J Virol 80(13): 6458-6468

LMP1 Strain Variants: Biological and Molecular Properties

Department of Microbiology-Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,^{Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599}²

^{Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: (919) 966-1701. Fax: (919) 966-9673. E-mail:}ude.cnu.dem@trn.

Received 2006 Jan 20; Accepted 2006 Apr 15.

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-κB. 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-κB 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.

Abstract

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the world's population. EBV infection is associated with a wide array of malignancies, including nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma, posttransplant lymphoma, hairy leukoplakia, Burkitt's lymphoma, and others (24, 45, 62). EBV primary infection is usually asymptomatic, although at times it results in the benign lymphoproliferative disease infectious mononucleosis (40). Viral reactivation may occur in latently infected memory B cells, and virus frequently can be detected in saliva (3, 49, 50). Viral infection is considered latent in EBV malignancies, and in NPC and Hodgkin's lymphoma, viral expression is restricted to EBNA1, latent membrane protein 1 (LMP1), LMP2, and the EBER and BART RNAs (24).

LMP1 is considered the EBV oncogene, as it transforms NIH 3T3 and Rat-1 fibroblasts. It is essential for EBV-induced B-lymphocyte transformation (22, 24, 32, 52, 58) and is frequently expressed in EBV malignancies (15, 62). LMP1 consists of 386 amino acids with a short 23-amino-terminal cytoplasmic tail, six transmembrane domains, and a long carboxyl-terminal tail that contains the two signaling domains carboxyl-terminal activating region 1 (CTAR1) and CTAR2. LMP1 self-associates in the plasma membrane due to its hydrophobic transmembrane domains and thus acts as a constitutively active growth factor receptor that does not require a ligand for its activation. The tumor necrosis factor receptor-associated factor associates with LMP1, which signals similarly to CD40 and to the type II tumor necrosis factor receptor (9, 21, 39). As such, LMP1 activates a wide array of signaling pathways through CTAR1 and CTAR2, including the NF-κB, mitogen-activated protein kinase, c-Jun N-terminal kinase, and phosphatidylinositol 3-kinase (PI3K)-Akt pathways (7, 12, 14, 20, 32, 42, 46). LMP1 also induces the deregulation of the cell cycle through manipulation of various cell cycle-regulatory molecules, including the cyclin-dependent kinase inhibitor (CDKI) p27^{, p16, inhibitor of differentiation 1 (Id1), Id3, retinoblastoma (Rb), and cyclin-dependent kinase 2 (CDK2) (}14, 27, 41).

Six LMP1 sequence variants, termed Alaskan, China 1, China 2, Mediterranean+ (Med+), Med−, and North Carolina (NC), have been isolated from clinical specimens (10, 35). These variants are distinguished by various signature amino acid changes from the prototypic LMP1 (B95.8), although changes are not found in CTAR1 or CTAR2. In addition, China 1 and Med+ have a 10-amino-acid deletion between CTAR1 and CTAR2 (10). Although this 10-amino-acid deletion has been linked to augmented transforming ability (28), several studies have indicated that LMP1-containing CTAR1 is essential and sufficient for Rat-1 fibroblast and Madin-Darby canine kidney (MDCK) cell transformation (25, 32). The abilities to induce NF-κB and the transforming properties of the B95, Med+, and CAO variants have been compared (4, 6, 16, 17, 29, 34). Slight enhancements in the abilities of the CAO variant, which is the most similar to China 1, and the Med+ variant to induce NF-κB signaling were mapped to the transmembrane domains. LMP1 also interacts with the E3 ubiquitin ligase component homologue of Slimb (HOS), a member of the β-TrCP/Fbw1 subfamily of F-box proteins which regulates the ubiquitin-dependent destruction of IκBα and β-catenin (34, 47, 53, 60).

Several studies have revealed that individuals can be infected with multiple EBV variants, that the LMP1 variants differ in abundance between throat wash samples and peripheral blood samples, and that their detectabilities and abundances vary over time (49-51). It is possible that these differences in abundance and detectability reflect different biological properties or cellular tropism. Recently, computational analysis of the variants revealed key amino acid changes in potential human leukocyte antigen (HLA)-restricted epitopes, suggesting a potential immune-modulated selection mechanism (11). Although all variants have been detected in various malignancies, the CAO/China 1 LMP1 variant is significantly more prevalent in NPC tumors from the region of endemicity in southern China and is predicted not to be presented by individuals who are A2 or A24, the most common HLA types found in patients with NPC from this region (11).

In this report, the biological properties of the LMP1 variants were characterized through their abilities to transform Rat-1 fibroblasts, affect epithelial cell motility, and induce homotypic adhesion of B lymphocytes. The LMP1 variants also were evaluated for their potentials to activate the PI3K-Akt and NF-κB signaling pathways as well as their abilities to alter cell cycle markers that have been associated with G₁/S-phase progression. All the variants were capable of transforming Rat-1 fibroblasts, as measured by blockage of contact-inhibited and anchorage-independent growth, and induced homotypic adhesion in BJAB cells. The PI3K-Akt signaling cascade was activated by all variants, and this activation was necessary for LMP1-induced Rat-1 transformation. Changes in cellular markers that are frequently associated with cell cycle deregulation were also induced by all variants. Finally, the Alaskan, China 1, and Med+ variants had increased NF-κB reporter activity compared to the other variants, in conjunction with decreased binding to HOS. These similarities in biological and molecular properties suggest that all of the variants have equal pathological potentials, in agreement with their detection in multiple types of cancer.

Acknowledgments

We thank David Everly and Betsy Edwards for critical reviews of the manuscript, Michele Chang for aid in cloning the LMP1 variants into pcDNA3, Kathy Shair for help with Western blots using the rat monoclonal antibodies, Serge Fuchs for the HOS plasmids, Albert Baldwin and Derek Holmes for the NF-κB reporter plasmid, and Natalie J. Thornburg for help with the statistical analysis.

This work was supported by National Institutes of Health grants CA 19014 and CA 32979 to N.R.-T.

Acknowledgments

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