Identification of latent membrane protein 2A (LMP2A) specific targets for treatment and eradication of Epstein–Barr virus (EBV)-associated diseases

Michelle Swanson-Mungerson, Masato Ikeda, Lori Lev, Richard Longnecker* and Toni Portis

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA

Keywords: antivirals, Epstein–Barr virus, tyrosine kinases, microarray analysis


    Introduction
 Top
 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
Epstein–Barr Virus (EBV) is a human herpesvirus that establishes a lifelong latent infection in B lymphocytes. EBV is associated with the development of several malignancies of lymphoid origin, including Burkitt’s lymphoma, Hodgkin’s disease, and lymphoproliferative disorders arising in immunocompromised patients.1 There are also diseases of epithelial origin associated with EBV infection, including nasopharyngeal carcinoma and oral hairy leucoplakia.1

In vitro, EBV can transform infected lymphocytes into continuously proliferating lymphoblasts. In fact, most functional studies of EBV proteins have been carried out using lymphoblastoid cell lines (LCLs) established in vitro.2,3 Expression of viral latency-associated transcripts in vivo appears to be tightly regulated. Whereas viral Epstein–Barr nuclear antigen 1 (EBNA1), EBV-encoded RNAs (EBERs), latent membrane protein 1 (LMP1) and LMP2A transcripts have been detected in tumour cells from patients with EBV-associated malignancies, LMP2A is one of the most consistently identified in vivo in B cells from healthy individuals harbouring a latent EBV infection.1 This indicates that LMP2A plays an important role in viral latency and persistence, which is necessary for the development of EBV-associated diseases.


    LMP2A and B cell receptor (BCR) signalling
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 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
In latently infected B lymphocytes, LMP2A has been shown to localize to small glycolipid-enriched microdomains in the plasma membrane.4 It is thought that by localizing to membrane microdomains, LMP2A may mimic an activated B cell receptor (BCR).5 Interestingly, EBV-transformed LCLs demonstrate a block in protein tyrosine phosphorylation, calcium mobilization and nuclear gene transcription following BCR activation.6,7 Studies have demonstrated that BCR activation in LMP2A-expressing B cells fails to activate the downstream signalling molecules Lyn, Syk, phosphatidylinositol 3-kinase (PI3-K), phospholipase C-{gamma}2 (PLC{gamma}2), Vav, Shc and mitogen-activated protein kinase (MAPK). Instead, Syk, PI3-K, and Vav are constitutively phosphorylated, albeit at a low level, in LMP2A- expressing cells.7 In these cells, the amino-terminal domain of LMP2A is tyrosine phosphorylated and associated with the Src family tyrosine kinase Lyn as well as Syk.7,8 Mutational analyses have indicated that tyrosines 74 and 85 [the immunoreceptor tyrosine-based activation motif (ITAM)] in LMP2A bind Syk whereas tyrosine 112 binds Lyn. All of these residues have been shown to be essential for the LMP2A-mediated block in BCR signal transduction.9,10

The first indication that LMP2A provides a survival signal to B cells was supported by data using an in vivo mouse model in which developing B cells expressing a LMP2A transgene failed to express surface IgM. Bone marrow B cells from these mice were shown to undergo Ig light chain, but not heavy chain, gene rearrangement, indicating that LMP2A signalling can bypass the requirement for Ig recombination and allow IgM negative cells, which would normally undergo apoptosis, to colonize peripheral lymphoid organs.11 The LMP2A-mediated alterations in development were abolished in transgenic mice expressing LMP2A with tyrosines 74 and 85 mutated to phenylalanines, supporting the proposal that the ITAM and Syk binding are required for LMP2A-mediated developmental and survival signals in B cells in vivo.12 Additionally, studies utilizing mice deficient in the BCR signalling molecules B cell linker protein (BLNK, SLP-65) and Bruton’s tyrosine kinase (BTK) have demonstrated that LMP2A utilizes these molecules for its effects on B cell development and survival.13,14

From these studies, we have identified several proteins that may be amenable for use as therapeutic targets for eradicating latent EBV infection, which precedes transformation of B cells into tumour cells. In the next section, we discuss recent findings that further define potential LMP2A targets involved in EBV latency.


    DNA microarray analysis of LMP2A-expressing cells
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 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
DNA microarray analysis has provided a wealth of information regarding alterations in gene expression associated with viral infection and tumorigenesis. We have utilized this technology to study changes in gene expression that occur in B lymphocytes upon LMP2A expression. A comparison of gene expression in B cells from LMP2A transgenic mice and non-transgenic littermates has determined that LMP2A interferes with global transcription factor regulation necessary for proper B cell development when expressed during B lymphopoiesis.15 In B cells from LMP2A transgenic mice, decreased expression of many genes associated with normal B cell development as well as reduced levels of the transcription factors that regulate their expression was observed. In particular, expression levels of the transcription factors E2A, early B-cell factor (EBF), Pax-5 and PU.1 were down-regulated in B cells from LMP2A transgenic mice. Additionally, transcriptional alterations in upstream signalling molecules that regulate the expression and/or activity of these transcription factors were observed, including effectors in the Notch and Ras/MAPK pathways.15 The ability of LMP2A to interfere with B cell transcription factor regulation has important implications regarding its role in EBV latency.

It is interesting to note that similar changes affecting global gene transcription have been described in Reed-Sternberg cells from Hodgkin’s lymphoma. Approximately 40–50% of Hodgkin’s lymphomas are EBV positive and express LMP2A.16,17 Reed-Sternberg cells are presumed to be derived from germinal centre B cells that should have been eliminated due to a lack of surface Ig expression.18 Recent reports have described characteristics of Reed-Sternberg cells similar to those of LMP2A-expressing B cells, including reduced expression of the transcription factors E2A, EBF, Pax-5 and PU.1.19,20 Similar findings indicating the activation of Notch have been observed in Reed-Sternberg cells and B cells from LMP2A transgenic mice.15,21 Our research strongly suggests that LMP2A is responsible for many of the transcriptional changes identified in Reed-Sternberg cells and may provide information regarding the importance of LMP2A in Hodgkin’s lymphoma. Microarray studies have been particularly helpful for identifying specific cellular pathways that may be altered by LMP2A during EBV infection. Utilizing the LMP2A transgenic mouse model, specific genes identified in these types of studies can be targeted utilizing functional inhibitors to determine their contribution to LMP2A-mediated cell survival.


    Functional inhibitors to study LMP2A signalling
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 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
As a result of the global changes in transcription induced by LMP2A, it is difficult to imagine a therapeutic target that could eradicate EBV latency at the level of gene expression. However, changes in gene transcription may help to identify the signal transduction molecules responsible for LMP2A-mediated survival. The pharmacological inhibition of proteins activated by LMP2A not only provides insight into the function of LMP2A in viral latency, but also may allow for the design of therapeutics to eradicate latently-infected B cells.

Based upon this premise, our laboratory recently set out to determine whether pharmacological inhibitors could block LMP2A function using in vitro assays with bone marrow cells from our LMP2A transgenic mice.22 These data demonstrated that a protein tyrosine kinase inhibitor, piceatannol, inhibited LMP2A function in vitro, which is not surprising since the ITAM in the cytoplasmic domain of LMP2A is required to provide B cells with development and survival signals in vivo.12 However, other inhibitors that blocked the activity of PI3-K and BTK did not affect LMP2A-mediated effects in vitro.22 Therefore, these data indicate that protein tyrosine kinases such as Lyn and Syk that can be inhibited by piceatannol or other specific inhibitors may be potential targets for therapeutics designed to eliminate latently-infected B cells. Furthermore, these data indicate that Syk does not activate PI3-K or BTK to mediate the effects of LMP2A in our experiments and indicate that other downstream targets of Lyn and Syk may be more likely to serve as targets for therapeutic strategies.

The concept of using therapeutics to target latent EBV infection in healthy individuals is not unreasonable. For example, the inhibitor that was shown to abrogate the LMP2A-dependent expansion of developing B cells in vitro, piceatannol,22 is the metabolite of resveratrol,23 which has anti-leukaemic properties.24 Thus, if clinical trials begin to address the efficacy of resveratrol or piceatannol in the treatment of cancers, then it may be possible to address the ability of this drug to inhibit EBV latent infection and development of associated cancers. Also, previous data demonstrate that LMP2A alters the regulation of members of the Ras/MAPK signalling pathway.15 Recently, a Phase I clinical trial has been completed with the Ras inhibitory drug R115777, which was shown to have anti-leukaemic activity.25 Therefore, it is possible that such an inhibitor may also be utilized to treat latent EBV infections, as well as EBV-associated malignancies.

LMP2A also contains two proline-rich PY motifs in its amino-terminal cytoplasmic region, which likely recruit proteins with Src homology 3 (SH3) or WW domains.26 These types of proteins have been shown to be important for regulating cellular signalling events. Recent studies have demonstrated that LMP2A associates with Nedd4 family ubiquitin protein ligases and that this results in the degradation of LMP2A and Lyn and possibly other signalling molecules, which may allow LMP2A to effectively modulate B cell signal transduction for maintenance of EBV latency.27,28 Therefore, by applying therapeutics that effectively increase the degradation of LMP2A by increasing the activity of Nedd4 family ubiquitin protein ligases in latently infected B cells, it may be possible to block survival signals induced by LMP2A and eradicate latent EBV infection in healthy individuals. Figure 1 depicts signalling components and downstream factors induced by LMP2A that can be used as potential targets to abrogate LMP2A function.



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Figure 1. Activated signalling components and induced factors utilized by LMP2A. (A) LMP2A is a membrane protein containing 12 transmembrane domains and an N-terminal cytoplasmic tail domain with eight tyrosines. Tyrosine 112 binds Lyn and tyrosines 74 and 85 (the ITAM) bind Syk. Two PY motifs bind Nedd4 family ubiquitin ligases and mediate internalization and degradation of LMP2A and its associated proteins. (B) LMP2A activates various signalling proteins that can serve as potential therapeutic targets for eradication of EBV latency and EBV-associated diseases. (C) Targeting upstream signalling molecules can prevent downstream effects of LMP2A signalling, such as altered expression of transcription factors, apoptosis inhibitors and cell cycle regulators.

 
In addition to the possibility of eradicating latent EBV infection in healthy individuals, there is also a therapeutic basis for eliminating LMP2A signals in EBV-associated malignancies. As described above, Reed-Sternberg cells of Hodgkin’s lymphoma typically exhibit a down-regulation in surface BCR expression. Presumably, signals emanating from LMP2A may provide survival signals that protect these cells from apoptosis, which normally occurs in BCR-negative peripheral B cells.29 Thus, the treatment of Hodgkin’s lymphoma patients with pharmacological inhibitors that target LMP2A-induced survival signals may lead to a decrease in the burden of cancerous cells in these patients.


    Summary and concluding statement
 Top
 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
In summary, utilization of DNA microarray technology and the use of specific inhibitors have helped to identify several cellular components or processes affected by LMP2A in vivo. Understanding which specific pathways are critical for LMP2A function will facilitate the design of effective therapeutics to prevent the development of and effectively treat EBV-associated diseases.


    Acknowledgements
 
M.S.-M. is supported in part by a training grant from the National Institutes of Health (CA09560). M.I. and T.P. are special fellows of the Leukemia and Lymphoma Society of America. R.L. is a Stohlman Scholar of the Leukemia and Lymphoma Society of America and supported by the Public Health Service grants CA62234 and CA73507 from the National Cancer Institute and DE13127 from the National Institute of Dental and Craniofacial Research.


    Footnotes
 
* Corresponding author. Fax: +1-312-503-1339; E-mail: r-longnecker{at}northwestern.edu Back


    References
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 Introduction
 LMP2A and B cell...
 DNA microarray analysis of...
 Functional inhibitors to study...
 Summary and concluding statement
 References
 
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