Delivery of the HSV-1 genome through the nuclear pore complex
Final Report Abstract
Within the course of the project, we developed an experimental assay enabling a comprehensive investigation of the nuclear envelope and nuclear pores from diverse perspectives. The assay can be used under both physiological and pathophysiological conditions, and its reliability was demonstrated. Applying this assay, we could determine the structural arrangement of the NPC permeability barrier. Moreover, we demonstrated that a dissociation of NPC proteins is essential to force a significant increase of its permeability. We presented a potent chemical, trans-cyclohexane-1 2-diol (TCHD), which leads to controlled and reversible dissociation of the NPC barrier forming NPC proteins from the NPC channel. The finding concerning the NPC barrier may help design novel strategies for non-viral gene therapy. The same experimental assay mentioned above can be used to study in a comprehensive fashion nuclear delivery mechanism of viral genomes as shown exemplarily using HSV-1. Our results indicate an involvement of hydrophobicity for HSV-1 capsid opening. Furthermore, the viral DNA is released as a single thread. The initial stage of DNA release out of the capsid is very likely pressure-driven. We assume that it penetrates the NPC in this conformation. It is compacted by the host intranuclear proteins once it reaches the interior of the nucleus. Our proposed experimental strategy can be easily extended to other viruses. Finally and along the same line as TCHD, the observations made concerning HSV-1 genome delivery through the NPC may help design novel strategies for non-viral gene therapy. The ultimate goal is to develop non-viral nano-particles mimicking HSV-1 capsid and containing therapeutic DNA. The idea behind this reasoning is to develop nano-particles, which target the therapeutic DNA safely and specifically to the NPC for subsequent nuclear delivery.
Publications
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(2011) Exceptional structural and mechanical flexibility of the nuclear pore complex. J Cell Physiol. 226(3), 675-82
Liashkovich, I., Meyring A., Kramer, A., and Shahin, V.
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(2011) Nuclear delivery mechanism of herpes simplex virus type 1 genome. J. Mol. Recognit. 24:414-21
Liashkovich, I., Hafezi, W., Kühn, J.E., Oberleithner, H., and Shahin V.
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(2012) Structural organization of the nuclear pore permeability barrier. J Contol Release, 160(3):601-8
Liashkovich, I., Meyring, A., Oberleithner, H., Shahin, V.
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(2013) Nanovisualization of viral DNA breaching the nucleocytoplasmic barrier. J Control Release, 173:96-101
Meyring-Wösten, A., Hafezi, W., Kühn, J., Liashkovich, I., Shahin, V.
