Pharmacological Manipulation of UPR: Potential Antiviral Strategy Against Chikungunya Virus

Abstract

Abstract

Viruses invade the host cells and maneuver the cellular translation machinery to translate the viral proteins in substantial amounts, which may disturb Endoplasmic Reticulum homeostasis leading to induction of Unfolded Protein Response (UPR), a host response pathway involved in viral pathogenesis. Here, we investigated the effect of UPR pathways on the pathogenesis of chikungunya virus infection. We observed that chikungunya virus mediated the modulation of UPR. A positive modulation was observed in the activation of IRE1 and ATF6 branch while the PERK branch of UPR observed suppressed upon virus infection. We further investigated the effect of the inhibition of UPR pathways on chikungunya virus replication using inhibitors for each branch. Cells treated with 3-ethoxy-5,6-dibromosalicylaldehyde (IRE1 inhibitor) and AEBSF (ATF6 inhibitor) significantly inhibits the viral replication process. This study has provided a novel perspective in designing antivirals against chikungunya virus.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01046-5.

Introduction

Chikungunya virus (CHKV) has been the causative pathogen of several episodes of epidemics in recent years, which have substantially impacted the global healthcare system. In the year 2022, more than 2 lac cases have been reported worldwide, majority of cases were reported from Brazil. Although the mortality rate associated with the virus is rare, the infection often leads to an unanticipated increase in the cases of crippling polyarthralgia which can last for weeks or even several years [1]. Despite the global prevalence of CHKV infections, there is no specific anti-viral treatment approved by FDA to date and the treatment is restricted to anti-inflammatory drugs that provide symptomatic relief only. Hence, there is a need for a better understanding of viral pathogenesis for designing alternative antiviral approaches.

Unfolded Protein Response (UPR) pathway is a host metabolic pathway that has potential to manipulate viral pathogenesis. UPR is a cytoprotective adaptive response, triggered during the accumulation of unfolded proteins in ER. Various physiological and pathological conditions, pharmacological agents, diseased states and viral infections such as dengue, hepatitis, influenza etc., cause ER dysfunction, culminating in ER stress [2]. During the accumulation of unfolded proteins in ER, the UPR sensors trigger the signaling cascade and assist in relieving the ER stress. UPR pathways restore ER homeostasis by up-regulation of the chaperones, attenuation of translation and degradation of misfolded proteins. The ER stress sensors, IRE1 and PERK, sense the ER stress and trigger a downstream signaling cascade initiated with auto-phosphorylation at the cytoplasmic domain of these effector proteins. The third ER stress sensor, ATF6 undergoes proteolytic cleavage during ER stress and act as a transcription factor for stress-responsive genes.

Several studies [3, 4] have emphasized the role of UPR during viral infection, wherein the host cell mobilizes UPR to restrict viral infection, whereas the virus impairs or orchestrates the UPR for its advantage [4]. Virus infection often leads to the disturbance of ER homeostasis by the accumulation of misfolded proteins. They hijack the host translation machinery to facilitate their replication by manipulating it to translate viral protein and exploit the endoplasmic reticulum-associated degradation (ERAD) process in degrading host cell proteins [3]. Various acute [3], chronic [4] and latent viruses [5] interact with host UPR in different ways to establish a favorable environment for the survival of the virus. UPR stimulates viral pathogenesis by attenuating the host translational machinery [2], regulating apoptosis [6], interferon response [7], autophagy [6] and modulation of host immune response [7]. Hepatitis viruses share molecular mimicry with UPR molecules and exploit UPR to set up lytic infection [4], whereas herpes viruses mobilize ERAD to degrade immune molecules and surface glycoproteins to evade immune response [5]. Similarly, in the case of the influenza virus, the IRE1 branch of UPR is induced, which is essential in viral replication [8] but blocks the replication in the case of respiratory syncytial virus [9].

In the current study, we studied the effect of chikungunya virus infection on UPR pathways, wherein IRE1 and ATF6 pathways were stimulated. We also established a direct connection between UPR response and chikungunya virus replication by blocking the individual branches of UPR. Since there are no available treatments against CHKV, alternative therapeutic interventions like modulating UPR pathways provide a new dimension that could be further explored for designing antivirals against chikungunya virus infection.

Methods

Cell Culture

The mammalian HEK293 cells and Vero cells were cultured in Dulbecco's Modified Eagle Media (DMEM, Gibco) supplemented with 10% FBS maintained at 37 °C incubator with 5% CO₂. The virus was cultured in C_6/36 cell line maintained in Minimum Essential Media (MEM; Gibco) supplemented with 10% FBS and incubated at 28 °C. The virus titer was calculated with the plaque assay in vero cells. In the viral experiments, the virus (MOI 1) was used to infect 70–80% confluent HEK293 monolayers at 37 °C.

Reagents

Tunicamycin (Cat#T7765), Dimethyl Sulfoxide (DMSO) (Cat#D2650), AEBSF (Cat#A8456) and 3-ethoxy-5,6-dibromosalicylaldehyde (Cat#SML0149) were purchased from Sigma Aldrich. The primary antibodies for western blotting: ATF6 (ab122897), GAPDH (ab9485), βeta-actin (ab8227) and IRE1-p (ab124945) were procured from Abcam and antibody eIF2α-p (Cat#9721S) was procured from Cell Signaling Technology (CST). The ATF4 antibody (PA5-27576) was procured from Invitrogen and chikungunya E1 antibody (NBP2-53111) was procured from Novus Biologicals. The HRP conjugated secondary antibodies anti-rabbit (Cat#7074) and anti-mouse (Cat#7076) were procured from Cell Signaling Technology.

Plaque Assay

Plaque assay was performed as described previously [10]. Different dilutions of viral supernatant were used to infect Vero cells for 1 h in 37 °C incubator. Post incubation, the virus was discarded and cells were washed with PBS. After washing, the cells were covered with overlay media. The plates were incubated in 37 °C incubator for 3–4 days till the plaques were visible. The overlay media was thereafter removed and the cells were fixed with 10% formaldehyde and stained with 1% crystal violet dye, followed by washing with distilled water. Finally, the number of plaques was counted corresponding to each viral dilution.

Quantitative Real-Time PCR

RNA was isolated from the HEK293 cells using the RNeasy Mini kit (Cat#74106, Qiagen). The isolated RNA was quantified by Qubit Fluorometer (Cat# Q33238, Thermo Fisher Scientific). The equal concentration (> 50 ng) of the RNA samples were reverse transcribed using high capacity reverse transcription kit (Cat#4368814, Invitrogen). The cDNA was used for gene expression study using ssofast evagreen supermix (Cat#1725202, Bio-Rad) using specific primers mentioned in supplementary Table 1. The cycling conditions were 90 °C for 2 min, followed by 35 cycles of 98 °C for 8 s, and 60 °C for 15 s. The PCR reaction was run in CFX96 Touch Real-Time PCR machine (Bio-Rad). The gene of interest was normalized to the GAPDH housekeeping gene and the data was expressed as fold change using 2^ΔΔCt.

Protein Isolation and Quantification

The total cellular protein was isolated posttreatment with 1X RIPA buffer (Cat#9806, CST) and protease inhibitor cocktail (Cat#5871, CST). Briefly, the cells were scrapped after addition of lysis buffer and the cell suspension was centrifuged at 14,000xg, 4 °C for 15 min in the refrigerated centrifuge. The protein in the supernatant was isolated and multiple aliquots were made and stored in −80 °C deep freezer. The protein sample was quantified with a BCA kit (Cat#23225, Pierce).

Western Blotting

Equal concentration of protein (30–40 ng) was mixed with 6X Laemmli Buffer and loaded on polyacrylamide gel. After the separation of proteins on the gel, the proteins were transferred on the PVDF membrane with a Semi-dry transfer system (Cat#1703940, Bio-Rad). The membrane was blocked with 5% BSA and probed with the primary antibody. Post-incubation, the membrane was washed with 1X Tris Buffer Saline with 0.01% Tween 20 (1X TBST) before probing with the HRP labelled secondary antibody. The blot was again washed with 1X TBST multiple times, before being developed using ECL substrate (Cat#1705061, Bio-Rad) and visualized using Chemidoc imaging system (Cat#1708370, Bio-Rad).

Statistical Analysis

All the experiments were performed in triplicate and data is represented as mean ± SD. Statistical comparison among the group was performed with the Student t-test. Statistical significance was based on p-value < 0.05.

Results

Chikungunya Virus Differentially Modulates UPR Pathways

We determined the growth kinetics of the virus (MOI 1) in HEK293 cells with real-time PCR at specific time points (24 h, 48 h), which was further confirmed by western blotting. The results confirmed the efficient infection at 48 h as determined by a significant increase in expression of chikungunya envelope (CHKVE1) at RNA (Supplementary Fig. 1A) and protein level (Supplementary Fig. 1B).

The effect of infection on UPR was studied by individually studying each branch of UPR at the above-mentioned time point (48 h). In the study, tunicamycin was considered as the positive control of UPR. Tunicamycin induces ER stress by impairing the N-linked glycosylation resulting in accumulation of misfolded proteins. The activation of UPR was first studied by analyzing the expression of downstream target genes of the IRE1 and PERK pathways. As shown in Fig. 1, the real-time data demonstrate the significant upregulation in the gene expression of ATF4 and CHOP genes (downstream mediators of PERK pathway), and a spliced variant of xbp1 (xbp1s), downstream mediators of IRE1 pathway during treatment with tunicamycin (4 h) in comparison to the control cells (Fig. 1A). However, during CHKV infection the increase in expression of xbp1s and marginal decrease in the expression of ATF4 and CHOP genes was observed, suggesting the induction of IRE1 and suppression of PERK branch of UPR (Fig. 1A).

Fig. 1.

Chikungunya virus modulates UPR pathways in HEK293 cells. A Gene expression analysis of ATF4(i), CHOP(ii) and spliced xbp1(iii) in response to virus infection and Tunicamycin treatment at indicated time points. The relative gene expression of a specific gene was normalized to GAPDH; the data represents the mean ± SD of two independent experiments performed in triplicates. B Western blots analysis of ATF4(i), eIF2α-p(i), ATF6(ii) and IRE1-p(iii) using GAPDH or β-actin as loading controls. The results are representative of three independent experiments

The stimulation of the ATF6 branch was substantiated by the increase in the intensity of the cleaved ATF6 fragment (pATF6), that translocate to the nucleus and acts as an active transcription factor (Fig. 1Bii). The induction of IRE1 branch and suppression of PERK branch of UPR observed transcriptionally was also confirmed at the protein level. The upregulation in the expression of phospho-IRE1 protein (Fig. 1Biii) and downregulation in the expression of ATF4 and phospho-eIF2α protein (Fig. 1Bi) validate the induction and suppression of IRE1 and PERK arm of UPR, respectively. Taken together, the above-mentioned results suggest the modulation of different branches of UPR by the chikungunya virus; IRE1 and ATF6 branches are upregulated, whereas the PERK branch is downregulated.

Antiviral Potential of UPR Inhibitors on Viral Replication

Differential modulation of different UPR branches by the CHKV led us to investigate the effect of inhibition of individual pathways on CHKV replication. The UPR inhibitors used in the study 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, 300 µM) and 3ethoxy-5,6-dibromosalicylaldehyde (DBSA,30 µM) inhibit ATF6 and IRE1 pathway respectively. The inhibitor of PERK pathway was not used in the study as the pathway was suppressed during virus infection. HEK293 cells were treated with inhibitors in independent experiments, followed by CHKV infection in the presence of inhibitors. The virus inoculum was removed, and the cells were replenished with complete media in the presence of inhibitors. The effect of inhibitors in the presence of the chikungunya virus was analyzed at a 48 h time point, as the previous data suggested optimal infection at this time point. The inhibitory potential of the inhibitor was simultaneously validated in the UPR induced by Tunicamycin; the positive control of UPR. We observed that AEBSF downregulates the expression of pATF6N (cleaved ATF6 fragment; Fig. 2Bii); and DBSA successfully inhibits the IRE1 pathway as confirmed by the decrease in the expression of xbp1s (Fig. 2A) and phospho-IRE1 (Fig. 2Bi). These results confirm the efficacy of inhibitors in blocking the UPR pathways induced during virus infection and tunicamycin treatment.

Fig. 2.

Efficacy of specific UPR inhibitors during ER stress and their effect on viral replication. The cells were treated with/without AEBSF and DBSA in separate 6-well plates, followed by co-treatment with tunicamycin (Tunica) or Virus. A The relative expression of xbp1s gene was assessed with real-time PCR which was normalized to GAPDH. The results represent the mean ± SD of two independent experiments performed in triplicates. B Protein expression profile of phospho-IRE1 and cleaved ATF6 fragment (pATF6N) was studied with western blotting. GAPDH was used as loading controls and the image is a representative of one of the replicates of triplicates run on the same gel. C The expression of viral E1 (CHKV E1) gene and protein was analyzed by real-time PCR(i) and western blotting(ii) respectively. The individual experiments were performed with inhibitors in triplicate. The real-time data is represented on the logarithmic scale

The effect of these inhibitors on viral replication was studied by analyzing the expression of CHKVE1 at gene and protein levels. These inhibitors interfere with the viral replication process, as observed by the substantial downregulation of the viral E1 gene at the mRNA level (Fig. 2Ci). The real-time data was further corroborated by the western blotting results, where the decrease in intensity of the band of CHKV E1 protein was observed in presence of inhibitors (Fig. 2Cii). Collectively, the above-mentioned data highlight the importance of modulating UPR pathways in regulating chikungunya virus replication inside the host cell.

Discussion

Unfolded protein response is a cytoprotective signaling cascade involved in the maintenance of cellular homeostatic. But in recent years, it emerged as an important cellular pathway involved in the pathogenesis of viral infections. Depending upon the type and intensity of stress, single or multiple branches of UPR may get activated. Therefore differential activation of UPR in various viral infection are observed, which in turn demonstrates the pro-viral role (Influenza [8], HCV [4], Dengue virus [11]) or anti-viral role (TGEV [12], HSV-1 [5]) in different viruses. The primary objective of UPR pathways is to relieve the stress of unfolded or misfolded proteins from ER and restore ER homeostasis. However, in case the cells are unable to restore the ER homeostatic state, the cells adopt apoptotic pathway [2].

In this study, we reported that the chikungunya virus triggers the ATF6 and IRE1 pathways, whereas the PERK pathway gets suppressed. The CHKV infection poses a translational burden on cells by directing the cells towards the overproduction of viral proteins. Therefore, the cleaved ATF6 (50 kDa) fragment observed during virus infection acts as a transcription factor for chaperone proteins and components of ERAD pathways, thereby relieving the ER stress by assisting in protein folding.

We also observed an elevated activation of the IRE1 pathway upon CHKV infection, which is also associated with the cellular processes involved in viral pathogenesis. The IRE1 is known to modulate the cellular type 1 interferon response; which is the first line of defense against viral infection [13]. The IRE1 also regulates cell survival and apoptosis via c-Jun N-terminal kinase (JNK) signaling [9]. Although the activity of IRE1 is highly balanced; but, the endoplasmic stress beyond the threshold limit promotes IRE1 mediated cellular apoptosis as a damage control mechanism. The pro-viral role of ATF6 and IRE1 has already been demonstrated in other virus infections: Hepatitis B Virus [14], Zika virus [15], West Nile Virus [16] etc. The earlier studies on the chikungunya virus on UPR also highlighted the similar pattern of UPR activation [17]. A recent study by Rathore et al. has shown that the nsp4 protein of the chikungunya virus contributes to the repression of PERK signaling [17]. All the three UPR pathways are highly interconnected with each other and with various other cellular signaling cascades, hence the final outcome of UPR activation is difficult to predict merely from the expression data of UPR mediators.

Despite some earlier data on the activation of unfolded protein response during chikungunya virus infection, there is no study evaluating the involvement of UPR signaling in the pathogenesis of chikungunya virus. We have investigated the role of the stimulated ATF6 and IRE1 signaling cascades in chikungunya virus replication. To this end, we have treated the cells with AEBSF and DBSA, the inhibitors of ATF6 and IRE1 pathways, respectively, to evaluate the role of individual UPR pathways in viral replication. We observed that inhibition of IRE1 signaling attenuates the CHKV replication, thus suggesting the pro-viral role of IRE1 pathway during chikungunya infection. A similar pro-viral potential of IRE1 pathway has been confirmed in other viruses like Influenza virus [8] and Hepatitis C Virus [18]. Similarly, the inhibition ATF6 cleavage by serine protease inhibitor, AEBSF, also knocks down the replication of chikungunya infection. AEBSF also inhibits the replication of other viruses like Dengue virus [19], Respiratory Syncytial Virus [20]. Therefore, the ATF6 mediated induction of chaperon protein seems crucial for efficient replication of the chikungunya virus. The modulation of viral replication by UPR signaling cascades could also be contributed by the molecular links of UPR with other downstream pathways like inflammatory responses [7], autophagy [6], apoptosis [6] etc.

This is the first study of its kind, which establishes the direct co-relation between UPR response and replication of the chikungunya virus. Notably, the antiviral strategies targeting the host cellular machinery have an advantage over the others, as they are less susceptible to the resistance imposed by the viruses. The strategy of inhibiting chikungunya virus infection by UPR modulators appears to be an attractive and practical strategy, since several UPR modulators are in clinical trials against cancer [21]. Our results provide a better understanding of virus-host interactions and emphasize an unexplored avenue for the designing of antiviral therapeutic against the chikungunya virus by targeting UPR pathways.

Conclusion

We have identified the implications of the UPR in chikungunya virus replication that has not been determined previously which could potentially lead to designing novel antivirals. We confirmed that chikungunya virus induces IRE1 and ATF6 branches of UPR and concluded that inhibiting these branches suppresses viral replication. The strategy of targeting the host cellular response mechanisms like UPR is a promising approach; as it will substantially reduce the chances of developing escape mutants. Our study provides a basic framework that needs to be further validated in the in vivo models of chikungunya virus infection. However, we speculate the effects to be more multifaceted than simply inhibiting viral replication, as the combined effect of the molecular pathways interlinked with UPR is challenging to predict and needs to be studied in depth in future studies.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

UPR

Unfolded protein response

CHKV

Chikungunya virus

ERAD

Endoplasmic reticulum-associated degradation

AEBSF

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride

DBSA

3-Ethoxy-5,6-dibromosalicylaldehyde

ATF6

Activating transcription factor 6

IRE1

Inositol requiring enzyme 1

PERK

PKR-like ER kinase

eIF2α

Eukaryotic initiation factor 2α

Author’s Contribution

MK and GD conceptualized and designed the study. NA performed the experiments and compiled the manuscript. SS helped in primer designing and writing the manuscript. NA, MK, GD and UD analyzed the data. MK, GD and UD reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding

The financial assistance was provided by Vallabhbhai Patel Chest Institute, University of Delhi, India.

Declarations

Conflict of interest

The authors declare they have no competing interests.

Ethical Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.