Flaviviruses are significant human pathogens that cause widespread mortality around the world. The genus encompasses several arthropod-borne viruses including Dengue (DENV), Yellow Fever (YFV), and Zika (ZIKV) (Bhatt et al., 2013; Daep et al., 2014; Weaver et al., 2016) . These viruses pose serious public health threats, particularly as increased global travel and climate change have caused an increase in the spread of both the virus and their mosquito vectors (A. albopictus and A. aegypti) (Tatem et al., 2012; Whitehorn and Yacoub, 2019) . It is estimated that 53% of the world population lives in areas suitable for Dengue transmission resulting in 50-100 million Dengue infections per year, of which 500,000 proceed to Dengue hemorrhagic fever and 22,000 are fatal (Bhatt et al., 2013; Messina et al., 2019) . ZIKV infection has been linked to serious neurological defects, including Guillain-Barré syndrome and microcephaly in newborns (Mendez et al., 2017) . While ZIKV diagnoses were relatively rare until the early 2000s, the 2015-2016 pandemic in the Americas highlighted the potential for rapid spread of the disease vectors (Weaver et al., 2016) .
Vaccines currently exist for only a limited range of flaviviruses (Collins and Metz, 2017 ). The first DENV vaccine, Dengvaxia, was approved by the FDA in early 2019 (Thomas and Yoon, 2019) . However, its use remains limited to children with confirmed prior infection. In addition, no post-exposure therapeutic options are available for patients infected with these viruses; current treatments only attempt to alleviate the symptoms (Bernatchez et al., 2020; Lim, 2019; Lim et al., 2013) . Antiviral strategies often focus on directly targeting viral proteins. While molecules inhibiting for instance the flavivirus RNAdependent RNA polymerase NS5 or the protease NS3 have been identified, the high mutation rate of the virus allows for resistance to be developed quickly (Bernatchez et al., 2020; Carrasco-Hernandez et al., 2017; Garcia et al., 2017) . One increasing area of exploration for alternative therapeutic approaches is the targeting of host factors that are critical for virus propagation (Kaufmann et al., 2018) . As these proteins are not under genetic control of the virus, development of resistance is much less likely (De Clercq, 2007; Geller et al., 2013 Geller et al., , 2007 . In addition, host-targeted therapeutics should be effective as broad-spectrum antivirals instead of targeting a single virus (Aviner and Frydman, 2020; Taguwa et al., 2019 Taguwa et al., , 2015 .
Flaviviruses contain a genome of positive-sense, single stranded RNA (+ssRNA) approximately 11kb in size. The viruses replicate and assemble around the endoplasmic reticulum (ER) membrane (Apte-Sengupta et al., 2014) . As the genome is translated after entry into the cell, the single polyprotein is inserted into the ER membrane and is co-and post-translationally processed into three structural (capsid, pre-membrane, and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins (Perera and Kuhn, 2008) . The host proteostasis network has been implicated in the maintenance of individual viral proteins and virus evolution (Aviner and Frydman, 2020; Phillips et al., 2017; Ravindran, 2018; Reid et al., 2018) . The proteostasis network is comprised of chaperones, co-chaperones and other protein quality control factors that control protein folding, assembly, post-translational modification, trafficking, and degradation. Genetic screens have identified many proteostasis factors as essential for DENV replication, including several components of the oligosaccharyl transferase (OST) complex, as well as the ER membrane protein complex (Lin et al., 2019; Marceau et al., 2016; Ngo et al., 2019; Savidis et al., 2016) . Aside from promoting biogenesis of viral proteins, these components also have additional roles in organizing viral replication centers at the ER membrane (Marceau et al., 2016; Ngo et al., 2019; Rothan and Kumar, 2019) . Additionally, diverse cellular chaperone and co-chaperone systems are required for all stages of the viral life cycle, including virus entry and disassembly, folding of individual viral proteins, as well as assembly and egress of new virions (Fischl and Bartenschlager, 2011; Heaton et al., 2016; Taguwa et al., 2019) . Pharmacologic inhibition of proteostasis factors, including the OST as well as cytosolic Hsp70 and Hsp90 chaperones, has been shown to be an effective strategy to reduce flavivirus infection in cell models (Heaton et al., 2016; Howe et al., 2016; Puschnik et al., 2017; Taguwa et al., 2019 Taguwa et al., , 2015 Yang et al., 2020) .
As flavivirus replication and polyprotein translation occurs at the ER membrane, infection results in expansion of the ER and remodeling of the ER proteostasis environment (Ravindran et al., 2016; Rothan and Kumar, 2019; Stohlman et al., 1975) .
DENV infection leads to modulation of the unfolded protein response (UPR), the adaptive stress-response that remodels the ER proteostasis environment to counter stress caused by accumulation of misfolded proteins (Peña and Harris, 2011; Perera et al., 2017) . The UPR consists of three overlapping but distinct signaling branches downstream of the stress-sensing receptors IRE1 (inositol-requiring enzyme 1), ATF6 (activating transcription factor 6), and PERK (Protein kinase R-like ER kinase) (Shoulders et al., 2013; Walter and Ron, 2011) . The first two branches primarily control the upregulation of chaperones and other proteostasis factors to expand the protein folding capacity of the ER. The PERK branch is responsible for translational attenuation via phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a) and subsequent activation of the integrated stress response. DENV has been shown to upregulate the IRE1/XBP1s and ATF6 branches of the UPR, while suppressing activation of the PERK branch (Peña and Harris, 2011) . The importance of UPR-dependent ER proteostasis remodeling for flavivirus infection has only been explored in knockout cell models. For example, the virus was less capable of propagating in IRE1 -/-MEF cells. In contrast, PERK knockout resulted in increased viral titers, while ATF6 knockout did not impact viral propagation. (Peña and Harris, 2011) .
Given the dependencies of DENV and other flaviviruses on UPR modulation and an enhanced ER proteostasis environment in the host cells during infection, we sought to explore whether these dependencies can be perturbed to impair viral infection.
Pharmacologic remodeling of ER proteostasis pathways has become an attractive strategy at correcting imbalances associated with diverse phenotypes related to protein stress and misfolding without affecting endogenous protein maturation or causing toxicity (Blackwood et al., 2019; Chen et al., 2014; Cooley et al., 2014; Kroeger et al., 2018; Plate et al., 2016) . In particular, compound 147, which was developed as a preferential activator of the ATF6 pathway, has broadly beneficial effects at reducing secretion of amyloidogenic proteins and protecting against oxidative organ damage from ischemia/reperfusion (Blackwood et al., 2019; Plate et al., 2016) .
Here, we demonstrate that ER proteostasis remodeling by compound 147 serves as an effective strategy to reduce flavivirus infection. We determine that the 147 mediated reduction in DENV replication and assembly is surprisingly not mediated through activation of ATF6. Instead, the activity is mediated by upstream covalent modifications of protein targets by 147. Prior work identified protein disulfide isomerases (PDIs) as critical targets for the 147-dependent ATF6 activation , however, our data suggests PDIs are not responsible for the antiviral activity. Finally, we show that 147 treatment can reduce proliferation of multiple DENV serotypes and several ZIKV strains, demonstrating that the pharmacologic agent could be a broadly effective strategy against flaviviruses and other viruses that depend on ER proteostasis processes.
DENV is known to activate the UPR in infected host cells and was previously found to specifically upregulate the ATF6 and IRE1/XBP1s signaling arms, which transcriptionally upregulate ER protein quality control factors and adjust proteostasis capacity (Fig. 1A ) (Peña and Harris, 2011; Shoulders et al., 2013) . To confirm the activation of ER proteostasis pathways by DENV, we performed an infection time-course with DENV serotype 2 (DENV2) in Huh7 liver carcinoma cells and measured transcript levels of an ATF6-regulated gene (PDIA4) and an XBP1s-regulated gene (ERDJ4) by quantitative RT-PCR. Both PDIA4 and ERDJ4 transcripts exhibited a time-dependent induction in response to DENV2 infection (Fig. 1B) . A smaller but similar time-dependent upregulation of ATF6 and XBP1s-regulated protein products could be observed by western blot (Fig.   S1A ). To corroborate these findings, we performed quantitative proteomics analysis of Huh7 cells at 24 and 36 hours post infection (hpi) with DENV2 to quantify global changes in protein expression ( Fig. S1B-C, Table S1 ). We found that DENV2 activated specific factors involved in ER proteostasis maintenance, such as DNAJC10, BiP, and HYOU1.
We also filtered previously defined genesets consisting of ~20 specific genes for each of the UPR signaling branches to quantify the cumulative activation of ER proteostasis pathways ( Fig. 1C ) (Grandjean et al., 2019) . This analysis indicated a mild stimulation of ATF6 and XBP1s-regulated proteins, while PERK-regulated proteins remained largely unchanged. In contrast, we did not observe upregulation of cytosolic proteostasis factors that are under control of the heat shock response (HSR) (Vabulas et al., 2010) . Overall, our results confirm that DENV2 preferentially remodels ER proteostasis pathways through ATF6 and IRE1/XBP1s-dependent upregulation of specific chaperones and protein folding factors.
Considering the dependencies of DENV and other flaviviruses on UPR activation, we determined the impact of the proteostasis regulators 147 (a preferential activator of the ATF6 branch), and Ceapin-A7 (Cp7) (a selective inhibitor of the ATF6 branch) on DENV infection ( Fig. 2A) . We pre-treated Huh7 cells with the respective compounds 16h prior to infection by DENV2 to induce or inhibit UPR-dependent proteostasis remodeling, and we re-treated immediately after infection. Quantification of DENV viral titers by a focus forming assay demonstrated a significant reduction in infection with compound 147 at 24 and 36 hpi (Fig. 2B) . In contrast, Cp7 treatment only resulted in a small reduction in viral titers at 24 hpi but the infection recovered at 36 hpi. Cell viability experiments indicated that the compounds did not induce a large amount of cell toxicity in Huh7 cells consistent with previous results in other cell lines (Fig. S2A ) (Plate et al., 2016) . We also conducted dose-response studies with 147 demonstrating that the compound was effective at reducing DENV titers at an IC50 of approximately 1 µM (Fig. S2B) . We observed that cell viability was maintained above 90% at this concentration (Fig. S2C) .
These results demonstrate that modulation of the ER proteostasis network with the preferential ATF6 activator 147 could represent an effective strategy to impair DENV2 infection.
The ER plays critical roles in several stages of the viral life cycle including replication of viral RNA at replication centers on the cytosolic side of the ER membrane, translation and proteolytic processing of the viral polyprotein in the ER membrane, as well as the folding, assembly and secretion of new virions Neufeldt et al., 2018) . To determine at what stage in the viral life cycle the compound treatment impaired viral propagation, we investigated the impact of compound 147 on viral RNA (vRNA) and protein levels ( Fig. 2C-D) . Quantification of vRNA by qPCR indicated a modest but significant reduction of 50% at 24 hpi, which was attenuated to 30% reduction at 36 hpi ( Fig. 2E) . Western blot quantification of NS3 and NS5 viral protein in Huh7 cell lysates from DENV2 infected cells showed a 35 -80 % reduction in viral non-structural proteins in response to 147 treatment (Fig. 2F, Fig. S2D ). This highlighted that the ER proteostasis regulator exerted a more pronounced effect on viral protein production relative to replication of vRNA. In contrast, infectious viral titers showed a far greater reduction reaching 98% at 12 hpi and sustaining 80% reduction at 36 hpi (Fig. 2G) . The intensified effect of 147 on viral titers compared to vRNA and protein levels suggests that the compound predominantly acts on a stage subsequent to translation of viral proteins, which would be consistent with disruption of viral maturation or secretion pathways through modulation of ER proteostasis. Lastly, we confirmed that 147 did not impair viral entry. Omission of the pretreatment prior to infection and treatment of Huh7 cells with the compound only after infection still resulted in a comparable reduction in viral protein and titers, confirming that the compound must act at a post-entry stage ( Fig. S2E-G) .
Given that flavivirus infection activates the ATF6 pathway, it seemed surprising that preactivation could reduce viral propagation. We therefore sought to investigate whether ATF6 activation and induction of ATF6-targeted proteostasis factors was required for the 147-dependent inhibition of DENV infection. We first confirmed by qPCR and quantitative Western blot analysis that treatment with 147 activated the ATF6 regulated gene HSPA5 (BiP) in Huh7 cells (Fig. S3A) . To probe the impact of ATF6 activation on the compound activity, we took advantage of the ATF6 inhibitor Cp7, which inhibits ATF6 . Cp7 mediates a neomorphic interaction between ATF6 and the peroxisomal membrane protein ABCD3, keeping ATF6 in a traffickingincompetent oligomeric state, which cannot be activated by 147 (Fig. 3A ) (Torres et al., 2019) . We confirmed by qPCR and Western blot that co-treatment with 147 and Cp7 could fully attenuate the 147-dependent induction of ATF6 target genes in Huh7 cells ( Fig. S3A) . We then investigated the addition of Cp7 on the 147-mediated reduction of DENV propagation. Co-treatment of Cp7 did not diminish the reduction in vRNA or NS3 protein ( Fig. 3B-C) . Furthermore, the addition of Cp7 only partially recovered the DENV2 viral titers at 24hpi (Fig. 3D) . These results highlight that the reduction in viral titers could not be fully attributed to the 147-mediated induction of ATF6 target genes.
To further probe whether the reduced viral propagation could be ascribed to activation of the ATF6 pathway, we took advantage of an orthogonal chemical genetic approach to selectively induce the ATF6 pathway independent of global ER stress. We transiently transfected a destabilized DHFR.ATF6 construct into Huh7 cells. This construct is constitutively degraded in the absence of a small molecule ligand, but can be stabilized through addition of trimethoprim (TMP) leading to accumulation of DHFR.ATF6 and selective induction of ATF6-regulated genes ( Fig. 3E ) (Shoulders et al., 2013) . We confirmed TMP-dependent upregulation of ATF6-regulated targets BiP, PDIA4, and GRP94 in Huh7 cells ( Fig. S3B-C) . Next, we pre-treated Huh7 cells with TMP to activate DHFR.ATF6, infected cells with DENV2, and quantified propagation of the virus by monitoring viral protein levels by Western blot and measuring infectious titers. Chemicalgenetic ATF6 activation did not lead to a measurable reduction in viral protein levels ( Fig.  3F ). At the same time, a moderate decrease in viral titers could be observed, but this reduction in viral propagation was much lower than seen with 147 treatment (Fig. 3G) .
Together, the results from the ATF6 inhibitor and chemical-genetic ATF6 activation indicate that induction of ATF6-regulated ER proteostasis fators does not impact vRNA replication or production of viral proteins, and only partially accounts for the reduction in DENV2 viral titers.
Considering that reduced viral propagation in response to 147 was only partially mediated by ATF6 activation, we sought to explore other mechanisms of how the molecule could impair the virus. Previous studies showed that 147 is a prodrug and requires metabolic activation in cells to generate a reactive p-quinone methide that can then form protein adducts with reactive cysteine residues ( Fig. 4A ) (Palmer et al., 2020; Paxman et al., 2018; Plate et al., 2016) . In several cell types, ER-resident protein disulfide isomerases (PDIs) were identified as the common protein targets of 147, and this modulation of PDIs was linked to the activation of ATF6 . To explore whether covalent targeting of reactive thiols by 147 is required for the reduction in DENV propagation, we blocked the covalent modifications through addition of an excess of the small-molecule thiol 2-mercaptoethanol (BME) to the cells treated with 147 (Fig. 4A) . We confirmed that this addition did not impair cell viability (Fig. S4B) , and that BME alone only had a minimal effect on viral protein levels (Fig. S4A) . When BME was added to DENV infected cells that were treated with 147, this resulted in a partial to complete recovery of NS5 protein at 24 and 36hpi, respectively (Fig. 4B) . Similarly, the reduction in DENV2 viral titers was partially attenuated by the addition of BME (Fig. 4C) . These results indicate that the targeting of cellular thiol groups is required for the inhibition of virus propagation.
We next explored whether targeting of specific PDIs proteins by 147 was required for the reduced viral replication. Previous studies determined the covalent targets of 147 in HEK293T, HepG2, and ALLC plasma cells . However, it was conceivable that the metabolic activation mechanism differs in cell types and could result in alternative targets. We therefore determined whether 147 could similarly target PDIs in Huh7 cells. We took advantage of the active analog 147-20, which contains an alkyne handle that enables further Click chemistry derivatization with desthiobiotin after protein labeling, followed by isolation of the targeted proteins on streptavidin resin (Fig. 5A) . The desthiobiotin probe also contained a TAMRA fluorophore that allowed gel-based visualization of the targeted proteins (Fig. 5B) . We confirmed that 147-20 retained activity reducing DENV titer levels and NS3 protein levels in infected cells ( Fig. S5A-B) . In contrast, treatment with 147-4, an inactive analog where generation of the quinone methide is blocked by the trifluoromethyl group, did not reduce infection. We probed for the presence of specific PDIs (PDIA4, PDIA1, PDIA6), which were identified as 147 targets in other cell lines. These proteins were clearly detectable at molecular weights of prominent labeled bands that were competed by 147, confirming them as targets in Huh7 cells (Fig. 5B, S5C ) . In DENV-infected cells that were treated with 147-20, no additional labeled protein bands were observed, indicating that viral proteins are not directly targeted by the compound (Fig. S5D) .
Considering the targeting of multiple PDI enzymes, we were interested in whether the inhibition of PDIs could have an important role in the reduction of viral propagation.
We created stable knockdown cell lines of several PDIs using shRNAs (Fig. S5E) . These cell lines were then infected with DENV2 and treated with 147 to assess the effect of PDI knockdown on propagation of the virus. Individual knockdown of PDIA1 (P4HB), PDIA4, or PDIA6 did not result in a decrease in viral titers indicating that none of these individual PDIs are essential for viral propagation (Fig. S5F) . Only knockdown of PDIA3 resulted in a small reduction in viral titers. Next, we measured viral infections in the knockout cell lines in the presence of 147 to determine whether particular PDIs are required for the 147-mediated virus inhibition. We observed no measurable attenuation in the reduced viral titers with any single PDI knockdown, including the PDIA3 knockdown, suggesting that no individual PDIs are responsible for the 147-mediated effect (Fig. S5F) .
To further explore whether PDI inhibition could be responsible for the reduction in DENV infection, we took advantage of several published small-molecule inhibitors of PDIA1 ( Fig. 5C ) (Banerjee et al., 2013; Cole et al., 2018; Hoffstrom et al., 2010) . Importantly, these molecules display different degrees of specificity towards PDIA1 relative to other PDIs, mirroring the polypharmacology observed with 147. We profiled the specific PDIs targeted by the compounds in Huh7 cells by modifying the alkyne handles on the molecule with a fluorophore. KSC-34 displayed the greatest preference for PDIA1, while Rb-11-ca also labeled PDIA4, PDIA6, and several other proteins (Fig. 5SG) . This is consistent with observations in other cell types and the development of KSC-34 as a more specific derivative of Rb-11-ca (Cole et al., 2018) . 16F16 does not contain an alkyne handle but previous studies show even broader reactivity towards PDIs than Rb-11-ca (Hoffstrom et al., 2010) . We then tested the effect of the compounds on DENV2 infection in Huh7 cells using the same treatment regime as for 147. KSC-34 and 16F16 were unable to reduce viral NS3 protein levels (Fig. 5D ). Rb-11-ca was able to lower viral protein production by 40%; however, this reduction did not translate into lower viral titers ( Fig. 5E) . Reduction in viral protein levels with Rb-11-ca could be attributed to compound toxicity, as we observed a 40% reduction in cell viability (Fig. S5H) . These results indicate that targeting of individual PDIs is not solely responsible for the reduced viral titers by 147 and that these existing PDI inhibitors are not effective at inhibiting DENV replication.
To identify additional mechanisms that could contribute to the 147-mediated reduction in DENV viral propagation, we turned to untargeted quantitative proteomics of the affinity-enriched samples to identify additional protein targets. We used tandem mass tags for relative quantification of proteins in the 147-20 enriched samples relative to the competition samples (147-20 treated with 3-fold excess 147) (Fig. 6A ). This analysis confirmed that PDIs were the most highly enriched proteins that were covalently targeted by 147-20 (Fig. 6B, Fig. S6A , Table S2 ). While labeling of PDIA4, PDIA6, and PDIA1 was confirmed above, we identified additional targeted PDIs (TXNDC5, PDIA3, TXM1) and two other highly enriched protein targets (GSTO1 and ALDH1A1). We compared these targets to previous datasets of 147-20 targets in HEK293T, HepG2, and ALMC-2 plasma cells (Fig. 6C, Fig. S6B ) . The additional proteins were targets specific to Huh7 cells and were not observed in other cell types, suggesting that they could have a unique role in the reduction in virus propagation in the Huh7 cells.
Studies until this point were carried out with Dengue serotype 2 BID-V533 (isolated in 2005 in Nicaragua) in Huh7 cells. To determine the scope of 147 antiviral activity, we tested the effect against DENV-2 propagation in HepG2 cells, another liver carcinoma cell line commonly used as an infection model. Prior data also showed that 147 treatment is non-toxic in HepG2 cells and can reduce amyloidogenic protein secretion (Plate et al., 2016) . When testing the impact on DENV titers in HepG2 cells, 147 treatment resulted in a significant reduction in DENV propagation 48 hpi ( Figure 7A) . A similar decrease in protein levels was also detected (Fig. S7A) . Next, we proceeded to test whether the compound could be more broadly effective against other DENV strains. We tested 147 against a different DENV-2 strain (16681, isolated in 1984 in Thailand) as well as against strains from DENV serotypes 3 and 4 (Kinney et al., 1997) . Treatment with compound 147 was able to reduce viral titers consistently greater than 95% (Fig. 7B-D) and protein levels were similarly reduced (Fig. S7B) . Finally, we sought to test whether compound 147 could be useful to treat other flaviviruses. We measured the effect against two different strains of Zika virus; MR766 (isolated from Eastern Africa in the 1950s), and PRVABC59 (isolated from Puerto Rico in 2015) (Dick and Kitchen, 1952; Lanciotti et al., 2016) . The molecule displayed similar activity at reducing ZIKV titers and protein levels for both strains (Fig. 7E-F, Fig. S7C-D) . These results provide evidence that compound 147 is broadly active against flaviviruses in multiple cell lines, thus facilitating its potential application as an extensive host-centered antiviral agent.
The continued health burden from arbovirus infections, such as Dengue and Zika, combined with the lack of effective vaccines and therapeutics against these viruses, highlights a need for the development of new antiviral strategies. Therapeutic methods which target host factors required for viral propagation provide a path unlikely to elicit drug resistance and may act broadly across several virus species (Aviner and Frydman, 2020; Geller et al., 2013 Geller et al., , 2007 . However, a challenge remains to target host factors that selectively impair viral replication without causing toxicity to the host cell and infected organism. Prior studies with 147 have already indicated that the compound has broad potential to safely ameliorate proteostasis imbalances that are associated a variety of disease conditions (Blackwood et al., 2019; Plate et al., 2016) . The compound is effective at reducing the secretion and aggregation of amyloidogenic proteins, such as immunoglobulin light chains and transthyretin (Plate et al., 2016) . In addition, selective remodeling of ER proteostasis pathways through an ATF6-dependent mechanism by 147 was shown to prevent oxidative organ damage in mouse models of ischemia/reperfusion (Blackwood et al., 2019) . The same study also showed that 147 could be safely administered to mice through IV injections and was able to activate ER proteostasis pathways in multiple tissues, including liver, kidney and heart (Blackwood et al., 2019) .
Here, we further expand the therapeutic potential for compound 147 by establishing that the small molecule serves as a promising strategy to target multiple flaviviruses without causing significant toxicity in the host cell.
Treatment with the small molecule proteostasis regulator, 147, resulted in a decrease in DENV vRNA, protein, and titer levels. The titer levels were the most affected, indicating that the compound was most active at preventing the assembly and secretion of mature infectious virions. This is consistent with the compound targeting critical proteostasis processes that could be required for production of infectious virions and are thus, crucial to the viral life cycle (Fischl and Bartenschlager, 2011; Heaton et al., 2016) .
147 was designed as a preferential activator of the ATF6 branch of the UPR. Given that DENV innately activates the ATF6 signaling pathway, it was unexpected that small molecule activation of ATF6 could impair virus propagation. However, addition of the ATF6 inhibitor Cp7 was not effective at reducing virus replication, indicating that ATF6 activation did not mediate the antiviral effect. Cp7 on its own also did not reduce virus infection, which is consistent with prior knockout of ATF6 not affecting DENV propagation (Peña and Harris, 2011) . These results prompted us to examine alternative mechanisms for the antiviral effects by investigating the specific protein targets of 147.
Prior work showed that 147 is a prodrug that is metabolically oxidized to an iminoquinone or quinone methide, which covalently targets nucleophilic cysteine residues on cellular protein (Palmer et al., 2020; Paxman et al., 2018; Plate et al., 2016) . We showed that the antiviral activity of 147 could be attenuated with BME treatment, demonstrating that the metabolic activation mechanism and covalent targeting of reactive thiols is similarly required to reduce virus infection. We repeated the chemoproteomic target identification in Huh7 cells using an analog of 147 and identified a similar set of PDIs, including PDIA1, PDIA4, and PDIA6, as targets in this cell line. However, knockdown of individual PDIs was not sufficient to reduce viral titers suggesting that these individual PDIs are not responsible for the inhibition of viral infection. This raises the possibility that 1) polypharmacologic targeting of multiple PDIs may be necessary to sufficiently impair virus propagation, or 2) alternative 147-targets are responsible for the antiviral effects.
We addressed whether inhibition of multiple PDIs could be required by testing the effect of existing PDI inhibitors that display varying selectivity towards PDIA1/P4HB and additional PDIs also modified by 147. None of the PDI inhibitor compounds significantly reduced DENV viral titers, suggesting that 147 elicits its antiviral activity through an alternative mechanism of action. Importantly, one of the compounds, Rb-11-ca, exhibited considerably more toxicity in Huh7 cells, even at the highest concentration that we tested for 147. Toxicity has also been observed for other PDI inhibitors (Langsjoen et al., 2017) .
This further highlights the unique properties of compound 147. The low toxicity could be explained by previous observations showing that less than 25% of the PDIA4 pool is labeled by the compound, indicating that PDIs are not globally inhibited by the molecule . Furthermore, endogenous protein secretion and formation of disulfide bonds in secreted proteins, including fully assembled immunoglobulins, is not affected by 147 (Plate et al., 2016) . This lack of perturbation to normal proteostasis processes and selective inhibition of viral propagation makes 147 an ideal candidate for a host-centered antiviral strategy.
To address the second possibility that alternative protein targets besides the most prominent PDIs could be responsible for reduction in virus propagation, we compared our identified targets in Huh7 cells to the prior list of covalently modified proteins in other cell lines . In addition to the PDIs studied above, additional members of the PDI family were identified (TXNDC5, TXM1), as well as two unique proteins that were identified only in Huh7 cells: ALDH1A1 and GSTO1. These new targets also have catalytic Cys residues that are likely modified by 147. Ongoing studies are directed at determining the potential role of these protein targets in the 147-mediated antiviral mechanism.
The recent COVID-19 pandemic has refocused attention to the need for broadspectrum antiviral agents that could inhibit future virus threats (Carrasco-Hernandez et al., 2017) . Targeting essential host pathways that are commonly hijacked by virus infection without causing significant host toxicity is recognized as one important strategy to combat future infection with unknown pathogens. We determined that compound 147 has broad antiviral activity against multiple DENV strains and serotypes and that the compound is also effective against multiple ZIKV strains. These results highlight broad utility of the proteostasis regulator compound to reduce flavivirus infection by targeting conserved host cell processes that are commonly exploited by the virus. Our results here now provide an entirely new therapeutic application of 147 as a broad antiviral agent to reduce flavivirus infections. 
The 3 rd generation system was used for production. Respective shRNA plasmids were transfected into HEK293T cells together with lentivirus packaging plasmids RRG, VSVG, and Rev using the calcium phosphate method as described . Media was changed 20 hours post-transfection. Supernatants were collected 3 days post transfection, and cleared at 3700xg for 10 minutes. To generate respective shRNA stable knockdown cell lines in Huh7 cells, lentiviruses prepared with pools of 2-3 shRNAs per target were added to Huh7 cells and media containing 4 µg/mL final concentration polybrene. Selection proceeded for 1 week under puromycin (5 µg/mL).
Confluent Vero cells in 96 well plates were inoculated with 10-fold serial dilutions of DENV or ZIKV in BA-1 diluent (1xM199 media, 5% BSA, 1x L-glutamine, 1x penicillin/streptomycin, 0.04% sodium bicarbonate, 50 mM Tris) for 2 hours. The cells were overlaid with a 1:1 mixture of 2x nutrient overlay (2x Earle's Balanced Salt Solution, 2x Ye-Lah Medium, 4% FBS, 0.4% sodium bicarbonate, 0.1 mg/mL gentamycin, 0.5 mg/mL amphotericin B) + 2.4% methylcellulose in water. After 2 days (ZIKV, DENV3, DENV4) or 3 days (DENV2), overlay was removed, and cells were fixed in ice cold 85% acetone for 30 minutes. Infectious foci were stained with a primary pan-flavivirus 4G2
antibody (1:1000 in 5% BSA, TBST) and secondary HRP antibody (1:1000 in 5% milk, TBST), then visualized using 400 µL 8 mg/mL 3-amino-9-ethylcarbazole (Sigma) in 10 mL 50 mM sodium acetate (Fisher) + 4 µL 30% H2O2 (Fisher) and exposed for 15-45 minutes until foci were visible.
Cell pellets were lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1%
Triton-X-100, 0.5% sodium deoxycholate) + Roche cOmplete protease inhibitor. Cells were left on ice for at least 10 minutes and lysate was cleared at 17,000xg for 10 minutes.
Cleared lysate concentrations were normalized and gel samples were separated by SDS-PAGE. Gels were transferred to PVDF membranes using standard settings on the TransBlot Turbo (BioRad). Blots were blocked in 5% non-fat dry milk in Tris-buffered saline (TBST, 50mM Tris pH 7,4, 150mM NaCl, 0.1 % Tween-20) for 30 minutes before applying antibodies. Primary antibodies (in 5% BSA, TBST) were applied at room temperature for 2 hours or at 4ºC overnight. Secondary antibodies (in 5% milk, TBST)
were applied at room temperature for 30 minutes or at 4ºC for 1 hour. All primary antibodies plus the anti-mouse HRP secondary were used at a 1:1000 dilution; all secondary antibodies were used at a 1:10,000 dilution.
RNA was prepared from cell pellets using the Zymo Quick-RNA miniprep kit. cDNA was synthesized from 500 ng total cellular RNA using random primers (IDT), oligo-dT primers (IDT), and Promega M-MLV reverse transcriptase. qPCR analysis was performed using 
Cell viability was determined using the CellTiter-Glo reagent (Promega). Huh7 cells were seeded into 96 well plates into 100 µL culture media. 100x dilutions of compounds were prepared in culture media. 11 µL of each compound dilution was added to wells in triplicate. 16 hours after treatment, media was removed and cells were infected as described above (for -DENV samples, no virus was added). Media was replaced 24 hours after infection and 100 µL Cell Titer Glo reagent was added. Plates were gently shaken for 10 minutes, then luminescence was measured for each well on a Synergy HT plate reader. Luminescence values for each condition and/or compound treatment were averaged and normalized to a DMSO control. Where multiple independent replicates are indicated in legends, samples were averaged, and standard error of the mean was propagated.
Huh7 cells were treated with 3 µM 147-20 or a combination of 3 µM 147-20 and 9 µM 147. Cells were harvested 16-18 hours post-treatment and lysed as described above.
Click chemistry using 100 µM Cy5 azide or TAMRA desthiobiotin azide was performed with 1.6 mM BTTAA, 0.8 mM copper sulfate, and 5 mM sodium ascorbate for 1 hour at 37ºC, shaking at 500 rpm. All reagents were purchased from Click Chemistry Tools. For detection, samples from Cy5 reactions were run directly on SDS-PAGE gels for analysis.
For enrichment of proteins, excess reagents were removed via methanol/chloroform precipitation; protein was washed twice with methanol before resuspension in 6 M urea.
Samples were diluted in PBS and added to pre-washed (in PBS) streptavidin agarose beads (Thermo Scientific). Pulldowns were rotated for 1 hour at room temperature.
Supernatant was removed and beads were washed six times in PBS and 1% SDS.
Samples were eluted two times by incubation with 50 mM biotin, pH 7.2, in 1% SDS (PBS) for 10 minutes.
Protein lysate from DENV infected and non-infected Huh7 cells (10 µg of protein as measured using Bio-Rad protein assay dye reagent) was aliquoted and purified using methanol/chloroform precipitation. Streptavidin-enriched samples for target identification were used without normalization and purified by methanol/chloroform precipitation. Mass spectrometry samples were prepared as described previously (Blackwood et al., 2019) .
Briefly, protein was resuspended in 3 µL 1% Rapigest (Waters) and diluted with water and 0.5 M HEPES. Proteins were reduced with 5 mM TCEP (Thermo Scientific) and
subsequently alkylated with 10 mM iodoacetamide (Sigma). Proteins were digested in sequencing-grade trypsin (Thermo Scientific) overnight at 37ºC while shaking at 750 rpm.
Peptides were labeled for 1 hour at room temperature using 11-plex tandem mass tag (TMT) reagents (Thermo Scientific) resuspended in 40% acetonitrile. Labeling reactions were quenched with 0.4% ammonium bicarbonate for 1 hour at room temperature.
Samples were combined and acidified to pH 2 by addition of 5% formic acid. Pooled samples were concentrated using a SpeedVac and resuspended in 94.9% water/5% acetonitrile/0.1% formic acid. Rapigest cleavage products were pelleted by centrifugation at 14,000xg for 30 minutes.
Digested peptides (20 µg) were loaded onto a triphasic column filled sequentially with 1.5 or 2.5 cm layers of 5 µm 100 Å C18 resin (Phenomenex), Luna 5 µm 100 Å strong cation exchange resin (Phenomenex) and a second layer of C18 resin using a high pressure chamber (Fonslow et al., 2012 
Peptide and protein identification, and TMT reporter ion quantification was analyzed using Proteome Discover 2.4. Searches were carried out with the SEQUEST node using a human proteome database (UniProt) with DENV proteins added manually and the following parameters: 20ppm precursor mass tolerance, minimum peptide length of 6 amino acids, trypsin cleavage with a maximum of 2 missed cleavages, static modifications of +57.0215Da (carbamidomethylation at C) and +229.1629 (TMT6/11plex), and dynamic modification of +15.995 (oxidation at M), N-terminal methionine loss (-131.040) , and Nterminal acetylation (+42.011). Search results were filtered with Percolator using a decoy database of reversed sequences with a peptide false discovery rate of 1% and a minimum of 2 peptides for protein identification. TMT intensities were quantified using the reporter ion quantification node with TMT intensities from each channel being normalized using the total peptide amount, quantitative value correction enabled to correct for TMT impurities, and co-isolation threshold set to 25%. Intensities for each protein were A. Schematic overview of the UPR showing the three signaling branches: IRE1/XBP1s, PERK, and ATF6. Prior work showed that DENV increased activity of the IRE1/Xbp1s and ATF6 branches, while simultaneously preventing activation of the PERK branch (Pena and Harris, 2011) . B. Time course of qPCR data showing transcriptional upregulation of an IRE1/XBP1s target (ERdj4) and an ATF6 target (PDIA4) in response to DENV activation (MOI 3) in Huh7 cells. Error bars shows SEM from 2 to 3 biological replicates and pvalues from two-tailed unpaired student t-tests are displayed. C. Box plots of proteomics data showing the aggregate upregulation of IRE1/XBP1s and ATF6 protein targets at 24 and 36 hpi. PERK and cytosolic heat-shock response (HSR) targets are not affected. Huh7 cells were infected with DENV (MOI 3) for 24h or 36h. Genesets for UPR and HSR pathways were defined based on prior transcriptional profiles (Grandjean et al., 2019) . p-values from two-tailed Wilcoxon signed rank tests are indicated. Cell-wide proteomics data comparing DENV infected to non-infected Huh7 cells is shown in Fig. S1B -C.
A. Western Blot showing time course of ATF6 upregulation over the course of DENV infection. Cells were infected with DENV-2 strain BID-V533 at MOI of 3 for 3 hours. Media was replaced and samples were taken at indicated timepoints postinfection. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. Actin is included as a loading control.
B.-C. Waterfall plots of proteomics data from samples infected with DENV-2 for 24 hours (B.) and 36 hours (C.), showing the presence of DENV proteins and upregulation of IRE1/XBP1s, ATF6, and PERK branches of the UPR. Cells were infected with DENV-2 strain BID-V533 at a MOI of 3 for 3 hours. Media was replaced and samples were collected at 24 or 36 hpi. Gene upregulation was quantified using TMT11plex reagents and comparison is between DENV-infected cells vs. noninfected cells. Graph represents 4-7 independent biological replicates. Source data is included in Table S1 . Media and treatments were replaced, and viability was measured 24hpi using the Promega Cell Titer Glo reagent. Graphs represent six independent biological replicates across two days of measurement. Error bars represent SEM. B. A dose-response curve shows DENV is sensitive to 147 treatment with an IC50 of approximately 1 µM. Cells were treated with the indicated concentrations of 147 for 16 hours, then infected with DENV-2 strain BID-V533 at a MOI of 3 for 3 hours. Media and treatments were replaced, and cells were collected 24 hpi. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. Western blot intensities were normalized to a GAPDH loading control. Graphs represent 2-4 independent biological replicates. Error bars represent SEM. C. Increasing 147 concentration causes decreasing cell viability. No decrease in viability was seen with 147-4 or Ceapin-A7 (Cp7). Cells were pretreated with the indicated concentrations of 147 for 16h, then infected (where indicated) with DENV-2 strain BID-V533 at a MOI of 3 for 3 hours. Media and treatments were replaced, and viability was measured 24 hpi using the Promega Cell Titer Glo reagent. Graphs represent three biological replicates. Error bars represent SEM. D. Representative western blot showing 147 attenuates DENV protein levels. Cells were pretreated with 10 µM 147, 6 µM Cp7, or both for 16h, then infected with DENV-2 strain BID-V533 at MOI of 3 for 3 hours. Media and treatments were replaced, and cells were collected at indicated timepoints. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. GAPDH is included as a loading control. No visualization of DENV protein could be seen at 12 hpi. E. Eliminating the pretreatment step will determine if 147 blocks viral entry. Cells are only treated post-infection with 10 µM 147, and focus forming units are quantified 24 hpi. F. Quantification of western blots with treatment only post-entry shows similar phenotype to treatment pre-and post-entry. Cells were only treated post-infection with 10 µM 147, and cells were harvested 24 hpi. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. Normalization to GAPDH was included as a loading control. Graph represents three independent biological replicates. Error bars represent SEM and p-value from an unpaired t-test is shown. G. Graph showing reduction in viral titers for only post-entry treatment. Cells were treated as in E-F and viral titers were determined by focus forming assay. Error bars represent SEM and p-value from a ratio paired t-test is shown.
A. Ceapin-A7 (Cp7) acts as an ATF6 inhibitor downstream of 147 by tethering inactive ATF6 to the peroxisomal membrane protein ABCD3 preventing ATF6 trafficking to the Golgi and activation through S1/S2 cleavage. B. Bar graph showing the reduction in vRNA levels in response to 147 and Cp7 treatment measured by qPCR in Huh7 cells infected with DENV as outlined in Fig.  2D . Cp7 does not attenuate the 147-mediate reduction. Error bars correspond to SEM from 3 biological replicates and p-values from unpaired t-tests are shown. C. Bar graph showing the reduction in NS3 viral protein levels in response to 147 and Cp7 treatment measured by Western blot in Huh7 cells infected with DENV as outlined in Fig. 2D . Cp7 does not attenuate the 147-mediate reduction. Error bars correspond to SEM from 3 biological replicates and p-values from unpaired t-tests are shown. D. Bar graph showing the reduction in DENV viral titers in response to 147 and Cp7 treatment in Huh7 cells infected with DENV as outlined in Fig. 2D A. Schematic outlining the metabolic activation mechanisms of 147. After oxidation by P450 enzymes, the generated p-quinone methide can react with thiol nucleophiles such as cysteine residues on cellular protein targets. Addition of exogenous free thiols such as 2-mercaptoethanol (BME) can quench the active form of 147 before it reacts with protein targets. B. Graph showing reduction in DENV NS3 and NS5 protein levels in response to 147 treatment and addition of BME. Huh7 cells were pretreated with 147 and or 55µM BME 16 hours prior to DENV infection (MOI 3). Protein levels were quantified by Western blot. Error bars show SEM and p-values from unpaired t-tests are shown. C. Bar graph showing DENV viral titer levels in response to 147 treatment and rescue through addition of BME. Huh7 cells were pretreated with 147 and or 55µM BME A. Western blot data showing BME does not reduce DENV propagation in Huh7 cells. Cells were pretreated for 16h, then infected (where indicated) with DENV-2 strain BID-V533 at MOI of 3 for 3 hours. Media and treatments were replaced, and cells were collected 24hpi. Cells were lysed, and protein samples were run out on SDS-PAGE gels and visualized using PVDF membranes. B. Cell viability data showing 147/BME treatment only modestly affects Huh7 cell viability over the course of treatment. Cells were pretreated for 16h, then infected (where indicated) with DENV-2 strain BID-V533 at MOI of 3 for 3 hours. Media and treatments were replaced, and viability was measured 24hpi using the Promega Cell Titer Glo reagent. Graphs represent three to six biological replicates. Error bars represent SEM.
A. Illustration of the chemoproteomic workflow for target identification using chemical derivatives of 147. Cells are treated with 147-20, an alkyne analog of 147 which retains activity and can covalently label proteins as outlined in Fig. 4A . The alkyne handle enables derivatization with either a fluorophore or biotin azide for detection or affinity purification of protein targets. 147-4 is an inactive analog of 147 used to determine specificity in competition experiments. B. Fluorescence SDS-PAGE image identifying proteins labeled by 147-20 in Huh7 cells. Huh7 cells were treated with 147-20 (3µM) and 3-fold excess competitor (active 147, or inactive 147-4) for 18 hours. Labeled proteins in cell lysates were derivatized with a TAMRA-desthiobiotin azide, proteins resolved on SDS-PAGE and a fluorescence image of the gel is shown. The pattern of labeled proteins reveals similar targets as seen in other cell lines . The location of PDIA4, PDIA6, and PDIA1/P4HB is indicated. Fig. S5C shows images of Western blot overlays probing for individual PDI targets. C. A. Illustration of the workflow for the chemoproteomic target identification. Huh7 cells were treated with 3µM 147-20 alone or in competition with 9µM 147 for 18 hours. Cell lysates were labeled with a TAMRA-desthiobiotin probe, labeled proteins were isolated on streptavidin beads, eluted and digested with trypsin. Individual samples were then labeled with tandem mass tags, pooled and subjected to LC-MS/MS for identification of proteins and quantitative comparison of proteins in the 147-20 treated compared to the 147-20/147 competition. B. Graph showing the most highly enriched proteins targets in the 147-20 treated samples compared to the competition with 147. The full data is shown in Fig. S6A .
High-confidence targets were filtered that displayed a log2 enrichment ratio greater than two standard deviations of the distribution of enrichment ratios across four biological replicates. C. Venn diagram showing the comparison of high-confidence protein targets of 147-20 in Huh7 cells and other cells lines (HEK293T, HepG2 and ALMC-2) identified in a prior study . Fig. S6B shows the overlapping targets with individual cell lines.
A. Chemoproteomic target identification for 147-20 shows an enrichment of several PDIs compared to the 147-competition control. Quantitative proteomics using TMT-11plex reagents was used to compare log2 intensities across 4 replicates of probe (147-20 alone) versus probe in competition with excess parent compound 147. Targets were ordered from most enriched (high log2 fold change) to least enriched (low log2 fold change). Protein disulfide isomerases (PDIs, highlighted as red dots) were among the most enriched targets identified. B. Venn diagram of highly enriched targets found in Huh7 cells in this study compared to HEK293T, HepG2, and ALMC-2 cells in a previous study . Previous target ID studies were comparable to the study conducted herein. E.-F. Compound 147 is similarly active at reducing infection of Zika virus. Huh7 cells were pretreated with 147 (10µM) for 16 hours prior to infection with ZIKV strain MR766 (E) or ZIKV strain PRVABC59 (F). Viral titers were determined 24 hours hpi by focus forming assay. All error bars show SEM from 3 biological replicates and p-values from ratio paired t-tests are shown.
A. Representative western blot showing similar phenotype in HepG2 cells as in Huh7 cells. HepG2 cells were pretreated with 10 µM 147 for 16h, then infected with DENV-2 strain BID-V533 at MOI of 3 for 3 hours. Media and treatments were replaced, and cells were collected at 24 and 48 hpi. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. GAPDH is included as a loading control. B. Representative western blot showing attenuation of DENV-2 16681 viral protein levels on treatment with 147. Cells were pretreated with 10 µM 147 for 16h, then infected with DENV-2 strain 16681 at MOI of 3 for 3 hours. Media and treatments were replaced, and cells were collected at 24hpi. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. GAPDH is included as a loading control. C. Representative Western Blot showing attenuation of ZIKV PRVABC59 viral envelope (E) protein levels on treatment with 147. Cells were pretreated with 10 µM 147 for 16h, then infected with ZIKV strain PRVABC59 at MOI of 0.5 for 1 hour. Media and treatments were replaced, and cells were collected at 24hpi. Cells were lysed, and protein samples were run on SDS-PAGE gels and visualized using PVDF membranes. GAPDH is included as a loading control. D. Representative Western Blot showing attenuation of ZIKV MR766 viral E protein levels on treatment with 147. Cells were pretreated with 10 µM 147 for 16h, then infected with ZIKV strain MR766 at MOI of 0.5 for 1 hour. Media and treatments were replaced, and cells were collected at 24hpi. Cells were lysed, and protein