Chapter 4: Discussion 4.1 Preface Egress from the infected cell is perhaps one of the most ill defined parts of the DENV life cycle. Previous work has demonstrated that this process occurs through exocytosis and is hence dependent on expression of the exocyst complex (Chen et al, 2011). This thesis clarifies that the expression of the exocyst complex is regulated at the translational level, at least in part, by the UPR (Figure 4-1). To ensure successful completion of its life cycle, DENV relies on the proteasome or increased expression of components of the UPP to alleviate ER stress, the inhibition of which leads to the decoupling of infectious virus production from RNA replication.   113     Figure 4-1. Schematic diagram of findings. 1. Proteasome inhibitors prevent polyubiquitylated misfolded proteins to be degraded by the proteasome. 2-3. Accumulation of misfolded proteins stresses the cell and activates the PERK pathway. 4. Activation of this pathway phosphorylates eIF2α. 5. Translational attenuation occurs and results in decreased levels of TC10 and EXOC7. 6. Dengue egress is inhibited due to the reduction in protein levels of these host factors necessary for virus egress.   114     4.2 Unfolded protein response during flavivirus infection The ER is an extensive membranous network that serves different specialized functions, such as intracellular signal transduction and calcium storage. In addition, most secreted and transmembrane proteins enter the lumen of the ER prior to being secreted for maturation. To maintain homeostasis in the ER, the ER has evolved three mechanistically distinct pathways collectively called the UPR. Under ER stress, ER chaperone immunoglobulin heavy chain binding protein (BiP), plays a central role in the activation of PERK, activating transcription factor (ATF6) and the ER transmembrane protein kinase/endoribonuclease (IRE-1) pathways (Ron & Walter, 2007; Schroder & Kaufman, 2005; Shen & Prywes, 2005). PERK is an ER-localized type I transmembrane protein and the PERK pathway functions as a first-response mechanism induced by various stressors and attenuates global protein synthesis via phosphorylation of eIF2α (Wek et al, 2006). PERK activation also induces the activation of C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein (GADD34). In the case of sustained ER stress, CHOP is responsible for apoptosis of the cells. Alternatively, CHOP can also function as a pro-survival transcription factor leading to induction of GADD34, a subunit of protein phosphatase 1c (PP1c) that targets the dephosphorylation of phosphorylated eIF2α (eIF2α-P) (Rutkowski et al, 2006). Failure to suppress ER stress leads to activation of the ATF6 and IRE1 pathways, which act to alleviate the accumulation of misfolded proteins by up-regulating host factors that increase the capacity of the ER to handle the synthesis of nascent proteins   115     (Ron & Walter, 2007). The life cycle of DENV and other members of the Flaviviridae family depend heavily on the host ER to translate, replicate, and package their genome (Lindenbach et al, 2007). If ER stress hampers the completion of its life cycle, DENV must thus be able to modulate the ER stress response to survive. Indeed, several studies have shown that DENV infection modulates the ER stress response in a time-dependent manner (Paradkar et al, 2011; Pena & Harris, 2011; Umareddy et al, 2007) (Yu et al, 2006). Early DENV2 infection has been shown to trigger and then suppress PERK-mediated eIF2α phosphorylation, and the IRE1 and ATF6 pathways were then activated in the later stages of infection (Pena & Harris, 2011). Consequently, inhibition of the proteasome could induce additional UPR that then overcomes the ability of DENV to regulate ER stress. Critically, inducing ER stress with an agonist without inhibiting proteasome function recapitulated the observed down-regulation of EXOC7 and TC10 protein levels along with the decoupling of infectious virus production from viral RNA replication. Supporting our data, salubrinal, a drug that inhibits eIF2α dephosphorylation, thereby increasing phosphorylated eIF2α levels was previously shown to reduce the production of infectious viruses (Umareddy et al, 2007). The activation of individual branches and components of the UPR by other members of the Flaviviridae family have also been reported. Studies with hepatitis C virus have demonstrated activation and suppression of the PERK pathway in a time-dependent manner (Pavio et al, 2003). Also, infection with Japanese encephalitis virus was shown to induce the IRE pathway, protecting cells from virus-induced cytopathic effects (Yu et al, 2006), whereas WNV induction of the UPR was shown to lead to   116     CHOP-mediated apoptosis (Medigeshi et al, 2007). Taken together, these studies suggest that flaviviruses have evolved to manipulate the UPR, rendering the UPR as a potential anti-viral host target. However, no therapeutic that specifically targets the three sensors of the unfolded protein response, has been licensed, likely due to the cellular toxicity it may cause. 4.3 Repurposing proteasome inhibitors as an anti-flaviviral therapeutic On the other hand, proteasome inhibitors are currently indicated in the treatment of certain hematological malignancies. The much greater sensitivity of myeloma cell lines and mantle cell lines to proteasome inhibition compared with normal peripheral blood mononuclear cells is poorly understood. The UPP plays a critical role in regulating ER stress to enable DENV to complete its life cycle by egressing cells through exocytosis. Perturbing this pathway is utilized by the Ae. aegypti midgut to inhibit continued infectious DENV production without harm to itself and this same approach could potentially be exploited as a therapeutic strategy in dengue. Indeed, the potency of proteasome inhibition as an anti-dengue strategy is suggested by the low nanomolar EC50 of bortezomib, one of the first FDA-approved proteasome inhibitor, in DENV-infected primary monocytes. Similarly, bortezomib treatment in an immunocompetent mouse model was able to reduce plasma leakage, the degree of thrombocytopenia as well as the pro-inflammatory responses. At a mechanistic level, we have demonstrated that proteasome inhibition could inhibit virus egress and hence spread within mammals. With a good understanding of its antiviral action, the potential of bortezomib or other proteasome inhibitors to serve as an anti-dengue or   117     even anti-flaviviral therapy will need to be explored in clinical safety and efficacy trials. Indeed, a known side effect of bortezomib is thrombocytopenia although this is only observed in multiple myeloma patients after weeks of continuous treatment. As treatment for dengue would not exceed a week, the side effects observed only after prolonged therapy may not be clinically relevant for dengue. Therefore, it is also unlikely that bortezomib would exacerbate the situation in patients who present with the symptoms of thrombocytopenia.  One limitation, however, is that bortezomib is given subcutaneously or intravenously to patients. This is not ideal for any antidengue therapeutics, as injections are not recommended for dengue patients having the tendency to bleed. Opportunely, this problem can be circumvented by the recent introduction of ixazomib, the first oral proteasome inhibitor that is currently undergoing Phase clinical studies (Moreau, 2014). Table 4-1 lists the current development of various proteasome inhibitors undergoing different phases of clinical trials. While we have demonstrated the inhibition of virus egress as the antiviral mechanism effected by proteasome inhibition, it is interesting that proteasome inhibitors may have other modes of antiviral action. The UPP has also been shown to be critical for the life cycle of Nipah virus. Inhibition of the proteasome led to impaired nuclear export of the viral matrix protein to the cytoplasm (Wang et al, 2010). Studies on retroviruses have also demonstrated that disruption of the proteasome function depletes the free ubiquitin pool (Mimnaugh et al, 1997), which is necessary for the ubiquitylation of late domain on Gag protein for proper viral budding (Patnaik et al, 2000; Schubert et al, 2000). While such mechanisms could contribute to our observed inhibition of DENV replication, our findings suggest another complementary   118     mechanism that impairs flaviviral life cycle. Altogether, proteasome inhibition could be a broad spectrum antiviral approach against viruses that egress cells via exocytosis or which requires ubiquitylation to regulate the functions of specific viral proteins.   119     Drug   Company   Binding   Route   Status   Bortezomib   (Velcade)   Millennium   Pharmaceuticals   Reversible   IV,  SC   FDA-­‐approved  for   multiple  myeloma   Carfilzomib     (Kyprolis)   Onyx   Pharmaceuticals   Irreversible   IV   FDA-­‐approved  for   multiple  myeloma   Oprozomib     (ONX  0912)   Onyx   Pharmaceuticals   Irreversible   Oral   Phase  I/II   Ixazomib  citrate     (MLN9708)   Millennium   Pharmaceuticals   Reversible   IV,  Oral   Phase  III   Marizomib   (NPI-­‐0052)   Nereus   Pharmaceuticals   Irreversible   Oral   Phase  I   Delanzomib   (CEP-­‐18770)   Cephalon  Inc.     Reversible   Oral   Phase  I/II   Table 4-1 Current developments of proteasome inhibitors undergoing different phases of clinical trials.   120     4.4 Learning from Ae. aegypti mosquito Unlike infection in humans, dengue infection in mosquitoes is nonpathogenic. It is interesting that the mosquito midgut down-regulates several components of the UPP naturally to inhibit persistent infectious DENV production. One explanation may be transcriptomic changes in the mosquito midgut upon ingestion of a bloodmeal or during its gonotrophic cycle. Both UBE2A and DDB1 act upstream of the proteasome in the UPP; the former belonging to the E2 ubiquitin-conjugating enzyme family (Jentsch et al, 1987), and the latter functioning as an adaptor molecule for the cullin 4ubiquitin E3 ligase complex (Higa et al, 2006). UBE2A targets several short-lived regulatory proteins for polyubiquitylation and subsequent turnover by the 26S proteasome (Jentsch et al, 1987). DDB1 has been shown to facilitate the ubiquitination and subsequent proteasome-mediated degradation of STATs for the Rubulavirus genus of Paramyxoviridae (Precious et al, 2005; Ulane et al, 2005). Our findings are concordant with previous studies in female mosquitoes where various UPP-specific genes such as TSG101 (AAEL012515), NEDD4 (AAEL002536) and SCF ubiquitin ligase (AAEL004691) have also been identified as critical host factors for DENV replication (Guo et al, 2010; Mairiang et al, 2013; Sim & Dimopoulos, 2010). Due to genetic variability across various mosquito strains and DENV-2 strains, as well as variation in the methodology of the experiments such as data analysis and time-points used, it is unsurprising that the UPP-specific genes detected in these studies were not the same individual genes. As these molecules function to signal for the activation of the effector of the UPP, the proteasome, we reasoned that DENV is dependent on the pathway rather than signaling intermediates   121     for successful completion of its life cycle. Down-regulation of several genes in the UPP, along with the midgut barrier (Gomez-Machorro et al, 2004), forms a major part of the mosquito’s response that restricts DENV infection. Multiple quantitative trait loci have been associated with the midgut barrier, but the operating mechanisms of specific genes involved remain to be fully determined (Black et al, 2002). Mosquito genes and physiological pathways related to innate immunity, redox activity, fat, protein and carbohydrate production and metabolism were found to be modulated in response to DENV infection (Behura et al, 2011; Tchankouo-Nguetcheu et al, 2010). These observations come from multiple studies of specific mosquito tissues and time points following infection, and used different combinations of Ae. aegypti strains and DENV-2 strains. It will be interesting to analyze the RNAseq data on the mosquito midgut transcriptome during DENV infection and compare these results with existing data available. Studying how these responses limit DENV infection without any apparent harm to the mosquito (Fragkoudis et al, 2009), which contrast with the human host response to DENV that is intimately linked with dengue pathogenesis (Whitehorn & Simmons, 2011), offers a hitherto unexplored opportunity for therapeutic discovery. 4.5 Beyond the anti-viral effects of bortezomib: Potential use as an adjuvant In the scope of this thesis, we show that bortezomib inhibited virus egress and induced apoptosis in primary monocytes. This raises the possibility that DENV ‘trapped’ in the cells could firstly, increase MHC presentation of viral antigens in infected cells and secondly, allow cross-presentation of viral antigen via apoptotic cells. The former is unlikely because bortezomib was demonstrated to down-regulate   122   Viral RNA was extracted from virus suspensions using the QIAamp® Viral RNA Mini kit (Qiagen) and a one-step qRT-PCR was performed using the SuperScript™ III Platinum® One –Step Quantitative RT PCR System (Invitrogen). The copy number detected was calculated by generating a standard curve from a plasmid that contained the region of interest. Total RNA was isolated using RNeasy® Mini kit (Qiagen) and reverse transcription was performed using the SuperScript™ III FirstStrand Synthesis System. qRT-PCR was carried out using SYBR Green PCR Master Mix and the LightCycler® 480 System (Roche) using the primers listed in table S2. Expression values were normalized against GAPDH and technical duplicates were run for each sample. Primary monocytes isolation Venous blood from the principal investigator was collected in BD sodium heparin vacutainers (Biomed Diagnostics). The blood was then diluted with volumes of 0.5% BSA (Sigma Aldrich) in phosphate buffered solution (PBS, 1st Base) (0.5% PBS/BSA) and carefully layered onto Ficoll-hypaque (GE Healthcare). The blood was then centrifuged at 750 × g, without brakes. The interphase cells containing the peripheral blood mononuclear cells (PBMCs) were aspirated and transferred to a clean tube. The PBMCs were washed three times with 0.5% PBS/BSA and resuspended in growth medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin). The cells were then seeded into T75 tissue culture flasks (NUNC) at ×107/flask and incubated at 37°C, 5% CO2 for 2.5 h, to allow plastic adherence of the monocytes to the flask surface. The adhered monocytes were washed five times with PBS to remove the non-adherent lymphocytes and replenished with fresh growth medium. These monocytes were allowed to recover overnight at 37°C, 5% CO2, before use in experiments. DiD labeling of DENV2 DiD labeling of DENV2 was performed as previously described (49). 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The UPP has also been shown to be critical for the life cycle of Nipah virus. Inhibition of the proteasome. changes in the mosquito midgut upon ingestion of a bloodmeal or during its gonotrophic cycle. Both UBE2A and DDB1 act upstream of the proteasome in the UPP; the former belonging to the E2 ubiquitin- conjugating