

Current understanding of this process focuses largely on these events at the NPC because investigation of nuclear import has conventionally used permeabilized cell systems with associated disruption of cytoplasmic structures. The movement of molecules >45 kDa generally requires active transport, where proteins conventionally interact via a nuclear localization sequence (NLS) with import proteins (importins) which mediate docking to and transit through the NPC ( Poon and Jans, 2005). All trafficking across the nuclear membrane occurs through the nuclear membrane embedded nuclear pore complex (NPC). The ability to exploit the cellular factors involved in nuclear targeting is of great therapeutic value as it permits the design of vehicles for the efficient delivery of drugs/therapeutic genes to the nuclear compartment ( Chan and Jans, 2002 Mastrobattista et al., 2006a, b). Nuclear protein transport is central to normal and aberrant cellular development and physiology, as well as the infectious cycles of intracellular pathogens ( Poon and Jans, 2005 Hearps and Jans, 2006). Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application.

This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import.

We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein.
