The Latent Membrane Protein 1 (LMP-1) protein of Epstein-Barr virus (EBV) is localized in the plasma membrane of the infected cell. LMP-1 possesses a hydrophobic membrane spanning domain, and charged, intracellular amino-and carboxy-termini. Two models have been proposed for the contribution of the aminoterminus to LMP-1's function: (i) as an e ector domain, interacting with cellular proteins, or (ii) as a structural domain dictating the correct orientation of transmembrane domains and thereby positioning LMP-1's critical e ector domains (i.e. the carboxy-terminus). However, no studies to date have addressed directly the structural contributions of LMP-1's cytoplasmic amino-terminus to function. This study was designed to determine if LMP-1's cytoplasmic amino-terminus (N-terminus) encodes information required solely for maintenance of proper topological orientation. We have constructed LMP-1 chimeras in which the cytoplasmic N-terminus of LMP-1 is replaced with an unrelated domain of similar size and charge, but of di erent primary sequence. Retention of the charged amino-terminal (N-terminal) cytoplasmic domain and ®rst predicted transmembrane domain was required for correct transmembrane topology. The absolute primary sequence of the cytoplasmic N-terminus was not critical for LMP-1's cytoskeletal association, turnover, plasma membrane patching, oligomerization, Tumor Necrosis Factor Receptor-associated factor (TRAF) binding, NF-kB activation, rodent cell transformation and cytostatic activity. Furthermore, our results point to the hydrophobic transmembrane domain, independent of the cytoplasmic domains, as the primary LMP-1 domain mediating oligomerization, patching and cytoskeletal association. The cytoplasmic amino-terminus provides the structural information whereby proper transmembrane orientation is achieved. Oncogene (2001) 20, 5313 ± 5330.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.