Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression.

BACKGROUND: The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. RESULTS: Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. CONCLUSIONS: The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

Steric-Deficient Oligoglycine Surrogates Facilitate Multivalent and Bifunctional Nanobody Synthesis via Combined Sortase A Transpeptidation and Click Chemistry.

Conferring multifunctional properties to proteins via enzymatic approaches has greatly facilitated recent progress in protein nanotechnology. In this regard, sortase (Srt) A transpeptidation has facilitated many of these developments due to its exceptional specificity, mild reaction conditions, and complementation with other bioorthogonal techniques, such as click chemistry. In most of these developments, Srt A is used to seamlessly tether oligoglycine-containing molecules to a protein of interest that is equipped with the enzyme's recognition sequence, LPXTG. However, the dependence on oligoglycine attacking nucleophiles and the associated cost of certain derivatives (e.g., cyclooctyne) limit the utility of this approach to lab-scale applications only. Thus, the quest to identify appropriate alternatives and understand their effectiveness remains an important area of research. This study identifies that steric and nucleophilicity-associated effects influence Srt A transpeptidation when two oligoglycine surrogates were examined. The approach was further used in complementation with click chemistry to synthesize bivalent and bifunctional nanobody conjugates for application in epithelial growth factor receptor targeting. The overall technique and tools developed here may facilitate the advancement of future nanotechnologies.

Increased In Vivo Exposure of N-(4-Hydroxyphenyl) Retinamide (4-HPR) to Achieve Plasma Concentrations Effective against Dengue Virus.

N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretinide) has promising in vitro and in vivo antiviral activity against a range of flaviviruses and an established safety record, but there are challenges to its clinical use. This study evaluated the in vivo exposure profile of a 4-HPR dosage regime previously shown to be effective in a mouse model of severe dengue virus (DENV) infection, comparing it to an existing formulation for human clinical use for other indications and developed/characterised self-emulsifying lipid-based formulations of 4-HPR to enhance 4-HPR in vivo exposure. Pharmacokinetic (PK) analysis comprising single-dose oral and IV plasma concentration-time profiles was performed in mice; equilibrium solubility testing of 4-HPR in a range of lipids, surfactants and cosolvents was used to inform formulation approaches, with lead formulation candidates digested in vitro to analyse solubilisation/precipitation prior to in vivo testing. PK analysis suggested that effective plasma concentrations could be achieved with the clinical formulation, while novel lipid-based formulations achieved > 3-fold improvement. Additionally, 4-HPR exposure was found to be limited by both solubility and first-pass intestinal elimination but could be improved through inhibition of cytochrome P450 (CYP) metabolism. Simulated exposure profiles suggest that a b.i.d dosage regime is likely to maintain 4-HPR above the minimum effective plasma concentration for anti-DENV activity using the clinical formulation, with new formulations/CYP inhibition viable options to increase exposure in the future.

Sortase A transpeptidation produces seamless, unbranched biotinylated nanobodies for multivalent and multifunctional applications.

Exploitation of the biotin-streptavidin interaction for advanced protein engineering is used in many bio-nanotechnology applications. As such, researchers have used diverse techniques involving chemical and enzyme reactions to conjugate biotin to biomolecules of interest for subsequent docking onto streptavidin-associated molecules. Unfortunately, the biotin-streptavidin interaction is susceptible to steric hindrance and conformational malformation, leading to random orientations that ultimately impair the function of the displayed biomolecule. To minimize steric conflicts, we employ sortase A transpeptidation to produce quantitative, seamless, and unbranched nanobody-biotin conjugates for efficient display on streptavidin-associated nanoparticles. We further characterize the protein-nanoparticle complex and demonstrate its usefulness in optical microscopy and multivalent severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigen interaction. The approach reported here provides a template for making novel multivalent and multifunctional protein complexes for avidity-inspired technologies.

Harnessing sortase A transpeptidation for advanced targeted therapeutics and vaccine engineering.

The engineering of potent prophylactic and therapeutic complexes has always required careful protein modification techniques with seamless capabilities. In this light, methods that favor unobstructed multivalent targeting and correct antigen presentations remain essential and very demanding. Sortase A (SrtA) transpeptidation has exhibited these attributes in various settings over the years. However, its applications for engineering avidity-inspired therapeutics and potent vaccines have yet to be significantly noticed, especially in this era where active targeting and multivalent nanomedications are in great demand. This review briefly presents the SrtA enzyme and its associated transpeptidation activity and describes interesting sortase-mediated protein engineering and chemistry approaches for achieving multivalent therapeutic and antigenic responses. The review further highlights advanced applications in targeted delivery systems, multivalent therapeutics, adoptive cellular therapy, and vaccine engineering. These innovations show the potential of sortase-mediated techniques in facilitating the development of simple plug-and-play nanomedicine technologies against recalcitrant diseases and pandemics such as cancer and viral infections.

The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

The cellular response to environmental stresses, such as heat and oxidative stress, is dependent on extensive trafficking of stress-signalling molecules between the cytoplasm and nucleus, which potentiates stress-activated signalling pathways, eventually resulting in cell repair or death. Although Ran-dependent nucleocytoplasmic transport mediated by members of the importin (IPO) super family of nuclear transporters is believed to be responsible for nearly all macromolecular transit between nucleus and cytoplasm, it is paradoxically known to be significantly impaired under conditions of stress. Importin 13 (IPO13) is a unique bidirectional transporter that binds to and releases cargo in a Ran-dependent manner, but in some cases, cargo release from IPO13 is affected by loading of another cargo. To investigate IPO13's role in stress-activated pathways, we performed cell-based screens to identify a multitude of binding partners of IPO13 from human brain, lung, and testes. Analysis of the IPO13 interactome intriguingly indicated more than half of the candidate binding partners to be annotated for roles in stress responses; these included the pro-apoptotic protein nuclear clusterin (nCLU), as well as the nCLU-interacting DNA repair protein KU70. Here, we show, for the first time, that unlike other IPOs which are mislocalised and non-functional, IPO13 continues to translocate between the nucleus and cytoplasm under stress, retaining the capacity to import certain cargoes, such as nCLU, but not export others, such as KU70, as shown by analysis using fluorescence recovery after photobleaching. Importantly, depletion of IPO13 reduces stress-induced import of nCLU and protects against stress-induced cell death, with concomitant protection from DNA damage during stress. Overexpression/FACS experiments demonstrate that nCLU is dependent on IPO13 to trigger stress-induced cell death via apoptosis. Taken together, these results implicate IPO13 as a novel functional nuclear transporter in cellular stress, with a key role thereby in cell fate decision.

Conservation of Importin α Function in Apicomplexans: Ivermectin and GW5074 Target Plasmodium falciparum Importin α and Inhibit Parasite Growth in Culture.

Signal-dependent transport into and out of the nucleus mediated by members of the importin (IMP) superfamily of nuclear transporters is critical to the eukaryotic function and a point of therapeutic intervention with the potential to limit disease progression and pathogenic outcomes. Although the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii both retain unique IMPα genes that are essential, a detailed analysis of their properties has not been performed. As a first step to validate apicomplexan IMPα as a target, we set out to compare the properties of P. falciparum and T. gondii IMPα (PfIMPα and TgIMPα, respectively) to those of mammalian IMPα, as exemplified by Mus musculus IMPα (MmIMPα). Close similarities were evident, with all three showing high-affinity binding to modular nuclear localisation signals (NLSs) from apicomplexans as well as Simian virus SV40 large tumour antigen (T-ag). PfIMPα and TgIMPα were also capable of binding to mammalian IMPß1 (MmIMPß1) with high affinity; strikingly, NLS binding by PfIMPα and TgIMPα could be inhibited by the mammalian IMPα targeting small molecules ivermectin and GW5074 through direct binding to PfIMPα and TgIMPα to perturb the α-helical structure. Importantly, GW5074 could be shown for the first time to resemble ivermectin in being able to limit growth of P. falciparum. The results confirm apicomplexan IMPα as a viable target for the development of therapeutics, with agents targeting it worthy of further consideration as an antimalarial.

Transcriptomic profile dataset of embryonic stem cells (Wild-type and IPO13-Knock Out) with and without oxidative stress.

The transcriptional response to cellular stress relies upon trafficking of regulators of transcription between the nuclear and cytoplasmic compartments, which occurs through action of members of the importin (IPO) superfamily. As a result of stresses such as oxidative or osmotic stress, one consequence is that importins become mislocalised, leading to inhibition of conventional nuclear transport. Here, we examine IPO13, which has a number of nonconventional characteristics, in the context of cell stress. We used Next Generation RNA Sequencing using the Illumina platform to compare the transcriptomes of Wild-type (WT) and IPO13-Knockout (KO) mouse embryonic stem cells in the absence and presence of oxidative stress. Differences in the mRNA expression profiles were observed between the cell lines in the absence and in the presence of stress. This data will be a key resource to enable characterization of the contribution of nuclear transporter IPO13 to cellular transcription in the absence and presence of oxidative stress, as well as more broadly, in the study of stem cell biology and effect of stress on embryonic stem cell transcription.

High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers.

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

High Throughput Screening Targeting the Dengue NS3-NS5 Interface Identifies Antivirals against Dengue, Zika and West Nile Viruses.

Dengue virus (DENV) threatens almost 70% of the world's population, with no effective therapeutic currently available and controversy surrounding the one approved vaccine. A key factor in dengue viral replication is the interaction between DENV nonstructural proteins (NS) 5 and 3 (NS3) in the infected cell. Here, we perform a proof-of-principle high-throughput screen to identify compounds targeting the NS5-NS3 binding interface. We use a range of approaches to show for the first time that two small molecules-repurposed drugs I-OMe tyrphostin AG538 (I-OMe-AG238) and suramin hexasodium (SHS)-inhibit NS5-NS3 binding at low µM concentration through direct binding to NS5 that impacts thermostability. Importantly, both have strong antiviral activity at low µM concentrations against not only DENV-2, but also Zika virus (ZIKV) and West Nile virus (WNV). This work highlights the NS5-NS3 binding interface as a viable target for the development of anti-flaviviral therapeutics.

Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus.

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP-host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp ß, and a complex of Imp α/ß but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp ß or Imp α/ß. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

The broad spectrum host-directed agent ivermectin as an antiviral for SARS-CoV-2 ?

FDA approved for parasitic indications, the small molecule ivermectin has been the focus of growing attention in the last 8 years due to its potential as an antiviral. We first identified ivermectin in a high throughput compound library screen as an agent potently able to inhibit recognition of the nuclear localizing Human Immunodeficiency Virus-1 (HIV-1) integrase protein by the host importin (IMP) α/ß1 heterodimer, and recently demonstrated its ability to bind directly to IMPα to cause conformational changes that prevent its function in nuclear import of key viral as well as host proteins. Cell culture experiments have shown robust antiviral action towards a whole range of viruses, including HIV-1, dengue, Zika and West Nile Virus, Venezuelan equine encephalitis virus, Chikungunya, pseudorabies virus, adenovirus, and SARS-CoV-2 (COVID-19). Close to 70 clinical trials are currently in progress worldwide for SARS-CoV-2. Although few of these studies have been completed, the results that are available, as well as those from observational/retrospective studies, indicate clinical benefit. Here we discuss the case for ivermectin as a host-directed broad-spectrum antiviral agent, including for SARS-CoV-2.

The nuclear transporter importin 13 is critical for cell survival during embryonic stem cell differentiation.

Nuclear transporter Importin (Imp, Ipo) 13 is known to transport various mammalian cargoes into/out of the nucleus, but its role in directing cell-fate is unclear. Here we examine the role of Imp13 in the maintenance of pluripotency and differentiation of embryonic stem cells (ESCs) for the first time, using an embryonic body (EB)-based model. When induced to differentiate, Ipo13-/- ESCs displayed slow proliferation, reduced EB size, and lower expression of the proliferation marker KI67, concomitant with an increase in the number of TUNEL+ nuclei compared to wildtype ESCs. At days 5 and 10 of differentiation, Ipo13-/- EBs also showed enhanced loss of the pluripotency transcript OCT3/4, and barely detectable clusters of OCT3/4 positive cells. Day 5 Ipo13-/- EBs further exhibited reduced levels of the mesodermal markers Brachyury and Mixl1, correlating with reduced numbers of haemoglobinised cells generated. Our findings suggest that Imp13 is critical to ESC survival as well as early post-gastrulation differentiation.

Ivermectin as a Broad-Spectrum Host-Directed Antiviral: The Real Deal?

The small molecule macrocyclic lactone ivermectin, approved by the US Food and Drug Administration for parasitic infections, has received renewed attention in the last eight years due to its apparent exciting potential as an antiviral. It was identified in a high-throughput chemical screen as inhibiting recognition of the nuclear localizing Human Immunodeficiency Virus-1 (HIV-1) integrase protein by the host heterodimeric importin (IMP) α/ß1 complex, and has since been shown to bind directly to IMPα to induce conformational changes that prevent its normal function in mediating nuclear import of key viral and host proteins. Excitingly, cell culture experiments show robust antiviral action towards HIV-1, dengue virus (DENV), Zika virus, West Nile virus, Venezuelan equine encephalitis virus, Chikungunya virus, Pseudorabies virus, adenovirus, and SARS-CoV-2 (COVID-19). Phase III human clinical trials have been completed for DENV, with >50 trials currently in progress worldwide for SARS-CoV-2. This mini-review discusses the case for ivermectin as a host-directed broad-spectrum antiviral agent for a range of viruses, including SARS-CoV-2.

Internalized Functional DNA Aptamers as Alternative Cancer Therapies.

Despite major advances, cancer remains one of the largest burdens of disease worldwide. One reason behind this is that killing tumor cells without affecting healthy surrounding tissue remains a largely elusive prospect, despite the widespread availability of cytotoxic chemotherapeutic agents. To meet these modern healthcare requirements, it is essential to develop precision therapeutics that minimise off-target side-effects for various cancer types. To this end, highly specific molecular targeting agents against cancer are of great interest. These agents may work by targeting intracellular signalling pathways following receptor binding, or via internalization and targeting to specific subcellular compartments. DNA aptamers represent a promising molecular tool in this arena that can be used for both specific cell surface targeting and subsequent internalization and can also elicit a functional effect upon internalization. This review examines various cancer targeting cell-internalizing aptamers, with a particular focus towards functional aptamers that do not require additional conjugation to nanoparticles or small molecules to elicit a biological response. With a deeper understanding and precise exploitation of cancer specific molecular pathways, functional intracellular DNA aptamers may be a powerful step towards more widespread development of precision therapeutics.

The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro.

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans.

Inhibitors of nuclear transport.

Central to eukaryotic cell function, transport into and out of the nucleus is largely mediated by members of the Importin (IMP) superfamily of transporters of α- and ß-types. The first inhibitor of nuclear transport, leptomycin B (LMB), was shown to be a specific inhibitor of the IMPß homologue Exportin 1 (EXP1) almost 20 years ago, but it has only been in the last five or so years that new inhibitors of nuclear export as well as import have been identified and characterised. Of utility in biological research, these inhibitors include those that target-specific EXPs/IMPs, with accompanying toxicity profiles, as well as agents that specifically target particular nuclear import cargoes. Both types of inhibitors have begun to be tested in preclinical/clinical studies, with particular focus on limiting various types of cancer or treating viral infection, and the most advanced agent targeting EXP1 (Selinexor) has progressed successfully through >40 clinical trials for a range of high-grade cancers and is approaching FDA approval for a number of indications. Selectively inhibiting the nucleocytoplasmic trafficking of specific proteins of interest remains a challenge, but progress in the area of the host-pathogen interface holds promise for the future.

Novel Flavivirus Antiviral That Targets the Host Nuclear Transport Importin α/ß1 Heterodimer.

Dengue virus (DENV) threatens almost 70% of the world's population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/ß1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5⁻IMPα/ß1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPß1, but can dissociate preformed IMPα/ß1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.