Kylie M. Wagstaff

The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-κB p65

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-κB, to the host cell nucleus. NF-κB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-κB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFα and IL-1β stimulated p65 nuclear translocation and IκB degradation, NleB inhibited the TNFα pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

Importins and Beyond: Non-Conventional Nuclear Transport Mechanisms

The movement of proteins between the cytoplasm and the nucleus conventionally involves the recognition of nuclear targeting signals by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear envelope‐embedded nuclear pore complexes (NPCs). It is becoming increasingly apparent, however, that distinct alternative pathways for nuclear transport exist and are relatively abundant. This review examines several of these novel pathways, including facilitation of Imp‐dependent transport by microtubule motors, and Imp‐independent pathways involving either other transport molecules such as the calcium‐binding protein calmodulin or through direct binding to the components of the NPC. The existence of these pathways and the fact that many proteins appear to possess separate Imp‐dependent and ‐independent nuclear import mechanisms ensure that the cell can function under conditions in which Imp‐dependent transport is inhibited and/or modulate the efficiency of Imp‐dependent transport itself, according to the need.

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.

Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer

Gene therapy, the correction of dysfunctional or deleted genes by supplying the lacking component, has long been awaited as a means to permanently treat or reverse many genetic disorders. To achieve this, therapeutic DNA must be delivered to the nucleus of cells using a safe and efficient delivery vector. Although viral-based vectors have been utilized extensively due to their innate ability to deliver DNA to intact cells, safety considerations, such as pathogenicity, oncogenicity and the stimulation of an immunological response in the host, remain problematical. There has, however, been much progress in the development of safe non-viral gene-delivery vectors, although they remain less efficient than the viral counterparts. The major limitations of non-viral gene transfer reside in the fact that it must be tailored to overcome the intracellular barriers to DNA delivery that viruses already master, including the cellular and nuclear membranes. In particular, nuclear transport of the therapeutic DNA is known to be the rate-limiting step in the gene-delivery process. Despite this, much progress had been made in recent years in developing novel means to overcome these barriers and efficiently deliver DNA to the nuclei of intact cells. This review focuses on the nucleocytoplasmic delivery of DNA and mechanisms to enhance to non-viral-mediated gene transfer.

An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.

Nuclear drug delivery to target tumour cells.

Cancer remains one of the leading causes of death worldwide. Although enticing, the concept of a chemotherapeutic treatment directed towards a single target that kills tumour cells, without any harmful side effects or death of neighbouring cells is probably naïve, due to the fact that tumour cells arise from normal cells and share many common biological features with them. Various means to damage/destroy tumour cells preferentially have been developed, but as yet, none are truly selective. However, by combining numerous tumour-specific/-enhanced targeting signals into a single modular multifaceted approach, it may prove possible some time in the future to achieve the desired outcome, without any unwanted bystander effects, with the delivery of cytotoxic drugs/DNA directly to the nucleus specifically within tumour cells of great interest in this context. To achieve this, modules such as the tumour-specific nuclear targeting signal of the chicken anemia virus Apoptin protein represent exciting possibilities. The present review discusses the question of nuclear delivery of bioactive molecules and its associated problems, as well as recent progress towards the development of tumour-specific modular recombinant transporters as viable anti-cancer therapeutics. In particular we focus upon the incorporation of tumour cell-selective nuclear targeting into these systems as a means to deliver cytotoxic genes or drugs to the very heart of tumour cells.

A Nuclear Transport Inhibitor That Modulates the Unfolded Protein Response and Provides In Vivo Protection Against Lethal Dengue virus Infection

BACKGROUND Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available. METHODS We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition. RESULTS We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model. CONCLUSIONS 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model.

The N-terminal basic domain of the HIV-1 matrix protein does not contain a conventional nuclear localization sequence but is required for DNA binding and protein self-association.

The HIV p17 or matrix (MA) protein has long been implicated in the process of nuclear import of the HIV genome and thus the ability of the virus to infect nondividing cells such as macrophages. While it has been demonstrated that MA is not absolutely required for this process, debate continues to surround the subcellular targeting properties of MA and its potential contribution to nuclear import of the HIV cDNA. Through the use of in vitro techniques we have determined that, despite the ability of MA to interact with importins, the full-length protein fails to enter the nucleus of cells. While MA does contain a region of basic amino acids within its N-terminus which can confer nuclear accumulation of a fusion protein, we show that this is due to nuclear retention mediated by DNA binding and does not represent facilitated import. Importantly, we show that the 26KK residues of MA, previously thought to be part of a nuclear localization sequence, are absolutely required for a number of MAs functions including its ability to bind DNA and RNA and its propensity to form high-order multimers/protein aggregates. The results presented here indicate that the N-terminal basic domain of MA does not appear likely to play a role in HIV cDNA nuclear import; rather this region appears to be a crucial structural and functional motif whose integrity is required for a number of other roles performed by MA during viral infection.

Global enhancement of nuclear localization-dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)‐containing proteins, but not their NLS‐mutated derivatives, is increased up to 7‐fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)‐transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/ MCF10CA1h cell pair. Nuclear accumulation of NLS/ nuclear export signal‐containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp‐1‐mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2‐ to 4‐fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5‐fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor‐specific drug nanocarriers in anticancer therapy.— Kuusisto, H. V., Wagstaff, K. M., Alvisi, G., Roth, D. M., Jans, D. A. Global enhancement of nuclear localization‐dependent nuclear transport in transformed cells. FASEB J. 26, 1181‐1193 (2012). www.fasebj.org

Nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce Venezuelan Equine Encephalitis Virus replication.

Targeting host responses to invading viruses has been the focus of recent antiviral research. Venezuelan Equine Encephalitis Virus (VEEV) is able to modulate host transcription and block nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins importin α/β1 and CRM1. We hypothesized that disrupting the interaction of capsid with importin α/β1 or the interaction of capsid with CRM1 would alter capsid localization, thereby lowering viral titers in vitro. siRNA mediated knockdown of importin α, importin β1, and CRM1 altered capsid localization, confirming their role in modulating capsid trafficking. Mifepristone and ivermectin, inhibitors of importin α/β-mediated import, were able to reduce nuclear-associated capsid, while leptomycin B, a potent CRM1 inhibitor, confined capsid to the nucleus. In addition to altering the level and distribution of capsid, the three inhibitors were able to reduce viral titers in a relevant mammalian cell line with varying degrees of efficacy. The inhibitors were also able to reduce the cytopathic effects associated with VEEV infection, hinting that nuclear import inhibitors may be protecting cells from apoptosis in addition to disrupting the function of an essential viral protein. Our results confirm that VEEV uses host importins and exportins during part of its life cycle. Further, it suggests that temporarily targeting host proteins that are hijacked for use by viruses is a viable antiviral therapy.