Monoclonal Antibodies to the Latent Membrane Protein of Epstein-Barr Virus Reveal Heterogeneity of the Protein and Inducible Expression in Virus-transformed Cells

Abstract

SUMMARYMonoclonal antibodies specific for the 'latent membrane protein' (LMP) of EpsteinBarr virus (EBV), one of the effector proteins of EBV-induced B cell transformation, have been generated from mice immunized with a fl-galactosidase fusion protein containing the carboxyl half of the B95.8 strain LMP sequence. Four monoclonal IgG1 antibodies, designated CS. 1, CS. 2, CS. 3 and CS. 4, which together recognized at least three different epitopes on the molecule, were used to examine various aspects of LMP expression in B cell lines transformed in vitro. The pooled CS. 1 to 4 reagent detected the LMPs encoded by each of 20 geographically distinct EBV isolates, despite a degree of inter-isolate heterogeneity in the size and antigenicity of the protein. In cell lines carrying the prototype B95.8 virus strain, particularly if these were virus producers, an additional lower molecular weight LMP was also detected; this appeared to correspond to the truncated form of the protein already predicted to exist from the analysis of B95.8 lytic cycle mRNAs. Attempts were made to identify an analogous truncated form of LMP in cell lines carrying other virus isolates after treatment with phorbol ester and/or sodium butyrate to induce virus production. Surprisingly these experiments showed that expression of the full length LMP molecule was itself strongly inducible by these agents; when monitored at the single cell level, this was a generalized response and was not restricted to cells entering a lytic cycle. Expression of LMP in EBVtransformed B cells therefore appears to be subject to a distinct type of regulation.

Epstein–Barr virus latent membrane protein-1 (LMP1) signalling is distinct from CD40 and involves physical cooperation of its two C-terminus functional regions

“…LMP1 and LMP1 encoding chimeras were detected using 1 mg/ml CS. 1 ± 4 (Rowe et al, 1987) in Blotto. All blots were incubated with 1 : 2000 dilution of rabbit antimouse IgG antibodies (Z0259: Dako) rabbit anti mouse diluted 1 : 2000 in Blotto followed by an incubation with goat anti-rabbit-alkaline phosphatase (Sigma; A-3812) diluted 1 : 10 000 in TBS-tween.…”

Epstein-Barr virus latent membrane protein-1 (LMP1) C-terminus activation region 2 (CTAR2) maps to the far C-terminus and requires oligomerisation for NF-κB activation

“…Expression of the chimeric proteins from the DNA construct was veri®ed by Western blotting and immuno¯uorescence after transient transfection into Jurkat cells. While the domains of the chimera originating from LMP1 were detected with the pooled monoclonal antibodies CS.1-4 which recognise di erent epitopes on the C-terminal cytoplasmic region of LMP1 (Rowe et al, 1987a), the extracellular domain of the chimerae derived from rat CD2 could be detected with the monoclonal antibody OX34 (Je eries et al, 1985).…”

“…Nitrocellulose transfers were blocked in blotto (5% w/v dried skimmed milk in TBS) for 2 h before binding primary antibody. LMP1 and LMP1 encoding chimerae were detected using 1 mg/ml CS.1-4 anti-LMP antibodies (Rowe et al, 1987a) or 1mg/ml OX34 anti-rat CD2 antibodies (Je eries et al, 1985) in blotto. All blots were incubated with Z0259 (Dako) rabbit anti-mouse diluted 1:2000 in blotto followed by an incubation with goat anti-rabbit-alkaline phosphatase (Sigma A-3812) diluted 1:10000 in TBS-tween.…”

Inhibition of in vitro proliferation of Epstein Barr Virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1

“…Figure 2 Immunoblot determination of LMP1 protein levels in B95.8 cells exposed to LMP1 antisense oligomers. Untreated B95.8 cells and cells exposed for 2 and 4 days to either Sc2 or AS2 oligomers at 20 mM were lysed in sample bu er (Rowe et al, 1987) at a concentration of 2610 7 cells/ml. Cell extracts corresponding to 4610 5 cells were subjected to polyacrylamide gel electrophoresis using a 5% stacking gel and a 10% resolving gel.…”