key: cord-0815060-39ai7a24
authors: Gagné, Bridget; Tremblay, Nicolas; Park, Alex Y.; Baril, Martin; Lamarre, Daniel
title: Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response
date: 2017-05-02
journal: Traffic
DOI: 10.1111/tra.12480
sha: 2ff61ec15104c7ff4107e9d4b0a71e82476ab603
doc_id: 815060
cord_uid: 39ai7a24

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon‐β (IFNB1) induction. Using a comprehensive microscopy‐based high‐content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A‐interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF‐κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown—a hub protein highly targeted by several viruses—decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus‐mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF‐κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad‐spectrum antiviral therapies. [Image: see text]

nuclear trafficking of the matrix protein has been reported to be critical for efficient production of viruses and to their pathogenesis. 14 Viruses also co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Influenza A virus, one of the few NSV to replicate in the nucleus, uses NS1 to form an inhibitory complex with nuclear RNA export factor 1 (NXF1) and NTF2-related export protein 1 (NXT1/p15) to block mRNA export, 15 while interaction of its nucleoprotein (NP) with NXT1 promotes the nuclear export of viral ribonucleoproteins (vRNPs) in an EXP1-dependent manner. 16 Viruses also interact with nucleocytoplasmic trafficking of transcription factors (TFs) of the immune responses as a viral strategy to promote viral growth. Highly pathogenic mononegavirale members such as the Nipah virus interacts with IMPα4 (KPNA3) and IMPα3 (KPNA4) for its viral protein nuclear localization to inhibit the activation of the IRF3-responsive promoter, 17 while Ebolavirus interacts with IMPα5 (KPNA1), IMPα6 (KPNA5) and IMPα7 (KPNA6) to disrupt their interaction with tyrosine-phosphorylated STAT1 for nuclear import, inhibiting IFN-stimulated gene (ISG) production. 18, 19 Similarly, positive-sense RNA poliovirus and rhinovirus are shown to disrupt nucleocytoplasmic trafficking by proteolytic cleavage of specific Nups that inhibit host antiviral defense pathways. [20] [21] [22] [23] Nidovirales like porcine reproductive and respiratory syndrome virus (PRRSV) degrades IMPα5 to block IFN-stimulated gene factor 3 (ISGF3) nuclear import, while SARS coronavirus (SARS-CoV) tethers IMPβ1 to ER and Golgi membranes, blocking STAT1 nuclear import. 24, 25 Finally, HCV NS3/4A interacts with IMPβ1 to prevent nuclear translocation of STAT1 and subsequent induction of ISGs. 1 Thus, viruses have evolved multiple strategies to exploit nucleocytoplasmic transport pathways to avert the host innate immune response and facilitate their replication.

NPCs are~60 to 120 MDa structures involved in the trafficking of macromolecules between the nucleus and the cytoplasm 26 that are composed of at least 30 different Nups. [27] [28] [29] These include the transmembrane Nups (POMs) that anchor to the NPC in the nuclear envelope, structural Nups that form the skeleton of the NPC, and the intrinsically disordered Nups that constitute the permeability barrier with their disordered phenylalanine-glycine-rich regions (FG-Nups).

The translocation of macromolecules (>40-60 kDa) across the NPC is facilitated by the IMP-β family proteins, which are nucleocytoplasmic transport receptors (NTRs) that primarily carry nuclear proteins and small RNAs as their cargoes through the nuclear pores. The human genome encodes 20 species of IMP-β family NTRs, of which 10 are nuclear import receptors, 7 are export receptors, 2 are bi-directional receptors and RanBP6 that the function is undetermined. 30 IMPβ1 is one of the best-studied member of the IMP-β protein family that mediate import of proteins into the nucleus either directly or indirectly through binding of adaptor proteins that belong to the IMP-α family. 30 These adaptors recognize proteins that contain a classical NLS and connect IMPβ1 and cargo molecules for their translocation in the nucleus. As such, IMPβ1 contains 19 tandem HEAT sequence repeats, which binds cargo-IMP-α complex through HEAT repeats 7-19, 31 RAN via HEAT repeats 1-3, 6, 7, 13 and 14 32, 33 and FG-Nups via HEAT repeats 5 and 6 for the translocation of cargo through NPCs. 34 Many proteins are also recognized directly by specific NTRs of the IMP-β family (TNPO1, TNPO2, TNPO3/IPO12) without the intervention of IMP-α, and often contain a proline-tyrosine NLS (PY-NLS) motif. 27 Conversely, EXP-1/CRM1, through interaction with NES, and others such as EXP-2/CSE1L, -5, -6, -7, -t and RanBP17 facilitate protein export to the cytoplasm. Finally, the direction of cargo transport is mainly controlled by GTP-binding nuclear protein Ran (RAN) across the nuclear envelope. RAN in the nucleus dissembles NTR-cargo import complexes, releasing the cargo and allowing NTRs to translocate back to the cytoplasm, 35 whereas RAN bound EXP-cargo complexes in the cytoplasm are dissembled when RANGTP is hydrolyzed to RANGDP by GTPase-activating protein 1 -(RANGAP1) and RAN binding proteins. 36 In this study, HCV-host interactors previously identified by liquid chromatography-mass spectroscopy (LC-MS/MS) analysis are investigated for their effect on IFNB1 transcription in response to SeV infection by gene silencing. Factors with the greatest inhibitory effects are functionally associated with protein transport by Gene Ontology (GO) analysis. Notably, all candidates were determined to interact with NS3/4A protein, which is well-known for its role in evading the antiviral response. The effects of silencing proteins associated to nucleocytoplasmic trafficking were investigated for IFNB1 production through the nuclear translocation of IRF3 and nuclear factor-kappa B (NF-κB) p65 TFs upon viral infection. By a microscopy-based high-content screening combined to gene silencing, we identified several proteins that reduce the number of IRF3-and p65-positive nuclei within a 10 hours period post-infection decreasing IFNB1 production. We also identified proteins that delayed, increased or had a differential effect on IRF3 and NF-κB p65 nuclear translocation, and similarly reduced IFNB1 production upon gene knockdown. Depletion of the main import carrier IMPβ1 showed the most significant decrease in IFIT1 (ISG56) expression, which correlated with rapid increase of viral protein levels and virus-mediated apoptosis. We found that expression of NS3/4A triggers the cleavage of IMPβ1 to evade IFNB1 production in This HCV interactome study led to the identification of selective host interactors to one of the viral proteins Core, NS2, NS3/4A, NS4B, NS5A and NS5B. The biological significance of these host proteins with an RNAi silencing screen further revealed that majority of these interactors are not affecting virus replication. However, HCV replication is often monitored in the RIG-I deficient Huh7.5 cell line such that one cannot assess if the viral-host protein interactions benefit the virus through subversion of the innate immune response resulting in increased viral replication and pathogenesis. To test this hypothesis, we silenced 132 selective HCV-host interactors ( Figure S1 , Supporting Information) using~5 independent short hairpin RNA (shRNA)expressing lentiviruses, and measured the induction of IFNB1 promoterdriven firefly luciferase upon SeV infection of A549 human lung carcinoma cells and Human Embryonic Kidney (HEK)293T cells. We previously showed that SeV infection predominantly activates the RLR pathway in these cells, leading to the nuclear translocation of NF-κB and IRF3 TFs, induction of IFNB1 mRNA and secretion of IFN-β cytokine. 37 In parallel, cells harboring a luciferase gene driven by the nonimmune endogenous elongation factor 1 alpha (EEF1A1) promoter were used to assess the effects of shRNAs on basal transcription. The gene silencing screens revealed 35 modulators of antiviral response in HEK293T cells and 29 modulators in A549 cells (Figure 1 ). In total, 53 HCV-host interactors, with 11 identified in both cell lines, specifically altered IFNB1

reporter expression by at least 2 independent shRNAs without affecting the constitutive expression of the EEF1A1 promoter (Table S2 ). The effect of silencing these 53 genes on modulation of HCV replication and antiviral response is summarized in Figure S2 . Notably, 12 proteins significantly affected both viral replication and IFNB1 induction. Overall, the data provide evidence that viruses can hijack cellular processes that contribute to the innate immune response and viral replication giving a dual growth advantage.

GO enrichment was performed to determine whether these 53 virushost interactors associated with one another through a particular complex or cell process. Table 1 lists 10 statistically significant enriched terms (P < .05) in order of association, as well as the respective genes. The 2 most enriched terms are "protein import into the nucleus," "docking" (enriched 72-fold with 4 proteins: IMPβ1/KPNB1, TNPO1, EXP1/XPO1 and EXP2/CSE1L, and "nuclear pore" (enriched 23-fold with 6 proteins: IMPβ1, TNPO1, lamin-B receptor [LBR], EXP1, EXP2 and RAN). As previously indicated, HCV proteins such as NS3 contain an NLS that contributes to the compartmentalization of viral replication within NPC-containing virus-induced membranous webs to limit access of pathogen-recognition receptors (PRRs) to these sites. Moreover, except for LBR, all 5 proteins associated with the nucleocytoplasmic transport have been shown to interact with NS3/4A, which is the main player in the evasion of the innate immune response through the cleavage of important signaling adaptors, such as MAVS and TRIF in the RLRs and Toll-like receptors B A Results were normalized according to cells treated with control shRNA non target (NT) (set to 1-black) based on an average of 2 independent experiments. The following criteria were applied to select modulator hits: at least 2 shRNAs per gene with >25% effect on IFNB1 promoter activity without affecting the nonimmune elongation factor 1α (EF1α) promoter-driven luciferase activity. Hits are clustered by their corresponding HCV binding partners, and the last 2 digits correspond to the shRNA number of The RNAi Consortium (TRC).

(TLRs) pathways, respectively. [38] [39] [40] In addition, NS3/4A protein is known to interact with IMPβ1 to prevent STAT1 nuclear translocation in response to type I IFN-mediated JAK-STAT pathway activation in HeLa human cervical cancer cells. 1 2.3 | Microscopy-based high content screening of nuclear transport of IRF3 and NF-κB p65 upon viral infection

The interaction of a set of nuclear transport factors with HCV proteins and their combined effects on increased viral replication and subversion of innate immune response encourage further mechanistic studies. Owing to their physiological function, we hypothesized that the observed effects on IFNB1 induction are associated with the nuclear translocation of IRF3 and NF-κB p65 in response to a phosphorylation event upon viral infection. 41, 42 The nucleocytoplasmic shuttling of IRF3 and NF-κB have been reported to be mediated via IMP-α/β and EXP1-dependent pathways, [43] [44] [45] [46] while a more recent study indicated that the import of NF-κB p65 mainly relies on the canonical IMPα1/IMPβ1 pathway following tumor necrosis factor-α (TNF-α) stimulation. 46 The Nups involved in these processes, however, are yet to be determined. To test our hypothesis, we developed a microscopy-based screen to quantify the translocation of IRF3 and NF-κB p65 to the nucleus in a kinetic analysis of SeV infection. Figure 2B ). Graphical representations of these results are plotted using the percentage of positive nuclear staining for IRF3 or NF-κB p65 ( Figure 2C ). Over the course of a 10-hour SeV infection, we observed an increase in both IRF3 and NF-κB p65 nuclear staining culminating to~75% positive cells at 5 hours post-infection, followed by a decrease to~30% at 10 hours. The microscopy data are confirmed biochemically with cell fractionation and western blot analysis ( Figure 2D ). As expected, we observed that IRF3 phosphorylation on Ser386 began at 1 hour, culminated at 5 hours and then decreased at 10 hours post-infection, consistent with the amount of total nuclear IRF3 observed at these time points. The phosphorylated forms of IRF3 could also be observed using the anti-IRF3 antibody in both cytoplasmic and nuclear fractions. Moreover, phosphorylation of the NF-κB negative regulator NFKBIA on Ser32-which triggers its ubiquitination and degradation-gradually increased from 1 to 10 hours post-infection.

NFKBIA degradation then allowed for NF-κB p65 nuclear translocation, which began at 1 hour, culminated at 5 hours and subsequently decreased at 10 hours post-infection. Cytoplasmic and nuclear fraction purity was confirmed by exclusive histone 1 (H1) staining in the (1 representative of 9 field images). Scale bar is equal to 100 μM. C, Graphic representation of the microscopy imagebased analysis showing an increase in the percentage of cells with positive nuclear staining for IRF3 or NF-κB p65 culminating with~75% of positive cells at 5 hours postinfection followed by a decrease to~30% of positive cells at 10 hours. D, Immunoblot analysis of total cell lysates, cytoplasmic and nuclear extracts of A549 cells infected with lentivirus-encoding shRNA NT at a MOI of 10 for 3 days and infected with SeV for 0, 1, 3, 5, 8 and 10 hours prior to cell harvesting (IMPα3 and EXP1), and to differential effects on IRF3 and NF-κB p65 translocation for 2 proteins (IMPα6 and RANBP3). We further divided these 33 proteins into subgroups based on their function and localization, respectively.

2.4 | Analysis of IMP-β NTRs and Nups knockdown on virus-mediated IRF3/NF-κB p65 nuclear translocation and IFNB1 production

The analysis revealed that the silencing of IMP-α proteins (KPNA1-6) led to a distinct phenotype for 5 of the 6 adaptors, with a major decrease in NF-κB p65 nuclear translocation at 3 hours post-SeV infection, the only exception being with IMPα3 (KPNA4) knockdown cells (Figures 3 and S3) . Notably, the effect of these adaptors on IRF3 was more variable-but IMPα3 also increased IRF3 Results are presented as average of all shRNAs for each IMP-α. Individual shRNA results on IRF3 and NF-κB p65 nuclear translocation, IFNB1 promoter activity and cell proliferation and survival are described in Figure S3 ( Figure 8D ). NS3/4A protease expression greatly hindered IRF3 and Altogether, the data demonstrate that NS3/4A protease-mediated cleavage of IMPβ1 inhibits innate immune response to viral infection by restricting nuclear translocation of IRF3 and NF-κB.

The goal of the study was to elucidate novel proteins, families or processes that affected innate immune response from a subset of human proteins previously elucidated to be interacting with HCV proteins.

We found that majority of host interactors did not affect HCV replication, 1 although their knockdown was performed in the Huh7.5 cell line that is deficient for the RLR signaling pathway. Thus, we Several NTRs were previously associated to the nuclear trafficking of NF-κB p65 while there are virtually no reports for IRF3 during viral infection. In our study, the depletion of IMP-α family members had varying effects on IRF3/NF-κB p65 nuclear localization but IFNB1 production was significantly decreased when IMPα1, IMPα4

and IMPα6 are individually silenced (Figures 3 and S3) , which could be attributed to these 3 genes decreasing both IRF3 and NF-κB p65 nuclear translocation at 3 hours post-infection. NF-κB p65 was reported to be transported by IMPα3 and IMPα4 while a more recent study identified IMPα1 as the most critical adaptor for its nuclear translocation upon tumor necrosis factor-α (TNF-α) treatment. 43, 44, 46 Our results largely support these studies with a predominant role of IMPα1 and IMPα4, except for the depletion of IMPα3 that may be over-compensating by other adaptors causing the increase of NF-κB p65 during viral infection. In addition, the need for fast activation upon viral infection must not require a process that is dependent on IMPα3 as its knockdown has no impact on IFNB1 production. Our data also demonstrate that IMPβ1 is one of the main import receptor for IRF3 and NF-κB p65 import upon viral infection (Figure 7) , as previously reported for NF-κB p65. 46 However, other carriers have been identified for NF-κB p65 import including IPO8, 46 which transports this factor in a NLS-independent fashion after stimulation TNF-α. In our study, knockdown of IPO8 resulted in a decrease IRF3 and NF-κB p65 nuclear translocation during the entire time course of infection and reduction of IFNB1 production ( Figures 4A and S4) , suggesting that an NLS-independent import process for IRF3 and NF-κB p65 may take place during viral infection. We also identified the import carriers TNPO1 and IPO7 as their knockdown reduces nuclear translocation of both TFs (Figures 4A,B, S4, S5) , which supports the finding of a PY-NLS that is recognized by TNPO1. 52 Finally, knockdown of IPO4, TNPO2 and IPO12 affect irregularly or at later time NF-κB p65 and IRF3 nuclear translocation (Figures 4, S4 and S5) , suggesting a weak if any contribution to these processes in a virus-dependent manner. IMP-β family NTRs involved in protein export have significant effects for IRF3 and NF-κB p65 nuclear translocation and IFNB1

production, but also have pleiotropic effects on cell survival. Nevertheless, EXP1 was previously shown to bind IRF3 NES, and the use of EXP1 inhibitor leptomycin B further demonstrated an accumulation of IRF3 in the nucleus. 45 Our results support EXP1 as the main carrier for IRF3 export causing nuclear accumulation of IRF3 during the early phase of the infection upon it knockdown (Figures 4 and   S6 ). This is further supported by RANBP3 knockdown, an EXP1 cofactor for protein export, 53,54 which increases nuclear translocation of IRF3 at 8 hours of SeV infection but surprisingly without affecting IFNB1 production ( Figure S6 ). Among all EXP family members, only EXP1 systematically exported NF-κB p65 at all time confirming a previous study. 46 Despite the increased nuclear levels of IRF3 and NF-κB p65, EXP1 knockdown decreased IFNB1 promoter activity following SeV infection ( Figure S6 ). This may be explained by an effect on cell survival and/or by the fact that residual NF-κB p65 can reassociate with IκBα to reduce it transcriptional activity when sequestered in the nucleus. 55, 56 Interestingly, EXP2 knockdown causes a dramatic decrease of nuclear IRF3 and NF-κB p65 during the entirety of the SeV infection time course leading to a significant reduction of IFNB1 and cell fitness ( Figure S6 ). This is possibly due to its major role for the export of IMP-α adaptors to the cytoplasm as a recycling mechanism of import complexes. 57 We also identified NXF1 and NXT1 proteins that significantly reduced nuclear import of IRF3/NF-κB p65 and IFNB1 production ( Figures 4D and S7) . These proteins are exploited by several viruses to promote viral mRNA export and inhibit host mRNA trafficking. 20 Further studies will be required to demonstrate if the diminished amounts of IRF3 and NF-κB p65 proteins translated and able to enter the nucleus are due to the reduced export of their mRNAs to prevent antiviral response. The silencing of RAN, NUTF2 and RCC1 equally affected IRF3 and NF-κB p65 nuclear translocation and IFNB1 production, while only RAN silencing affected cells survival (Figures 4E and S8 ). As NUTF2 is responsible for recycling RANGDP back to the nucleus and RCC1 for exchanging the GDP to GTP, these data confirm that the lack of RANGDP or RANGTP in the nucleus affect nuclear transport with IRF3 being less affected than NF-κB p65. Nevertheless, the silencing of RAN components NUTF2 and RCC1 decreasing IFNB1 production without affecting cell fitness ( Figure S8 ) further corroborate that this process is critical for ensuring proper nucleocytoplasmic transport required for antiviral response. Finally, multiple Nups had a similar silencing phenotype with the reduced nuclear translocation of IRF3 and NF-κB and S10). The linker Nups, which are extremely important for the proper assembly of the NPC and recruitment of NUP62 for transport competency, also contribute to IRF3/NF-κB p65 nuclear transport ( Figures 5C and S11) . Indeed, NUP93 and NUP88 knockdown decrease the translocation of both TFs and IFNB1 production as previously reported. 37, 59 At last, central FG-Nups NUP54 and NUPL1

(encoding NUP58 and NUP45) also significantly reduce IFNB1 production, in contrast to the weak phenotype of NUP35 ( Figures 5D and S12).

Very importantly, our data revealed a unique phenotype of protein interaction with IMPβ1 can counteract IFNB1 production by disrupting IRF3 and NF-κB p65 nuclear transport upon induction of a cellular antiviral state. We previously reported that the silencing of IMPβ1 phenocopied HCV infection in preventing STAT1 from accumulating in the nucleus, thus providing strong evidence that the interaction between NS3/4A and IMPβ1 mediates the blocking of STAT1 nuclear accumulation to promote HCV replication. 1 We now provide 

DAVID database was used for functional annotation. 62,63 DAVID functional annotation chart tool was used to perform GO biological process and InterPro protein domain analysis. Terms with a P-value lesser than 5 × 10 −2 were considered as significantly overrepresented.

Elucidating novel hepatitis C virus-host interactions using combined mass spectrometry and functional genomics approaches

Identification of host proteins required for HIV infection through a functional genomic screen

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication

A host of factors regulating influenza virus replication

Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web

Hepatitis C virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication

A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport

Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration

Transportin 3 and importin alpha are required for effective nuclear import of HIV-1 integrase in virus-infected cells

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein

Molecular determinants for subcellular localization of hepatitis C virus core protein

The hepatitis C virusinduced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites

Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses: paramyxoviruses and beyond

Influenza virus targets the mRNA export machinery and the nuclear pore complex

NXT1, a novel influenza A NP binding protein, promotes the nuclear export of NP via a CRM1-dependent pathway

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus-and toll-like receptor 3-triggered signaling pathways

Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin alpha proteins with activated STAT1

Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1

Viral subversion of nucleocytoplasmic trafficking

Specific cleavage of the nuclear pore complex protein Nup62 by a viral protease

Differential targeting of nuclear pore complex proteins in poliovirus-infected cells

RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage

Porcine reproductive and respiratory syndrome virus Nsp1beta inhibits interferon-activated JAK/STAT signal transduction by inducing karyopherin-alpha1 degradation

Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane

The selective permeability barrier in the nuclear pore complex

Components and regulation of nuclear transport processes

Functional architecture of the nuclear pore complex

The structure of the nuclear pore complex

Biological significance of the importin-beta family-dependent nucleocytoplasmic transport pathways

Structure of importin-beta bound to the IBB domain of importin-alpha

Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport

Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking

Individual binding pockets of importin-beta for FG-nucleoporins have different binding properties and different sensitivities to RanGTP

Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins

Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses

MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus

Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation

Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport

NF-{kappa}B is transported into the nucleus by importin {alpha}3 and importin {alpha}4

NF-kappaB p52, RelB and c-Rel are transported into the nucleus via a subset of importin alpha molecules

Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1

XPO7 and IPO8 mediate the translocation ofNF-kappaB/p65 into the nucleus

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus

Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear pore-targeting complex formation with NPI-1, but not Rch1

Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity

Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection

Rules for nuclear localization sequence recognition by karyopherin beta 2

RanBP3 influences interactions between CRM1 and its nuclear protein export substrates

Ranbinding protein 3 is a cofactor for Crm1-mediated nuclear protein export

Signaling molecules of the NF-kappa B pathway shuttle constitutively between cytoplasm and nucleus

Cytoplasmic sequestration of rel proteins by IkappaBalpha requires CRM1-dependent nuclear export

Structural biology of nucleocytoplasmic transport

Integrated structural analysis of the human nuclear pore complex scaffold

Tumor marker nucleoporin 88 kDa regulates nucleocytoplasmic transport of NF-kappaB

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor

3Cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking STAT1/STAT2 nuclear translocation

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources

Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists

Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF-κB signaling of IFNB1 antiviral response

We thank K. Audette and J. Duchaine of the IRIC's screening facility.