Copyright by Choy Ming Ju Milly 2015     Abstract The mosquito-borne dengue virus (DENV) is a cause of significant global health burden. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remain ill defined. We show here that a functional UPP is critical for virus egress in both Aedes aegypti mosquitoes and human monocytic cell lines. Using RNA inference studies, we show in vivo that knockdown of ubiquitin proteasome pathway-related genes, including proteasomal subunits, ß1, ß2 and ß5 decouples RNA replication from infectious titer production in the mosquito midgut. Mechanistically, inhibition of proteasome function prevented virus egress by exacerbating endoplasmic reticulum (ER) stress through the unfolded protein response (UPR). UPR-induced translational repression reduced overall protein levels of the exocyst components needed for exocytosis. This mechanism also appears to be amenable for clinical translation as inhibition of UPP in primary monocytes with the licensed proteasome inhibitor, bortezomib, inhibited DENV titers even at low nanomolar drug concentration. Furthermore, we show in vivo in a wild type mouse model that DENV replication and spread in the mouse spleen is exquisitely sensitive to proteasome inhibition. The mechanism of action suggests that such a therapeutic approach may apply to other viruses that rely on exocytosis for virus egress to complete their life cycle.   iv     Acknowledgements I would like to express my heartfelt gratitude to Professor Duane J Gubler and Associate Professor Ooi Eng Eong for their mentorship throughout the course of my study. I thank them for being ever so generous in sharing their wealth of knowledge and experiences with me. My sincere appreciation is extended to my thesis advisory committee members, Professor Subhash Vasudevan and Assistant Professor Ashley St John for their invaluable advice and critical suggestions. Special thanks to Summer Zhang, Tan Hwee Cheng, Mah Sook Yee, Angelia Chow, Brett Ellis, Jason Tang, October Sessions and all my colleagues at Duke-NUS Graduate Medical School for helping me in one way or another. Lastly, I would like to acknowledge my loved ones, especially my mum and husband for their encouragement all these years. My elder sister, Julieanne is also instrumental in sparking my interest in science at a very young age. I am glad to be able to share my successes and failures with them. This body of work is dedicated to my little baby, Ethan, who is the main source of my inspiration.   v     Contents Title Page      i   Abstract  Signature    ii   Copyright    iii   Abstract   .  iv   Acknowledgements    v   Table  of  Contents   .  vi   List of Tables    x   List of Figures   .  xi   Chapter 1. INTRODUCTION    1   1.1 Dengue . 1.1.1 Dengue – a disease of global significance 1.1.2 Dengue structure and genome 1.1.3 Clinical symptoms and disease manifestations 1.1.4 Laboratory diagnosis of dengue . 12 1.1.4.1 Immunoglobulin M (IgM) detection 12 1.1.4.2 Immunoglobulin G (IgG) detection . 12 1.1.4.3 DENV isolation and propagation 12 1.1.4.4 Quantitative real time polymerase chain reaction (qRT-PCR) 14 1.1.4.5 NS1 antigen detection 15 1.2 Role of host and viral factors in dengue pathogenesis 17 1.2.1 Host factors . 17 1.2.1.1 Epidemiological evidence for secondary infection and severe dengue . 18 1.2.1.2 T cell response . 18 1.2.1.3 Antibody-dependent enhancement (ADE) 19 1.2.2 Viral factors 23   vi     1.2.2.1 Genotype differences . 23 1.2.2.2 Glycosylation of E and NS1 proteins 24 1.2.2.3 Other viral factors 25 1.3 Animal models for dengue 26 1.3.1 Non-human primates 26 1.3.2 Laboratory mice 26 1.4 DENV infection in hosts 29 1.4.1 Life cycle of DENV 29 1.4.2 DENV infection in human 33 1.4.3 DENV infection in mosquito vector . 33 1.5 Prevention and control of dengue 37 1.5.1 Ae. aegypti – a public health scourge . 37 1.5.2 Vector control . 37 1.5.3 Vaccine development and progress 40 1.5.3.1 Live attenuated vaccines and chimeric live attenuated vaccines . 40 1.5.3.2 Bivalent or trivalent vaccines – a possibility? . 42 1.5.4 Antiviral drugs 44 1.5.4.1 Therapeutic antibodies . 44 1.5.4.2 Viral protein inhibitors 44 1.5.4.3 Antiviral therapies for supportive management of dengue . 46 1.5.4.4 Host factors inhibitors . 46 1.5.4.5 Celgosivir . 47 1.6 Ubiquitin proteasome pathway in viral infection 50 1.7 Specific aims 53 Chapter 2. Materials and Methods . 54 2.1 Mosquitoes and cells . 54 2.2 Primary monocytes isolation . 54   vii     2.3 Virus stock . 55 2.4 Plaque assay . 55 2.5 qRT-PCR . 56 2.6 DENV-2 infection in mosquitoes 56 2.7 Gene silencing assays 57 2.8 Generation of whole-transcriptome cDNA library 58 2.9 RNAseq analysis 58 2.10 DENV-2 infection in monocytes . 59 2.11 DiD labeling of DENV-2 . 59 2.12 Alexa Fluor labeling of DENV-2 60 2.13 MTS cell viability assay 60 2.14 Western blots . 61 2.15 Immunofluorescence . 61 2.16 Transmission Electron Microscopy . 62 2.17 RNase treatment of β–lactone treated cells . 62 2.18 Annexin V staining of primary monocytes 62 2.19 Bortezomib treatment in DENV-infected C57BL/6 mice . 63 2.20 Immunohistochemistry analysis of mouse spleen . 63 2.21 Measurement of hematocrit level and platelet count in whole blood 64 2.22 MCPT-1, TNF-α and IFN-γ quantification . 65 2.23 Viral load quantification in mouse spleen . 65 2.24 Statistical analysis 66 Chapter 3. Results    67   3.1 Establish a mosquito infection model at Duke-NUS Graduate Medical School . 67 3.1.1 Comparison of mosquito inoculation technique and qRT-PCR to measure DENDENV concentration in mosquitoes, vertebrate and mosquito cell cultures, and h a and human sera . 67 3.2 Investigate the role of the UPP in DENV life cycle 77   viii     3.2.1 Functional UPP is required for infectious DENV-2 production in mosquito mmidguts 77 3.2.2 Regulation of UPP specific genes decouples infectious DENV-2 production f from viral RNA replication in mosquito midguts . 83 3.2.3 Proteasome inhibition with β-lactone did not alter virus entry at non-toxic lelevels 91 3.2.4 DENV-2 egress is dependent on proteasome function . 93 3.3 Proteasome inhibition exacerbates ER stress and represses translation of E EXOC7,TC10 and EXOC1 98 3.4 Potential use of bortezomib, a proteasome inhibitor, as an anti-flaviviral drug 102 3.4.1 Bortezomib inhibits infectious DENV production in primary monocytes . 102 3.4.2 Bortezomib reduced viral load and signs of dengue pathology in C57BL/6 mmice . 108 3.5 Summary of results 112 Chapter 4. Discussion 113 4.1 Preface . 113 4.2 Unfolded protein response during flavivirus infection 115 4.3 Repurposing proteasome inhibitors as an anti-flaviviral therapeutic 117 4.4 Learning from Ae. aegypti mosquito . 121 4.5 Beyond the anti-viral effects of bortezomib: Potential use as an adjuvant . 122 4.6 Conclusion . 123 References . 124 Appendix A 147 Appendix B 223 Biography 226   ix     List of Tables Table 3.1: Comparative titration of C6/36 cell culture virus supernatants by qRT-PCR and mosquito inoculation . 73 Table 3.2: Percentage of DENV-2-infected mosquitoes after knockdown of proteasome subunits (p-value; Fischer’s Exact test) . 82 Table 3.3: Summary of Illumina HighSeq 2000 RNA-sequencing using Partek Genomic Suite v6.6 . 85 Table 4.1: Current developments of proteasome inhibitors undergoing different phases of clinical trials 120     x     this protein required mono-ubiquitylation for nuclear export. Concomitantly, depletion of free ubiquitin through proteasome inhibition inhibited the Nipah virus life cycle with exquisite sensitivity (Wang et al, 2010). Likewise, 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). For DENV, host factors belonging to the UPP required for successful infection have been identified previously in both patients and mammalian cell lines (Fink et al, 2007; Kanlaya et al, 2010; Raghavan & Ng, 2013). For instance, a profound difference in gene expression and protein levels of key components of the UPP were detected in DENV-infected cell lines and patients (Fink et al, 2007; Kanlaya et al, 2010; Sessions et al, 2013). Concordant with this, both large-scale siRNA-screens identified components of the UPP as flaviviral replication promoting factors (Krishnan et al, 2008; Sessions et al, 2009). Pharmacological inhibition of the UPP, such as proteasome inhibition (Fink et al, 2007) or interference with the ubiquitin E1 activity (Kanlaya et al, 2010) led to a significant reduction in DENV production, in vitro. However, none of the licensed proteasome inhibitors have been tested in vivo due to uncertainty on the mechanism of action on the virus life cycle. One possibility is that proteasome inhibition blocks DENV entry via endocytosis (Chu & Ng, 2004; Krishnan et al, 2008), which is dependent on ubiquitylation (Hicke, 2001). However, this process is cell-type dependent (Fernandez-Garcia et al, 2011). Furthermore, when opsonized with non- or subneutralizing levels of antibody, DENV   51     is also able to bypass endocytosis and enter cells via proteasome independent Fc receptor-mediated phagocytosis (Booth et al, 2002). Thus, if proteasome inhibition only inhibits viral entry, it would not be a suitable therapeutic strategy for secondary dengue. The dearth of knowledge warrants future work to investigate if the UPP have a role in DENV life cycle besides virus entry using relevant models of dengue infection. Elucidating the mechanisms involved would allow us to repurpose existing proteasome inhibitors in the market as a broad-spectrum anti-flaviviral therapeutic.   52     1.7 Specific aims 1. Establish a mosquito infection model at Duke-NUS Graduate Medical School. 2. Investigate if the UPP plays a role in DENV life cycle besides viral entry during dengue infection in a relevant model, Ae. aegypti and human monocytic THP-1 cell line. 3. Elucidate the mechanisms involved during proteasome inhibition in DENV life cycle.   53     Chapter 2: Materials and Methods 2.1 Mosquitoes and cells A colony of Ae. aegypti was established in 2010 at Duke-NUS Graduate Medical School from field-caught specimens in Singapore and maintained at 28oC and 80% humidity. The colony was supplemented monthly with field-collected mosquitoes (10% of colony) to maintain genetic diversity. C6/36, Vero, BHK-21, and THP-1 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendation. Primary monocytes were isolated from the principal investigator. 2.2 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   54     replenished with fresh growth medium. These monocytes were allowed to recover overnight at 37°C, 5% CO2, before use in experiments. 2.3 Virus stock Different strains of DENV-1 (EDEN 872, EDEN 2402, EDEN 2928, EDEN 3300 and PVP41), DENV-3 (EDEN 803, EDEN 863 and EDEN 2930) and DENV-4 (EDEN 2270, EHI 69273 and EHI 16693) used in this study are clinical isolates from Singapore, while different strains of DENV-2 (ST, EDEN 3295, PR1940, PR2167, PR6913, PR8545 and PVP110) used are clinical isolates from Singapore and Puerto Rico. DENV was propagated and infectious titer was determined by plaque assay. DENV-1-4 strains used in this study were propagated in the Vero or C6/36 cell lines and harvested when 75% or more of the cells showed cytopathic effect. Infectious titer was determined by plaque assay. To obtain high virus titer, the virus was purified through 30% sucrose cushion as previously described (Chan et al, 2011). Virus pellets were resuspended in mM Hepes, 150 mM NaCl, and 0.1 mM EDTA (HNE) buffer and stored at −80 °C until use. Infectious titer was determined by plaque assay. 2.4 Plaque assay Serial dilutions (10-fold) of virus were added to BHK-21 cells in 24-well plates and incubated for hour at 37 °C. Media was aspirated and replaced with 0.8% methylcellulose in maintenance medium (RPMI-1640, 2% FCS, 25 mM Hepes, penicillin, and streptomycin). After days at 37 °C, cells were fixed with 20% formaldehyde at room temperature for 20 and washed with water, and mL of 1% crystal violet   55     was added for 20 min. The plates were washed and dried, and PFU/mL were calculated. 2.5 qRT-PCR 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 Appendix B. Expression values were normalized against GAPDH and technical duplicates were run for each sample. 2.6 DENV-2 infection in mosquitoes Three to four day-old mosquitoes were infected with DENV. To investigate virus kinetics, Ae. aegypti mosquitoes were inoculated with 100 MID50 of DENV and incubated at 28oC. Intra-thoracic inoculation of DENV was performed as previously described (Choy & Gubler, 2014; Rosen & Gubler, 1974). Infected mosquitoes were harvested at days 3, 7, 10, 14 and 17 post infection. Surviving mosquitoes were killed by freezing and stored at -80oC until assayed. For oral infection, purified DENV-2 ST (1 x 109 PFU/mL) was mixed 1:10 with commercially obtained pig blood. The blood   56     meal was maintained at 37°C for 10 prior to feeding three to four day-old female mosquitoes using an artificial feeding system. Mosquitoes were cold anesthetized to pick only fully engorged mosquitoes to be used for subsequent experiments. Infectious blood meal titers were measured using plaque assay and ranged from 7.5-9 x 107 PFU/mL. 2.7 Gene silencing assays RNA interference (RNAi)-mediated gene silencing in mosquitoes was performed as previously described (Garver & Dimopoulos, 2007). Briefly, three to four day-old female mosquitoes infected either through ingestion of DENV-2 spiked blood meal or inoculated intrathoracically with 100MID50 of DENV-2 were held until dpbm or dpi, at which time they were cold anesthetized and injected with µg of dsRNA per mosquito. Mosquitoes injected with dsRNA containing random sequences from pGEM T easy vector were used as controls. Surviving mosquitoes were harvested dpbm or dpi after which the midguts or heads/thoraces of these mosquitoes were removed respectively, and individually homogenized in DMEM (supplemented with 10% FCS, penicillin and streptomycin) using a high-speed homogenizer FastPrep®24 (MP Biomedicals). Each homogenized sample was centrifuged and the supernatant was removed and stored at −80°C until they were titrated by plaque assay and qRT-PCR. The dsRNAs used were synthesized using the HiScribe T7 in vitro transcription kit (New England Biolabs). The primer sequences used for dsRNA synthesis and primer sequences used to confirm gene silencing by qRT-PCR are presented in Appendix B respectively.   57     2.8 Generation of whole-transcriptome cDNA library DENV-2-infected and control mosquitoes were dissected at dpbm, and 100 midguts for each condition were pooled per replicate and stored in TRIzol reagent (Invitrogen). Total RNA from mosquito midguts was extracted using TRIzol and rRNA was removed by hybridization using RiboMinus™ Eukaryote Kit for RNA-Seq (Invitrogen). Polyadenylated mRNA was then isolated from mosquito midguts by one round of selection with the Dynabeads® mRNA Purification Kit (Invitrogen). Quality of mRNA was assessed by electrophoresis on the Bioanalyzer 2100 (Agilent). For RNAseq sample preparation, NEBNext mRNA Sample Prep Master Mix Set was used according to the manufacturer’s protocol (NEB). Briefly, 0.5ug mRNA was used for fragmentation and then subjected to cDNA synthesis using SuperScript™ III Reverse Transcriptase (Invitrogen) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleotide kinase and T4 polymerase), was ligated to Illumina paired end (PE) adaptors. Size selection was performed using a 2% agarose gel, generating cDNA libraries ranging in size from 275–325bp. Finally, the libraries were enriched using 15 cycles of PCR and purified by the QIAquick PCR purification kit (Qiagen). 2.9 RNAseq analysis cDNA libraries were sequenced on Illumina HiSeq 2000 (Duke-NUS Genome Biology Facility, Singapore). Resulting reads were mapped to the AaegL1 library built from the Aedes aegypti genome (Nene et al, 2007) using Tophat v1.3.0   58     (http://tophat.cbcb.umd.edu/index.html) (Trapnell et al, 2009) with the coveragesearch, microexon-search and butterfly-search options. Differential gene expression analysis was done using Cufflinks v1.3.0 (http://cufflinks.cbcb.umd.edu/)(Trapnell et al, 2010) with the multi-read-correct (Cufflinks), -r and -s (Cuffcompare; using the same annotation gtf and AaegL1 fasta files respectively as in Tophat) and the fragbias-correct (same AaegL1 fasta file used for Tophat) and multi-read-correct options. 2.10 DENV-2 infection in monocytes THP-1 cells were pretreated for h with DMSO or stated concentrations of β-lactone (Sigma Aldrich) or thapsigargin (Sigma Aldrich) before addition of DENV-2 (moi 10) opsonized with enhancing concentrations of humanized 3H5 antibodies (0.39 µg/mL). The mixture was then incubated for 20 on ice to synchronize entry and infection was performed for h at 37°C. The cells were then washed thrice in PBS to remove any inoculum that was not phagocytosed and cultured in maintenance media for another 46 h. Cells and supernatants were harvested for qRT-PCR using 3’UTR dengue primers and GAPDH as control, and plaque assay analyses. For bortezomib and epoxomicin experiment, primary monocytes were pretreated with stated concentrations of bortezomib diluted in PBS or epoxomicin diluted in DMSO and infected with DENV-1-4 (moi 10) opsonized with enhancing concentrations of humanized 4G2 antibodies (1.56 µg/mL). The supernatants were harvested at 48 h for plaque assay analyses. 2.11 DiD labeling of DENV-2   59     To test for virus entry, DiD labeling of DENV-2 was performed as previously described (Chan et al, 2011). Briefly, ~2.8 × 108 PFU DENV-2 was mixed with 800 nmol of DiD (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindodicarbocyanine, 4chlorobenzenesulfonate salt, Invitrogen) in DMSO (final DMSO concentration [...]... List of Figures   Figure 1. 1: Global distribution of dengue 3 Figure 1. 2: Distribution of DENVs 1- 4 in (A) 19 70 and (B) 2 011 4 Figure 1. 3: Flavivirus genome and polyprotein 7 Figure 1. 4: DENV structure 8 Figure 1. 5: Clinical signs and symptoms of dengue 11 Figure 1. 6: Viral load and antibodies quantification in primary dengue infection 16 Figure 1. 7: ADE in dengue. .. alleviated inflammatory responses in DENV infected C57BL/6 mice 11 1 Figure 4 .1: Schematic diagram of findings 11 4               xii     Chapter 1: Introduction 1. 1 Dengue 1. 1 .1 Dengue – a disease of global significance Dengue has emerged to be the most important arthropod-borne viral disease globally, causing an estimated 390 million infections annually (Bhatt et al, 2 013 ) The global... 1. 7: ADE in dengue infection 22 Figure 1. 8: DENV life cycle 31 Figure 1. 9: Schematic representation of the exocyst complex tethering a vesicle to plasma membrane 32 Figure 1. 10: Anatomy of Ae aegypti 36 Figure 1. 11: Ae aegypti distribution in the Americas during the 19 30s and in 19 70 and 2003 39 Figure 1. 12: Dengue vaccines under development... (B) 2 011 Compared to the 19 70s, the increased incidence and spread of all four dengue serotypes observed in 2 011 indicates the urgent need for DENV control Figure adapted from (Gubler, 19 98)   4     1. 1.2 Dengue structure and genome Dengue belongs to the genus Flavivirus family Flaviviridae (Kuno et al, 19 98; Solomon & Mallewa, 20 01) The Flaviviridae family includes several other viruses that pose a... is evident in three epidemics in Cuba where DHF/DSS occurred in patients who were infected with DENV -1 in 19 77 and then infected with DENV-2 in 19 81 or 19 97 (Guzman et al, 2002) For the 19 97 outbreak, silent infections occurred for people with primary infection of DENV In the same outbreak, a very high proportion of DENV -1- immune adults (from 19 77 epidemic) infected with DENV-2 developed either DF or... serum or tissues   10     Figure 1- 5 Clinical signs and symptoms of dengue WHO case definition of (A) DF, (B) DHF, and (C) DSS (D) The incubation period before signs of symptoms develop can range from day 3 to day 14 , average 4-7 Figure adapted from (Whitehead et al, 2007)   11     1. 1.4 Laboratory diagnosis of dengue 1. 1.4 .1 Immunoglobulin M (IgM) detection The efficacy of clinical diagnosis, surveillance,... cytokine storm and immunopathogenesis of DHF/DSS (discussed in 1. 2 .1. 2) Lastly, cells of the mononuclear cell lineage are capable of supporting dengue virus infection via an antibody-dependent enhancement (ADE) process (discussed in 1. 2 .1. 3), which can increase disease severity (Halstead, 19 88; Halstead & O'Rourke, 19 77a; Halstead & O'Rourke, 19 77b; Kliks et al, 19 89) Supporting evidence for each of these... synthesized as the precursor (prM), functions as a chaperone during maturation of the viral particle The E protein can be divided into three structural or functional domains: the central domain; the   5     dimerization domain, which presents a fusion peptide; and the receptor-binding domain (Figure 1- 4) The highly basic C protein packages the RNA genome and is surrounded by 18 0 monomers of E protein... Sangkawibha et al, 19 84; Thein et al, 19 97) For dengue, the immune status of the host may modify the disease in two directions – toward infections accompanied by mild or no disease because of partial or complete protection, or in the direction of increased disease severity (Halstead, 19 88; Sabin, 19 52) This could be attributed to the following reasons Firstly, as the four serotypes of DENV have evolved... to 1% or less (Kalayanarooj, 2 014 ; Trung & Wills, 2 014 ; WHO, 19 97) Many dengue infections present as viral syndrome and may be missed or mistaken for   9     influenza or another viral infection A definitive diagnosis of dengue infection can be made only in the laboratory and relies on detecting specific antibodies in the patient’s serum, isolating the virus, or detecting viral antigen or RNA in . 11 1 Figure 4 .1: Schematic diagram of findings 11 4 ! ! ! ! ! ! ! ! 1! Chapter 1: Introduction 1. 1 Dengue 1. 1 .1 Dengue – a disease of global significance Dengue has emerged to be the. !vi! List of Tables! !x! List of Figures! !xi! Chapter 1. INTRODUCTION! !1! 1. 1 Dengue 1 1. 1 .1 Dengue – a disease of global significance 1 1. 1.2 Dengue structure and genome 5 1. 1.3 Clinical. results 11 2 Chapter 4. Discussion 11 3 4 .1 Preface 11 3 4.2 Unfolded protein response during flavivirus infection 11 5 4.3 Repurposing proteasome inhibitors as an anti-flaviviral therapeutic 11 7