High molecular weight complex analysis of Epstein-Barr virus Latent Membrane Protein 1 (LMP-1): structural insights into LMP-1's homo-oligomerization and lipid raft association.
LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1's TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers.
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, United States.
Christopher M
CM
Wrobel
Timothy R
TR
Geiger
Rebecca N
RN
Nix
Aaron M
AM
Robitaille
Sandra
S
Weigand
Alfredo
A
Cervantes
Miguel
M
Gonzalez
Jennifer M
JM
Martin
D004591:Electrophoresis, Polyacrylamide Gel; D004854:Herpesvirus 4, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D021962:Membrane Microdomains / Q000821:virology*; D008970:Molecular Weight; D055503:Protein Multimerization; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D014763:Viral Matrix Proteins
BMH; BN-PAGE; Complex formation; EBV; EGS; Epstein–Barr virus; IFNα; LMP-1; Latent Membrane Protein-1; Lipid raft; N-ethylmaleimide; NEM; Oligomerization; TMEA; TRAF; Tumor Necrosis Receptor Associated Factor; bismaleimidohexane; blue native polyacrylamide gel electrophoresis; ethylene glycol bis[succinimidylsuccinate; interferon alpha; tris-[2-maleimidoethyl]amine