerts an anti-viral infl uence [6,36]. Recent studies from our laboratory have established a role of the XBP1 and ATF6 ER stress-sensors for JEV- mediated autophagy induction in neuronal cells [7]. Further, the IRE1-dependent decay (RIDD) pathway is also activated in JEV- infected neuronal cells and benefi ts virus replication [37].
The link between oxidative stress and autophagy has been established in several studies [38e40]. ROS has been shown to

Fig. 4. Effect of DPI and NAC on JEV titres. JEV titres at 24 hpi, as determined by plaque assays in JEV infected Neuro2a cells treated with DPI/NAC as described in Material and Methods. *P < 0.05.

specifi cally regulate ATG4 activity for autophagosome elongation [40]. Oxidative stress can also upregulate transcriptional factors such as HIF-1, p53, FOXO3 and NRF2 which have been linked to autophagy activation in different studies [39,41]. DPI has been shown to block autophagy in several independent studies [42e46]. Further many studies have hypothesised and extrapolated the requirement of ROS for autophagy based on treatment with anti- oxidants like NAC and DPI [43e46].
To examine the role of oxidative stress in JEV- induced auto- phagy, we tested two widely used anti-oxidants DPI and NAC in our experimental set-up. We observed that while DPI treatment showed an autophagy block in JEV- infected neuronal cells, it was not because of inhibition of oxidative stress but due to the ER stress pathways. Further, DPI treatment augmented oxidative stress in JEV

Fig. 3. Effect of DPI and NAC on Thapsigargin induced ER stress. Neuro2a cells were treated with vehicle control/Thapsigargin, followed by treatment with DPI/NAC. At 8 h cell lysates were blotted with the indicated antibodies (upper panel), and total RNA was amplifi ed using Xbp1 primers to check for Xbp1 splicing (lower panel). Blots shown are from one representative experiment and similar results were observed in three independent experiments. (B) Neuro2a cells were co-transfected with plasmids p5xATF6-GL3 and pCI-Neo- hRluc at 24 h post-transfection were treated with vehicle control/Thapsigargin followed by treatment with DPI/NAC. Firefly and renilla luciferase activities were quantified in the cell lysates. Graph shows mean relative luciferase activity (firefly/renilla) normalized to mock infection from three independent experiments.

M. Sharma et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6 5

infection in various cell types.
DPI has been extensively used as an inhibitor of NADPH oxidase (NOX) [18,19,47]. However because of its wide inhibitory potential for all Flavin-dependent enzymes its specificity for use as a NOX inhibitor has been questioned [48e50]. Data on regulation of intracellular ROS by DPI are highly controversial as several studies have also shown that DPI enhances glutathione efflux from cells, generates ROS and augments oxidative stress [51e54]. DPI can also induce mitochondrial superoxide production and can sensitize cells to different apoptotic insults [52,55,56]. In our study, we observed that DPI did not inhibit but further enhanced ROS levels in JEV infection of different cell types-neuronal, fi broblast and epithelial cells. The anti-oxidant NAC that is known to enhance glutathione biosynthesis, was effective in quenching pharmacologically (DMF) and JEV- induced oxidative stress.
We observe that NAC can partially, and, DPI can completely, block JEV- induced autophagy in neuronal cells. While NAC treat- ment quenched ROS it did not exert any effect on the activation of ER stress markers like CHOP production, Xbp1 splicing, however ATF6 activation was suppressed significantly. Previous studies from our lab have clearly demonstrated that RNA intereference- mediated depletion of ATF6 and XBP1 protein significantly blocks JEV- induced autophagy independently of each other. Thus the partial block of autophagy observed in NAC treatment could be mediated through inhibition of the ATF6 arm of ER stress. DPI did not inhibit but rather enhanced ROS in both mock- and JEV- infected cells. DPI further completely abrogated the ER stress re- sponses in JEV- infected cells suggesting that DPI is acting at a yet undetermined step crucial for the induction of UPR. Studies have shown that the fl avonol Quercetin, activates the yeast ER stress- induced kinase-endonuclease IRE1's RNAase activity that is essential for Xbp1 splicing. Structural studies showed that quercitin bound a ligand binding pocket at the dimer interface of IRE1's ki- nase extension nuclease domain, suggesting that endogenous cytoplasmic ligands can function alongside stress signals from the ER lumen to modulate IRE1 activity [57]. The complete inhibition of autophagy induction seen in our study in DPI treated JEV-infected cells, suggests the involvement of DPI sensitive (fl avin dependent/
independent) enzymes in the activation of ER stress signalling pathways during JEV infection.

Funding

This work was supported by a DST grant (SERB/EMR/2015/
001506) to MK. Transparency document
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.149.

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