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. 2006 Jul;80(13):6458-68.
doi: 10.1128/JVI.00135-06.

LMP1 strain variants: biological and molecular properties

Bernardo A Mainou  1 , Nancy Raab-Traub
Affiliations

LMP1 strain variants: biological and molecular properties

Bernardo A Mainou et al. J Virol. 2006 Jul.

Abstract

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-kappaB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med-, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-kappaB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.

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Figures

FIG. 1.
FIG. 1.
LMP1 variants block contact-inhibited growth of Rat-1 cells. Rat-1 fibroblasts were transduced with pBabe and pBabe-LMP1 variants, maintained for 10 to 14 days in full serum, fixed, stained with the anti-LMP1 antibody CS1-4, and subsequently imaged with phase-contrast and immunofluorescent microscopy for focus formation. Alaskan immunofluorescent staining is not provided.
FIG. 2.
FIG. 2.
Anchorage-independent growth of Rat-1 fibroblasts is induced by LMP1 variants. Rat-1 cells stably expressing the LMP1 variants or the vector control (pBabe) were resuspended in soft agar, maintained for 21 days, and then imaged with phase-contrast microscopy for anchorage-independent growth.
FIG. 3.
FIG. 3.
LMP1 variants increase motility of HFK cells. Monolayers of HFK cells stably expressing the LMP1 variants or the vector control (pBabe) were scratched and assayed for increased motility 24 and 48 h postscratch by phase-contrast microscopy. White lines delineate approximate locations of scratch boundaries.
FIG. 4.
FIG. 4.
Activation of the PI3K-Akt signaling pathway by LMP1 variants. (A) LMP1 variants and the vector control (pBabe) expressed stably in Rat-1 cells were assayed for LMP1 expression with CS1-4 and HA antibodies (asterisks identify LMP1-specific bands), phosphorylated and total Akt, and GSK3β, PTEN, mTOR, and total E-cadherin by Western blot analysis. Equal protein loading was confirmed with Ponceau S staining and an actin Western blot (not shown). (B) Quantitative analysis of LMP1 (white bars), phosphorylated Akt (checkered bars), phosphorylated GSK3β (striped bars), and E-cadherin (black bars) protein levels. LMP1 levels were normalized to B95.8, whereas phospho-Akt, phospho-GSK3β, and E-cadherin levels were normalized to pBabe.
FIG. 5.
FIG. 5.
LY294002 blocks LMP1-induced focus formation. Rat-1 cells stably expressing the vector control (pBabe) or the LMP1 variants were seeded in six-well plates and maintained for 10 to 14 days in media with the PI3K inhibitor LY294002 or the vehicle control (DMSO), stained with crystal violet, and observed for focus formation with phase-contrast microscopy.
FIG. 6.
FIG. 6.
LMP1 variants regulate cell cycle progression protein markers. (A) Rat-1 cells stably expressing pBabe or the LMP1 variants were assayed for LMP1 expression (asterisks indicate LMP1 variants) and p27 and Id1 protein levels by Western blot analysis. Equal loading was confirmed with an actin Western blot. (B) Quantitative analysis of LMP1 (white bars), p27 (black bars), and Id1 (striped bars) protein levels. LMP1 expression was normalized to B95.8, whereas p27 and Id1 levels were normalized to pBabe. (C) HFK cells stably expressing pBabe or the LMP1 variants were assayed for LMP1 expression, phosphorylated and total levels of Rb and CDK2, and p21/Waf1. Equal loading was confirmed with the actin blot.
FIG. 7.
FIG. 7.
Homotypic adhesion of BJAB cells is induced by LMP1 variants. (A) Equal numbers of BJAB cells stably expressing pBabe or the LMP1 variants were seeded and observed for homotypic adhesion every 24 h by phase-contrast microscopy. Representative fields at 3 days postseeding are shown. (B) LMP1 variants activate Akt in BJAB cells. BJAB cells stably expressing pBabe or the LMP1 variants were assayed for LMP1 expression and phosphorylated and total levels of Akt. Equal loading was confirmed with an actin blot.
FIG. 8.
FIG. 8.
Differential binding of HOS by LMP1 variants. (A) Sequence alignment of the LMP1 variants from amino acids 207 to 370 of LMP1 B95.8 showing residues important for HOS-LMP1 binding, including the destruction box, a serine residue at amino acid 350, and a serine residue at amino acid 366. The 10-amino-acid deletion found in the China 1 and Med+ variants is represented by del. Asterisks indicate amino acids important for HOS-LMP1 binding. B95.8 contains all residues required for HOS-LMP1 binding. (B) 293T cells cotransfected with pcDNA3 (vector control), with the LMP1 variants and HOS, or with HOS alone were harvested 48 h posttransfection and analyzed for HOS-LMP1 binding through coIP. HOS was immunoprecipitated with anti-HA-conjugated beads, and Western blot analysis for LMP1 was carried out with anti-c-myc. Western blot analysis of total protein (total load) confirmed expression of the LMP1 variants, HOS, IκBα, β-catenin, and actin to confirm equal loading.
FIG. 9.
FIG. 9.
All LMP1 variants activate NF-κB. 293T cells were triply transfected with pcDNA3 (vector control) or the LMP1 variants, with pRL-SV40, and with pGL3-Basic (vector control) or pGL3-3NF-κB (NF-κB reporter). Forty-eight h posttransfection, cells were harvested, and relative luciferase activity was assessed by comparing the firefly luciferase activity of the NF-κB reporter to the control Renilla luciferase activity (pRL-SV40). The Tukey-Krammer test was used to calculate P values. All LMP1 variants had P values of less than 0.001 compared to pcDNA3.
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References

    1. Arvanitakis, L., N. Yaseen, and S. Sharma. 1995. Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells. J. Immunol. 155:1047-1056. - PubMed
    1. Atkinson, P. G., H. J. Coope, M. Rowe, and S. C. Ley. 2003. Latent membrane protein 1 of Epstein-Barr virus stimulates processing of NF-kappa B2 p100 to p52. J. Biol. Chem. 278:51134-51142. - PubMed
    1. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV persistence in memory B cells in vivo. Immunity 9:395-404. - PubMed
    1. Blake, S. M., A. G. Eliopoulos, C. W. Dawson, and L. S. Young. 2001. The transmembrane domains of the EBV-encoded latent membrane protein 1 (LMP1) variant CAO regulate enhanced signalling activity. Virology 282:278-287. - PubMed
    1. Cantley, L. C., and B. G. Neel. 1999. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA 96:4240-4245. - PMC - PubMed

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