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. 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. 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. 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. 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. 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. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application. INTRODUCTIONNuclear protein transport is central to normal and aberrant cellular development and physiology, as well as the infectious cycles of intracellular pathogens Hearps and Jans, 2006). 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). All trafficking across the nuclear membrane occurs through the nuclear membrane embedded nuclear pore complex (NPC). The movement of molecules Ͼ45 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 . 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. Thus, it is only through recent studies with live cells that it has become clear that nuclear import can involve additional levels of regulation in the cytoplasm involving the microtubule (MT) cytoskeleton (Giannakakou et al., 2000;Lam et al., 2002;Roth et al., 2007), a network composed of ϩ/Ϫ polarized polymers of tubulin with the negative end at the MT organizing center (MTOC) located near the nucleus of nonpolarized cells. MTs are suggested to act as tracks for cargo delivery by molecular motors including the multiprotein complex motor, dynein, which mediates net movement of cargoes toward the MTOC (retrograde movement),...

Nucleocytoplasmic distribution of the rabies virus phosphoprotein is implicated in the evasion of cellular antiviral mechanisms by rabies virus and has been reported to depend on an N-terminal nuclear export sequence and a C-terminal nuclear localization sequence. This paper identifies a second nuclear export sequence that is located between key residues of the nuclear localization sequence in the phosphoprotein C-terminal domain. The C-terminal domain confers predominantly nuclear localization in unstimulated transfected cells, indicating that the nuclear localization sequence is the dominant signal at steady state. However, protein kinase-C activation or mutagenesis to mimic protein kinase-C phosphorylation at a site proximal to the C-terminal nuclear localization/export sequences shifts the targeting activity of the C-terminal domain toward nuclear exclusion, indicating that the nuclear export sequence becomes the dominant signal in activated cells. Mapping of these sequences within the three-dimensional structure of the C-terminal domain indicates that their activities may be coregulated by phosphorylation and/or conformational changes in the domain. The data are consistent with a model in which intimate positioning of the nuclear localization sequence, export sequence, and phosphorylation site within a single domain provides a switch mechanism to rapidly and efficiently balance the reciprocal import and export signals in response to cellular stimuli.