Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA
Keywords: antivirals, EpsteinBarr virus, tyrosine kinases, microarray analysis
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Introduction |
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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 EpsteinBarr 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.
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LMP2A and B cell receptor (BCR) signalling |
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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 Brutons 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.
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DNA microarray analysis of LMP2A-expressing cells |
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It is interesting to note that similar changes affecting global gene transcription have been described in Reed-Sternberg cells from Hodgkins lymphoma. Approximately 4050% of Hodgkins 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 Hodgkins 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.
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Functional inhibitors to study LMP2A signalling |
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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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Summary and concluding statement |
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Acknowledgements |
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Footnotes |
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References |
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21
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