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The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially ␣3␤1, ␣6␤1, ␣7␤1, and ␣6␤4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of ␣3 and ␣6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.

A contemporary goal of researchers in leucocyte signalling has been to uncover how cells physically organize and compartmentalize signalling molecules into efficient, regulated signalling networks. This work has revealed important roles of membrane microdomains that are characterized by their distinctive protein and lipid compositions. Recent studies have demonstrated that besides typical cholesterol- and glycosphingolipid-enriched 'rafts', leucocyte membranes are equipped with a different type of microdomain, made up of tetraspanin proteins. Tetraspanin proteins are involved in the organization of tetraspanin-enriched microdomains by virtue of their capacity to specifically associate with key molecules, including integrins, leucocyte receptors and signalling proteins. The aspects of leucocyte function influenced by tetraspanin microdomains include adhesion, proliferation and antigen presentation. However, the mechanisms by which tetraspanin complexes link to intracellular signalling pathways, are still largely unknown. This review discusses how tetraspanin microdomains might function to regulate signalling in lymphoid and myeloid cells, and how they relate to lipid rafts. In addition, we discuss new insights into the role of tetraspanins in malignant disease.

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.The host immune response to viral infection is a key factor in defining viral pathogenicity and the outcome of the infection. This depends not only on the capacity of the host to mount an innate and/or adaptive immune response against the virus but also on the ability of the virus to evade/subvert this response (22).The principal response of host cells to viral infection is the production of type I interferons (IFNs) (including alpha interferon [IFN-␣] and IFN-␤), which, on binding to IFN receptors on the cell surface, activate the JAK/STAT intracellular signaling pathway that culminates in the phosphorylation, heterodimerization, and nuclear translocation of the transcription factors signal transducer and activator of transcription 1 (STAT1) and STAT2. In the context of a complex called IFNstimulated gene factor 3 (ISGF3), the activated STATs bind to promoters in the DNA that contain an IFN-stimulated response element (ISRE) sequence, resulting in the transcription of a plethora of IFN-stimulated genes (ISGs) encoding antiviral proteins which act to establish the antiviral state in cells (reviewed in reference 22).To propagate efficiently in host cells, viruses have had to evolve multiple strategies to dampen the host IFN system, which appear to involve the expression of viral proteins with IFN antagonist functions. These IFN antagonists are reported to exert their effect by a variety of mechanisms, reflecting the diversity of host antiviral responses, but the STATs are known as common targ...

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.

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormonerelated protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110 Rb tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.Key words: actin filaments, FRAP, importins, microtubules, nuclear import, retinoblastoma protein Transport into the nucleus through the nuclear envelopelocalized nuclear pore complexes (NPCs) is dependent on modular nuclear localization sequences (NLSs) (1-3) within cargoes that are typically recognized by members of the importin (IMP) superfamily. Most commonly, the IMPa/b1 heterodimer or IMPb1 alone (4-6) act to effect NLSdependent translocation through the NPC, and release within the nucleus. Understanding of the detailed mechanisms of nuclear protein import, however, is largely based on the study of semi-intact cell systems (7-10), where cytoskeletal elements are either lacking or severely damaged, meaning that the role of the cytoskeleton has been largely overlooked. Many viruses are known to exploit the microtubule (MT) network for efficient nuclear targeting (11), while there is evidence for negative regulation of the nuclear import of several viruses through association with the actin cytoskeleton (12). Intriguingly, IMPa has been demonstrated to associate with MTs in mammalian and plant cells (13,14), while NLS-comprising peptides have been reported to invoke active transport along MTs (15). Significantly, a role for the MT network in facilitating nuclear import (16) is strongly implicated in the case of the tumor suppressor protein p53 and the cancer-related signaling molecule parathyroid hormone-related protein (PTHrP) (17,18). Treatment with the MT-depolymerizing agent nocodazole (NCZ) reduces both p53 and PTHrP nuclear accumulation in vivo (17,18), with application of the flu...

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),...