Abstract
Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/β-recognized nuclear localization signal [Pryor et al. Traffic 8:795–807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795–807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/β using an AlphaScreen protein–protein binding assay.
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Acknowledgments
This work was supported by the Australian National Health and Medical Research Council (Fellowship and Project grants ID#333013/384105 and #143710, respectively, to D.A.Jans) and the ARC (Discovery Project/Fellowship #110104437, to K.M.Wagstaff). We thank Cassandra David for assistance with tissue culture.
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Fraser, J.E., Rawlinson, S.M., Wang, C., Jans, D.A., Wagstaff, K.M. (2014). Investigating Dengue Virus Nonstructural Protein 5 (NS5) Nuclear Import. In: Padmanabhan, R., Vasudevan, S. (eds) Dengue. Methods in Molecular Biology, vol 1138. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0348-1_19
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DOI: https://doi.org/10.1007/978-1-4939-0348-1_19
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