The role of the unfolded protein response in dengue virus pathogenesis

3.1 The inflammatory pathways activated in response to viral infections

Viral infections induce a potent inflammatory response in the infected cells. The complex network activates type 1 interferons (IFNs) and other antiviral mediators to eradicate the offending virus and limit its dissemination (Chang, Liao, & Lin, 2006). Proinflammatory mediators such as tumour necrosis factor α (TNFα), interleukin (IL) 6, IL-8, and RANTES recruit other cell types, induce oxidative stress, and contribute to inflammation (Valadao, Aguiar, & de Arruda, 2016). In addition, several cell death pathways are activated in the infected cells to limit infection. Viral components are detected by toll-like receptors (TLRs) and cytoplasmic helicases (Nasirudeen et al., 2011; Olagnier et al., 2014; Valadao et al., 2016). The ensuing cascade of events (Dalrymple, Cimica, & Mackow, 2015; Green, Beatty, Hadjilaou, & Harris, 2014) is shown schematically in Figure 2.

3.2 The role of UPR in the inflammatory response against viral infection

There is evidence that the UPR plays a crucial role in inflammatory mediator production during viral infections (Grootjans et al., 2016). It potentiates the production of inflammatory mediators, which limit viral replication and facilitate cell survival. The UPR and its downstream inflammatory cascade is activated by virally induced TLR signaling, NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS) produced during viral infection, and ER stress related to misfolded and unfolded proteins. Type 1 IFN and IFN-stimulated gene (ISG) produced in response to viral infection play a major role in limiting dissemination of the virus. Here, we have summarized the role of the UPR in activating inflammatory pathways in response to viral infections. We also highlight how certain viruses utilize the UPR in a proviral manner to inhibit IFN signaling.

Viral infection-induced TLR signaling can play a role in UPR activation. TLR signaling activates the IRE1 pathway leading to splicing of XBP1 (Figure 1a). XBP1s is crucial in inducing the production of TNFα, IFN-β, and IL-6 in macrophages following TLR stimulation (Kim et al., 2015; Martinon, Chen, Lee, & Glimcher, 2010). Macrophages deficient in XBP1 have an impaired cytokine response to pathogens (Martinon et al., 2010). In addition, IRE1 is able to increase IL-1β directly by inducing glycogen synthase kinase 3β (Kim et al., 2015). The IRE1-TRAF2 complex activates JNK (Figure 1a) and recruits IκB kinase to induce nuclear factor-κB (NF-κB) activation (Walter & Ron, 2011; Figure 2). Translational activation of retinoic acid-inducible gene I (RIG-I) by the IRE1-RIDD pathway further activates NF-κB-induced inflammation (Lencer, DeLuca, Grey, & Cho, 2015; Figure 2). Activation of the PERK pathway and the subsequent attenuation of protein translation increases the ratio of NF-κB/IκB resulting in increased NF-κB activity (Tam, Mercado, Hoffmann, & Niwa, 2012). Both JNK and NF-κB lead to induction of cytokines and inflammatory mediators. ER stress potentiates induction of TNFα, IL-1, IL-6, and chemokines such as CXCL1 and CXCL2. ATF6 also plays a role in activating NF-κB (Grootjans et al., 2016; Figure 2). GADD34 is important in overcoming the translation inhibition induced by phosphorylated eIF2α (Figure 1). The recovery of protein synthesis by expression of GADD34 plays a crucial role in producing IFNβ in response to viral double-stranded RNA sensing (Clavarino et al., 2012; Dalet et al., 2017). GADD34 deficiency inhibited IFNβ production and increased chickungunya viral replication in mouse embryonic fibroblasts. Despite such evidence on the role of GADD34 in enhancing innate immunity, reversing the translation attenuation could be hijacked by viruses to produce viral proteins. This will be further discussed in subsection 4.1 in the context of DENV.

In addition, viral infections produce NOX2-derived ROS. The oxidative stress cause accumulation of misfolded and unfolded proteins resulting in ER stress and activation of UPR. ROS are essential for XBP1-induced cytokine production (Grootjans et al., 2016). Concomitant activation of TLR and UPR pathways has the potential to amplify the cytokine response and determine cell survival during viral infections.

Although the UPR facilitates innate immune responses against viral infections, the literature reveals that viruses inhibit type 1 IFN signaling in a PERK-dependent manner (Liu et al., 2009). Activation of the PERK arm due to ER stress phosphorylates IFN-α/β-receptor 1 leading to its degradation. Liu et al. revealed that vesicular stomatitis virus and hepatitis C virus (HCV) infections cause PERK-dependent ubiquitination of IFN-α/β-receptor 1 and subsequent reduced type 1 IFN signaling.

4.1 The role of the UPR in facilitating viral replication in DENV infection

Increased production of viral proteins in dengue infection leads to accumulation of unfolded and misfolded proteins in the ER (Diwaker et al., 2015). This results in ER stress and thus activation of the UPR as a host response. The ensuing cascade of events described above relieves ER stress, determines cell survival, and mounts an inflammatory response to eliminate the virus. DENV, similar to many other viruses, has been found to manipulate the host UPR pathways to enhance its survival. In this section, we describe how DENV facilitates viral protein production and inhibits apoptosis to ensure enhanced viral replication.

Dengue infection induces the three arms of the UPR (Klomporn, Panyasrivanit, Wikan, & Smith, 2011; Peña & Harris, 2011; Umareddy et al., 2007). Activation of this host response is observed in immune (Klomporn et al., 2011) and nonimmune cells (Peña & Harris, 2011; Umareddy et al., 2007; Yu, Hsu, Liao, & Lin, 2006) infected with DENV. Activation of the UPR is time dependent (Peña & Harris, 2011) and also strain and serotype specific (Umareddy et al., 2007). The PERK arm is activated early in DENV infection followed by IRE1-XBP1 in midinfection and ATF6 later in the infection (Peña & Harris, 2011). Serotype-specific differences in UPR activation are seen in dengue-infected A549 cells and similarly in antibody-dependent enhancement (ADE) infection of THP-1 monocytic cells (Paradkar, Ooi, Hanson, Gubler, & Vasudevan, 2011; Umareddy et al., 2007). DENV 1 and 3 serotypes cause a stronger induction of BiP, phosphorylation of eIF2α, higher GADD34, and CHOP mRNA levels than DENV 1 and 4 (Paradkar et al., 2011). In addition, ADE infection of monocytes using different strains of DENV 2 clinical isolates (Indonesian outbreak) showed higher levels of BiP, CHOP, and GADD34 in strains causing severe disease (Paradkar et al., 2011). Fast replicating viral strains demonstrated higher ER stress. This suggests a possible role for UPR in DENV pathogenesis and severity.

Activation of the PERK arm is antiviral due to attenuation of protein translation. Peña et al. demonstrated that DENV transiently activated PERK-dependent eIF2α phosphorylation at 6 hr postinfection, which reversed rapidly causing suppression of eIF2α activation thereafter. DENV ensures continuous production of its proteins and facilitates virion production by rapidly reversing PERK activation (Figure 1b). This is presumed to be due to an increased expression of GADD34, which acts in a feedback loop to inhibit further phosphorylation of eIF2α (Peña & Harris, 2011; Umareddy et al., 2007). Pharmacological activation of the PERK arm (via nuclear transporter 4-HPR) or removing the GADD34-mediated dephosphorylation of eIF2α (using Salubrinal) results in significant inhibition of DENV viral replication (Fraser et al., 2014; Umareddy et al., 2007). It is apparent that DENV modulates the PERK pathway to overcome translation attenuation for survival, and pharmacological upregulation of this process results in enhanced antiviral activity.

Infection with DENV activates the IRE1-XBP1 pathway and a subset of XBP1-activated genes involved in ERAD, which helps to alleviate ER stress (Yu et al., 2006). XBP1 pathway activation is induced by ER-associated DENV glycoproteins (prM, E, and NS1) and smaller hydrophobic ER-anchored proteins (NS2A, NS2B, and NS4B). In addition, DENV is able to inhibit apoptosis mediators downstream of IRE1-XBP1 (Figure 1a), which results in increased cell survival and enhanced viral replication (Peña & Harris, 2011). Inhibition of XBP1 using small interfering RNA, in conjunction with DENV infection, results in reduced ER expansion, increased cytopathic effects of the virus, and increased levels of the apoptosis marker procaspase 3 (Yu et al., 2006). This is further evidence that activation of the IRE1-XBP1 pathway in dengue infection protects the cells against apoptosis and alleviates ER stress contributing to DENV pathogenesis.

4.2 Modulation of autophagy via the UPR facilitates DENV production and inhibits cellular apoptosis

Autophagy is a cellular catabolic process which promotes formation of autophagic vacuoles to degrade and recycle cellular constituents. It plays an important role in regulating cell growth and development and is implicated in certain disease pathologies (Lee et al., 2008). Dengue NS4A is an important mediator of autophagy (McLean, Wudzinska, Datan, Quaglino, & Zakeri, 2011). Activation of this antiviral response promotes removal of viral constituents and facilitates viral antigen presentation. However, DENV is known to modulate the process in a proviral manner. DENV induces autophagy resulting in protection of infected cells against apoptosis (Datan et al., 2016; Fischl & Bartenschlager, 2011; Lee et al., 2008). Lee et al. demonstrated that activation of autophagy during dengue infection results in an increase in intracellular and extracellular viral load. Double membrane vesicles formed during autophagy provide a platform for viral replication (Jain, Chaturvedi, & Jain, 2014). In addition, autophagy promotes viral transmission by evasion of antibody-dependent neutralisation (Wu et al., 2016). DENV-induced autophagy is able to facilitate β-oxidation of cellular lipids to provide adenosine triphosphate for viral replication (Heaton & Randall, 2011). It is evident that autophagy is utilised by DENV for its advantage.

Two of the mediators activating autophagy in DENV infection are ER stress and the UPR (Datan et al., 2016). The PERK pathway activated in response to ER stress upregulates ROS production and increases autophagy turnover during DENV infection. Although the contribution of ROS to autophagosome formation is modest, PERK plays an important role in maintaining high autophagy levels and contributes to production of mature and infective viral particles (Datan et al., 2016). Thus, the UPR plays an important role in mediating and activating autophagy, which facilitates viral replication.

4.3 The role of the UPR in DENV-induced inflammation

DENV, similar to many viruses, activates signaling pathways leading to production of antiviral mediators. These proinflammatory cytokines and chemokines aid eradication of DENV. Evasion of these defenses by DENV will result in enhanced viral replication. Simultaneously, overexpression of these mediators can cause harm to the host (Jaiyen, Masrinoul, Kalayanarooj, Pulmanausahakul, & Ubol, 2009; Valadao et al., 2016) and possibly contribute to the cytokine storm, which is associated with increased vascular permeability seen in severe dengue disease. The UPR is an important mediator of virus-induced inflammatory signaling. However, the role of the UPR in DENV-mediated inflammation is underinvestigated. The literature supporting this area is complicated to interpret due to the different cell systems used and the pathways investigated. We bring together what is currently understood to summarize for the field how interactions between DENV and the UPR can affect, with both proviral and antiviral outcomes, innate immune pathways such as IFNα/β, NF-κB, IFN regulatory factor 3 (IRF3), and TNF-α (Table 1).

Activation of the UPR by VER-155008 (VER) treatment in DENV infection activates innate immune factors such as PKR, IRF3, IL-1β, and NF-κB (Diwaker et al., 2015). VER causes pharmacological inhibition of GRP78 (BiP) and the enhanced innate immune activation achieved by upregulation of IRE1, PERK, and ATF6 results in increased DENV clearance. This suggests that the UPR can potentiate innate immune responses in DENV to facilitate viral clearance.

A critical antiviral mechanism against DENV is the type 1 IFN response. Patients with severe disease have lower serum IFNα levels and suppression of ISG during early DENV infection, which highlights the role of type 1 IFN in dengue pathogenesis (De La Cruz Hernandez et al., 2014; Medina et al., 2015; Singla et al., 2016). DENV, similar to many viruses, has developed strategies to evade IFN activation, signaling, and effector mechanisms. DENV inhibits IFN induction and its signaling pathways to facilitate its replication and survival (Green et al., 2014). Both TLR-induced inflammatory pathways and RIG-I/MDA-5-induced pathways are inhibited by DENV in an attempt to reduce type 1 IFN and cytokine production (Borsa et al., 2015; Chang et al., 2012; Dalrymple et al., 2015). One host response exploited by DENV to evade innate immunity is the UPR-autophagy pathway. The role of UPR-autophagy in suppressing innate immunity is well studied in infection with HCV, another Flavivirus. Activation of the UPR is required for upregulating autophagy in HCV-infected cells, and this activation significantly reduces the HCV pathogen-associated molecular pattern-mediated cytoplasmic RIG-I signaling. RIG-I activation leads to phosphorylation, dimerization, and nuclear translocation of IRF3 (Figure 2). which induces IFNβ promoter activation. Ke et al. demonstrated that UPR-autophagy mediators (CHOP, ATG5, LC3B, IRE1, PERK, and ATF6) negatively regulated HCV pathogen-associated molecular pattern-mediated IFNβ mRNA levels and IFNβ promoter activation. Interference with the UPR-autophagy pathway by stable gene knockdown (ATG5 and CHOP gene silencing) in human hepatoma cells led to increased IFNβ promoter activation and IFNβ mRNA levels in HCV. Furthermore, IFN-mediated downstream signaling detected by IFNα-stimulated ISG response element promoter activity and IFN-induced protein with tetratricopeptide repeats 1 (IFIT1/ISG56) expression was upregulated in ATG5 and CHOP gene knockdown cells in HCV infection (Ke & Chen, 2011). In the same study, the authors showed that the negative effect of UPR-autophagy on innate immune reponses is not limited to HCV but extended to DENV. IFNβ promoter activity and ISG56 expression were enhanced in cells following ATG5 and CHOP gene silencing and in Atg5−/− mouse embryonic fibroblasts in DENV infection (Ke & Chen, 2011). It is evident that UPR-autophagy plays a crucial role in inhibiting type 1 IFN activation and its downstream signaling cascades in DENV infection. Further investigation of the role of UPR-autophagy pathway in DENV-mediated inflammation is useful as repression of this cascade is an attractive antiviral therapeutic option.

DENV infection induces production of proinflammatory mediators, such as TNFα, implicated in causing severe disease (Cardier et al., 2005; Chakravarti & Kumaria, 2006). Activation of the PERK arm of the UPR in DENV infection was recently shown to upregulate Nrf2-CLEC5A-mediated TNFα production (Cheng et al., 2016). Pharmacological inhibition of Nrf2 in vivo results in reduced TNFα production in the brain of DENV-infected mice and a better clinical outcome.

The UPR has complex interactions with the anti-inflammatory and proinflammatory cascades during DENV infection. Manipulation of induction of UPR pathways during DENV infection shows that they can mediate suppression and induction of innate immune and inflammatory pathways, affecting both viral replication and immunopathology. Further investigation of these interactions would lead to a better understanding of DENV pathogenesis and help explore antiviral therapeutic targets.