Epstein-Barr virus (EBV) causes infectious mononucleosis and is causally associated with two kinds of B-cell lymphomas and carcinoma in humans. In cell culture EBV infects and immortalizes human B-lymphocytes efficiently. It is likely that EBV's pathogenicity at least for its associated lymphoid diseases results from its capacity to immortalize B-cells. In this grant application we propose to refine and extend our genetic analysis of EBV's immortalizing functions. We have developed a method to study EBV genetically (1) that uses EBV-derived vectors that contain all the viral cis-acting signals required for them to participate in the lytic phase of the viral life cycle. Introduction of these vectors into appropriate cells followed by induction of the lytic cycle of an endogenous EBV allows the vectors to be amplified and packaged. These packaged vectors can infect human B-lymphocytes (1). This scheme will be optimized to yield maximum levels of the packaged vectors or maximum levels of recombinants between the vectors and the endogenous, helper EBV. The optimized scheme will be used to identify viral genes in addition to that encoding the EBV nuclear antigen-2 (EBNA-2) that are required for immortalization. Three extensions of this genetic scheme will be used to make these identifications. In addition, two approaches will be pursued to make helper cells - cells that provide helper viral functions but fail to pack-age their endogenous viral DNA. Helper cells would greatly facilitate a genetic analysis of EBV. Finally we propose to use our scheme to analyze the EBNA-2 gene which has been shown to be required for the immortalization of human B-lymphocytes by EBV (1).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI029988-01
Application #
3145025
Study Section
Virology Study Section (VR)
Project Start
1990-09-01
Project End
1995-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Gires, O; Zimber-Strobl, U; Gonnella, R et al. (1997) Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule. EMBO J 16:6131-40
Brielmeier, M; Mautner, J; Laux, G et al. (1996) The latent membrane protein 2 gene of Epstein-Barr virus is important for efficient B cell immortalization. J Gen Virol 77 ( Pt 11):2807-18
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Kempkes, B; Pich, D; Zeidler, R et al. (1995) Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA. J Virol 69:231-8
Mitchell, T; Sugden, B (1995) Stimulation of NF-kappa B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J Virol 69:2968-76
Gahn, T A; Sugden, B (1995) An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene. J Virol 69:2633-6
Kempkes, B; Pich, D; Zeidler, R et al. (1995) Immortalization of human primary B lymphocytes in vitro with DNA. Proc Natl Acad Sci U S A 92:5875-9

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