These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.

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.

Chicken anemia virus viral protein 3 (VP3 or apoptin) localizes more efficiently in the nucleus of transformed than nontransformed cells. Although previous studies implicate the C-terminus of apoptin as being responsible, the molecular basis is controversial, and the extent to which altered nuclear transport efficiency in tumor cells may influence VP3 differential targeting unclear. Here we establish that the C-terminus of VP3 (residues 74-121), out of the context of the full-length protein, is indeed sufficient for tumor cell-enhanced nuclear targeting through phosphoinhibition of VP3 (74-121)-mediated nuclear export occurring exclusively in tumor cells. Importantly, we show that VP3 (74-121) is unique in showing tumor cell-enhanced nuclear targeting in that other NLS-containing proteins fail to show differential localization in human osteosarcoma cells compared to their normal isogenic counterparts. Thus, the C-terminus of VP3 represents a unique tumor cellenhanced nuclear targeting module with potential application in tumor cell-specific drug delivery. ' 2008 Wiley-Liss, Inc.Key words: apoptin; VP3; tNTS; NLS; NES; tumor specific; nuclear import; nuclear export; phosphorylation Shuttling of macromolecules such as proteins between the nucleus and cytoplasm is a central feature of eukaryotic cell function and generally occurs in a facilitated fashion through nuclear envelope-embedded multi-protein channels, known as nuclear pore complexes (NPCs). Nuclear protein import is mediated by a family of soluble cellular transport receptors called importins (IMPs), of which there are 2 types, a and b. IMPb, either directly or through the adapter protein IMPa, recognizes short modular sequences called nuclear localization signals (NLSs) on proteins carrying them and subsequently mediates translocation of the IMP:cargo complex through the NPC into the nucleoplasm, where the complex dissociates.1 Facilitated nuclear protein export occurs in an analogous fashion, whereby nuclear export signals (NESs), generally short hydrophobic (leucine-rich) patches, are recognized by IMPb homologues known as exportins, of which CRM-1 is the most well characterized.2 Nucleocytoplasmic shuttling can be modulated by a number of distinct mechanisms, with phosphorylation close to an NLS or NES being one of the most common mechanisms of regulating nucleocytoplasmic shuttling of NLS/ NES-carrying cargoes, by enhancing or inhibiting recognition by IMPs or EXPs, respectively (see Refs.3 and 4). Nuclear transport efficiency can also be modulated by altering the levels of expression of the individual components of the nuclear transport machinery, including IMPs/Exportins, which commonly occurs during development/differentiation or during tumorigenesis. 4 The chicken anemia virus viral protein 3 (VP3 or apoptin) is a 121 amino acid (aa) nucleocytoplasmic shuttling protein, which has the ability to localize to a higher extent in the nucleus of tumor as compared to normal cell types.5 Several studies to date have reported on the tumor cell-enhanced n...

We previously reported that overexpression of members of the Importin (Imp) superfamily of nuclear transporters results in increased nuclear trafficking through conventional transport pathways in tumour cells. Here we show for the first time that the extent of overexpression of Impβ1 correlates with disease state in the MCF10 human breast tumour progression system. Excitingly, we find that targeting Impβ1 activity through siRNA is >30 times more efficient in decreasing the viability of malignant ductal carcinoma cells compared to isogenic non-transformed counterparts, and is highly potent and tumour selective at subnanomolar concentrations. Tumour cell selectivity of the siRNA effects was unique to Impβ1 and not other Imps, with flow cytometric analysis showing >60% increased cell death compared to controls concomitant with reduced nuclear import efficiency as indicated by confocal microscopic analysis. This hypersensitivity of malignant cell types to Impβ1 knockdown raises the exciting possibility of anti-cancer therapies targeted at Impβ1.

Investigating the localisation of proteins within live cells via fluorescence microscopy typically involves the fusion of the protein of interest to a large fluorescent protein such as green fluorescent protein (GFP). Alternate fluorescent labelling technologies such as the fluorescent biarsenical dye molecules (e.g. FlAsH, ReAsH) are preferable to the use of large fusion proteins in many respects and allow greater flexibility in terms of the location of the labelling site. We assessed the ability of the FlAsH-derived biarsenical dye molecule Lumio to label a range of tetracysteine containing proteins within live cells and report that although in some circumstances Lumio is capable of positively detecting such proteins, the sensitivity and specificity of labelling is significantly reduced, making the Lumio-labelling system unsuitable for the detection of a wide range of protein within live cells.

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