The cytoplasmic amino-terminus of the Latent Membrane Protein-1 of Epstein-Barr Virus: relationship between transmembrane orientation and effector functions of the carboxy-terminus and transmembrane domain.
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 amino-terminus to LMP-1's function: (i) as an effector 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 effector 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 different primary sequence. Retention of the charged amino-terminal (N-terminal) cytoplasmic domain and first 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-kappaB 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.
Coffin
W F
WF
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Box 347, Boulder, Colorado 80309, USA.
Erickson
K D
KD
Hoedt-Miller
M
M
Martin
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002918:Chymotrypsin / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D003902:Detergents; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018841:HSP90 Heat-Shock Proteins; D016328:NF-kappa B; D009856:Oncogene Proteins, Viral; C092306:TRAP1 protein, human; D014763:Viral Matrix Proteins; D017830:Octoxynol; D002918:Chymotrypsin; D014357:Trypsin; D011402:Pronase