Epstein–Barr virus latent membrane protein-1 (LMP-1) and lytic LMP-1 localization in plasma membrane-derived extracellular vesicles and intracellular virions

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Abstract
Epstein–Barr virus (EBV) is a human herpesvirus associated with a number of malignancies. EBV establishes a latent infection in human B cells in vitro, and infected lymphoblastoid cells proliferate indefinitely as a result of virus activation of cellular signalling pathways. Latently infected cells express a viral oncoprotein called the latent membrane protein-1 (LMP-1). LMP-1 signals both proliferative and survival signals to the infected B cell. The switch from latency to lytic replication is associated with upregulation of an N-terminally truncated LMP-1, called lytic LMP-1 (lyLMP-1). To understand better the relationship between LMP-1 protein function and the virus life cycle, LMP-1 and lyLMP-1 were precisely localized in infected B cells. Immunoelectron microscopy of latently infected cells revealed LMP-1 localized in discrete patches in the plasma membrane. Unexpectedly, immunogold-labelled LMP-1 was found in vesicles budding from the plasma membrane into the extracellular space and in small membrane vesicles accumulating in conditioned medium from infected cells. LyLMP-1 immunolabelling was observed only in B95-8 cells harbouring detectable intracellular virus particles and was abundant in the nuclear membrane early, and in the plasma membrane late, following lytic cycle induction. LyLMP-1 immunoreactivity was also observed at sites of virus budding and associated with intracellular virions, suggesting that lyLMP-1 might be incorporated into cytoplasmic virions when budding through the nuclear membrane.