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Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines
Published on: November 8, 2011
Localization of the Epstein-Barr virus protein LMP 1 to exosomes
James Flanagan1, Jaap Middeldorp2, Tom Sculley3,1
1The Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Queensland 4006, Australia.
Insights
Epstein-Barr virus latent membrane protein (LMP 1) is found in intracellular compartments and on exosomes. These LMP 1-containing exosomes can inhibit immune cell proliferation, suggesting a role in immune evasion by EBV-associated tumors.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Epstein-Barr virus (EBV) latent membrane protein 1 (LMP 1) is a key viral oncoprotein.
- LMP 1 acts as a constitutively active signaling molecule, influencing cellular processes.
- Its precise localization and function within infected cells, particularly in relation to immune evasion, require further elucidation.
Purpose of the Study:
- To investigate the subcellular localization of EBV LMP 1.
- To determine if LMP 1 is associated with exosomes.
- To assess the impact of LMP 1-containing exosomes on immune cell function.
Main Methods:
- Immunofluorescent confocal microscopy to visualize LMP 1 distribution.
- Co-localization studies with Golgi markers (GS15) and MHC-II.
- Immunoelectron microscopy for high-resolution localization.
- Exosome isolation from lymphoblastoid cell lines.
- Assays to measure peripheral blood mononuclear cell proliferation.
Main Results:
- LMP 1 exhibits heterogeneous distribution within cells, localizing to intracellular compartments beyond the plasma membrane.
- Intracellular LMP 1 partially co-localizes with the Golgi apparatus.
- LMP 1 is present on exosomes derived from EBV-infected cells.
- LMP 1-containing exosomes inhibit the proliferation of peripheral blood mononuclear cells.
Conclusions:
- EBV LMP 1 localizes to intracellular compartments and is released on exosomes.
- Exosomal LMP 1 possesses immunoregulatory functions by inhibiting immune cell proliferation.
- This mechanism may contribute to immune evasion in EBV-associated malignancies like nasopharyngeal carcinoma and Hodgkin's disease.
Abstract:
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.
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