Localization of the Epstein–Barr virus protein LMP 1 to exosomes James Flanagan, 1 Jaap Middeldorp 2 and Tom Sculley 1,3 Correspondence James Flanagan jamesF@qimr.edu.au 1 The Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Queensland 4006, Australia 2 Department of Pathology, VU Medical Center, Free University, Amsterdam, The Netherlands 3 Department of Pathology, University of Queensland, St Lucia, Brisbane, Queensland 6067, Australia Received 4 November 2002 Accepted 21 February 2003 The Epstein–Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane. Co-labelling of cells with both an LMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin’s disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system. INTRODUCTION LMP 1 is a 63 kDa membrane protein that very closely resembles a constitutively active membrane receptor. LMP 1 has a hydrophilic amino terminus, six highly hydrophobic 20 aa transmembrane domains separated by five less hydrophobic 8 aa segments and a long hydrophilic cyto- plasmic tail containing two active domains (Liebowitz et al., 1986). An intronic start codon results in a smaller 45 kDa truncated LMP 1 protein, termed ‘lytic LMP 1’, which is expressed only in some EBV strains, specifically during virus replication (Baichwal & Sugden, 1987; Hudson et al., 1985). The lytic LMP 1 protein shares little or no functional similarity to the full-length form, in that it does not trans- form rodent fibroblasts, it does not alter the B-lymphocyte phenotype and is dispensable for transformation (Wang et al., 1985, 1988). LMP 1 protein has been shown to associate with the cytoskeleton and aggregates to form patches on the cell membrane, a function that, like other cellular receptors, is necessary for its activation of NF-kB (Gires et al., 1997; Mann et al., 1985). The domains involved in forming these aggregated patches have been defined as the 25 aa cytoplasmic amino terminus and the first two membrane-spanning domains (Bloss et al., 1999). A signi- ficant proportion of LMP 1 is detergent insoluble, and as such, was originally defined as cytoskeleton associated (Liebowitz et al., 1986). More specifically, it binds to the cytoskeleton protein vimentin and can up-regulate expres- sion of this protein (Liebowitz & Kieff, 1989; Liebowitz et al., 1987). Glycosphingolipid-rich domains or lipid rafts are discrete domains within the plasma membrane that are detergent insoluble and provide a clustering point for signal transduction proteins such as G-proteins as well as LMP 1 and its interacting protein, TRAF3 (Ardila-Osorio et al., 1999; Clausse et al., 1997). One study has proposed that LMP 1 may also accumulate in the lysosomal compartment (Laszlo et al., 1991). Lysosomes are thought to form by fusion of pinocytotic vesicles to yield endosomes which, in turn, transform first into multivesicular bodies (MVB) and then into mature lysosomes (Morales et al., 1999). Haematopoietic cells have been shown to use lysosomal compartments to store and release their secretory products. The direct fusion of lysosome-related compartments with the plasma membrane in haematopoietic cells has been associated with the release of exosomes (Andrews, 2000). Non-structured intracellular aggregation of LMP 1 in a fraction of lymphoblastoid cells of different origin was noted 0001-8944 G 2003 SGM Printed in Great Britain 1871 Journal of General Virology (2003), 84, 1871–1879 DOI 10.1099/vir.0.18944-0