Epstein-Barr Virus Molecular Biology

Staff
Funding
Collaborators
Student Projects
Key Publications
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Lab Head: Dr Tom Sculley
Tom.Sculley@qimr.edu.au

Epstein-Barr virus (EBV) infects more than 90% of the human population, and following primary infection with EBV all individuals retain the virus for life. EBV is the aetiological agent of infectious mononucleosis and is associated with a variety of lymphoid and epithelial cancers such as Burkitts' lymphoma (BL), Hodgkin's disease, T cell lymphomas, post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC) and a number of lymphoepithelioma-like carcinomas such as gastric carcinoma. The oncogenic potential of EBV is reflected in its ability to efficiently transform and immortalise human B cells in vitro. Despite the presence of the complete viral genome in EBV immortalized B lymphocytes, only a limited number of viral genes are expressed.

The primary focus of our research involves understanding how EBV is able to transform and immortalize B cells. The EBNAs-3 and -6 proteins of EBV are required for immortalization of primary human B-lymphocytes in vitro. These two EBNA proteins are targeted exclusively to the cell nucleus, and localize to specific sites within the cell nucleus. Although it has been known for more than 10 years that EBNAs -3 and -6 associate with discrete nuclear structures, neither the components of these structures, nor their functions, have yet been determined.

EBNA-3 and EBNA-6 proteins co-localize in the nucleus of cells

In one study we have mapped the region of EBNA-6 responsible for its association with the sub-nuclear structures and have demonstrated that EBNA-3 and EBNA-6 co-localize on the same nuclear structures. We have shown that the cellular protein SMN (survival of motor neurons protein) is also found on the same structures as EBNA-3 and EBNA-6. Nuclear SMN appears to mediate recycling of pre mRNA splicing factors and may also have a function related to gene regulation suggesting that EBNA-6 may utilize SMN to affect either RNA processing or transcriptional regulation. Other DNA viruses have already been shown to express proteins capable of influencing RNA processing as part of their transcription and replication strategies. Understanding what these proteins do in cells is the first step in developing compounds to treat EBV-associated diseases.

In a separate study cell cycle analyses have demonstrated that EBNAs -3, and -6 are all capable of disrupting the G2/M checkpoint response in cells. Co-immunoprecipitations experiments have demonstrated that the EBNA-3 protein of EBV was capable of interacting with chk2, a kinase implicated in the G2/M DNA damage and replication checkpoint responses. The function of EBNAs -3 and -6 proteins now appears to be far more complex than anticipated and this data suggests a role for these proteins in disrupting the host cell cycle machinery. If we could determine how the EBNA-3 protein overcomes cell cycle checkpoints we may be able to interfere with this process and prevent EBV from causing diseases.

Immunoelectron micrograph of cells labelled with MHC II and LMP 1 antibodies

LMP 1 is an EBV encoded membrane protein that functions as a constitutively active signaling molecule and associates in lipid rafts clustered with other signaling molecules. Another study has shown that LMP 1 also localises to an intracellular compartment in cells. Co-labelling of cells with both an LMP 1 antibody and an antibody to the golgi protein GS15 revealed that the intracellular LMP 1 partly co-localised with the golgi apparatus in a proportion of cells. Confirmation of intracellular LMP 1 was obtained by immunoelectron microscopy using rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 was also found to be present on exosomes derived from an LCL. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells suggesting that LMP 1 maybe involved in immune regulation. This may be of particular relevance in EBV associated tumours such as nasopharyngeal carcinoma and Hodgkins disease, that express only EBNA 1 and LMP 1 as LMP 1 containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect allowing the tumour cells to evade the immune system.

Related work has arisen through the identification of a novel cellular gene product in EBV transformed B cells. The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. We have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin1alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin1 polyclonal antibody showed that full-length Sin1 and several smaller isoforms are widely expressed. Sin1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1alpha isoform also could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK.

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Staff

Funding

National Health and Medical Research Council
University of Queensland

Collaborators

• Prof Nancy Raab-Traub, Lineberger Comprehensive Cancer Center, University of North Carolina, USA
• Prof John Hancock, University of Queensland, Brisbane
• Prof Elizabeth Benson, ICPMR, Westmead Hospital, Sydney
• Assistant Prof G. Bushell, Griffith University, Brisbane
• Dr Kerstin Falk, Karolinska Institute, Stockholm, Sweden
• Dieter Maier, University of Hohenheim, Stuttgard, Germany
• Dr Brian Gabrielli, Dept of Pathology, University of Queensland
• Dr Graham Kay, QIMR, Brisbane
• Dr Nigel McMillan, Princes Alexanda Hospital, Brisbane
• Dr Jaap Middeldorp, Dept. Pathology, Free University Hospital, Amsterdam, The Netherlands
• Dr Sharon Milgram, Physiology Department, University of North Carolina, USA
• Professor Bill Sugden, McArdle Laboratory for Cancer Research, Madison, USA

Student Projects

The EBV Molecular Biology Laboratory has a number of projects available for students at BSc Honours and PhD level.

Key Publications

Schroder, W., Cloonan, N., Bushell, G. and SCULLEY, T. (2004) Alternative polyadenylation and splicing of mRNAs transcribed from the human Sin1 gene. Gene 339, 17-23.

Schroder, W., Bushell, G. and SCULLEY, T. (2005) Alternatively Spliced mRNAs Expressed from the Human Sin1 Gene Encode Isoforms of a JNK-binding Protein. Cellular Signaling 17(6), 761-7.

Krauer K, Buck M, Flanagan J, SCULLEY T, Burgess A and Gabrielli B. (2004) The EBNA-3 gene family proteins disrupt the G2M checkpoint. Oncogene 23:1342-1353 [pubmed abstract]

Krauer K, Buck M, Flanagan J, Belzer D and SCULLEY T. (2004) Identification of the Nuclear Localisation Signals within the Epstein-Barr virus EBNA-6 protein. J. Gen. Virol. 85:165-172 [pubmed abstract]

Krauer K, Buck M, Belzer D, Flanagan J, Chojnowski G and SCULLEY T. (2004) The Epstein-Barr Virus Nuclear Antigen (EBNA)-6 Protein co-localizes with EBNA-3 and survival of motor neurons protein (SMN). Virology 318:280-294 [pubmed abstract]

Blood A, Edwards CJ, Ishii HH, Bryson G, SCULLEY TB and Gobe GC. (2003) Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B-cell lymphoma cells: role of bcl-xl, bax and caspase protein. Arch.Virol. 149:289-302 [pubmed abstract]

Flanagan J, Middeldorp J and SCULLEY T. (2003) Localization of the Epstein-Barr virus protein LMP 1 to exosomes. J. Gen. Virol. 84:1871-1879 [pubmed abstract]

Krauer KG, Buck M and SCULLEY TB. (1999). Characterization of the Transcriptional Repressor RBP in Epstein-Barr Virus Transformed B-cells. J. Gen. Virol. 80(Pt 12):3217-26 [pubmed abstract]

Kienzle N, Buck M, Greco S, Krauer K and SCULLEY TB. (1999). Epstein-Barr virus encoded RK-BARF0 protein expression J. Virol 73:8902-8906 [pubmed abstract]

Kienzle N, Young DB, Liasakou D, Buck M and SCULLEY TB. (1999). Intron retention may regulate the expression of the Epstein-Barr virus nuclear antigen 3 family genes. J. Virol. 73:1195-1204 [pubmed abstract]

Buck M, Cross S, Krauer K, Kienzle N and SCULLEY TB. (1999). A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle. J. Gen. Virol. 80:441-445 [pubmed abstract]

Kienzle N, SCULLEY TB, Greco S and Khanna R. (1999) Cutting Edge: Silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: A novel implication of RNA processing for antigen presentation. J Immunol 162:6963-6966 [pubmed abstract]

Krauer KG, Belzer DK, Liaskou D, Buck M, Cross S, Honjo T and SCULLEY TB. (1998) Regulation of Interleukin-1-beta (IL-1b) transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP. Virology 252:418-430 [pubmed abstract]

Kienzle N, SCULLEY TB, Poulsen L, Buck M, Cross S, Raab-Traub N and Khanna R. (1998) Identification of a cytotoxic T lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: A critical role for antigen expression. J. Virol. 72:6614-6620 [pubmed abstract]

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