Induction of [p16.sup.INK4a] is the major barrier to proliferation when Epstein-Barr Virus transforms primary B cells into lymphoblastoid cell lines

To explore the role of [p16.sup.INK4a] as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding [p16.sup.INK4a] and [p14.sup.ARF]. Using recombinant EBV-BAC viruses expressing conditional EBNA3C...

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Bibliographic Details
Published in:PLoS pathogens 2013-02, Vol.9 (2)
Main Authors: Skalska, Lenka, White, Robert E, Parker, Gillian A, Sinclair, Alison J, Paschos, Kostas, Allday, Martin J
Format: Article
Language:English
Subjects:
B cells
Cell lines
Cell proliferation
Epstein-Barr virus
Genetic aspects
Genetic transformation
Health aspects
Physiological aspects
Viral genetics
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Summary:To explore the role of [p16.sup.INK4a] as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding [p16.sup.INK4a] and [p14.sup.ARF]. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active [p16.sup.INK4a] protein but expressing a functional 14ARF-fusion protein (p14/ p16). The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional [p16.sup.INK4a]. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in [p16.sup.INK4a] and growth arrest, EBNA3C inactivation in the [p16.sup.INK4a]-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the [p16.sup.INK4a]-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in [p16.sup.INK4a] expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are [p16.sup.INK4a]-null, functional EBNA3C is dispensable for the outgrowth of LCLs.
ISSN:1553-7366
1553-7374
DOI:10.1371/journal.ppat.1003187