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Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line
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Limited nucleotide pools restrict Epstein-Barr virus-mediated B-cell immortalization
A Y Hafez1, J E Messinger1, K McFadden1
1Department of Molecular Genetics and Microbiology, Center for Virology, Duke University School of Medicine, Durham, NC, USA.
Oncogenesis
|June 13, 2017
Summary
Epstein-Barr virus (EBV) infection causes B-cell hyper-proliferation, but limited purine deoxyribonucleotide triphosphate (dNTP) pools cause DNA damage. Supplementing nucleosides enhances EBV B-cell immortalization by reducing replicative stress.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Epstein-Barr virus (EBV) infection of B cells causes hyper-proliferation.
- Many EBV-infected cells undergo senescence due to ATM/Chk2-mediated growth arrest.
- DNA damage response is activated by oncogenes and tumor viruses.
Purpose of the Study:
- To investigate the role of DNA replicative stress and nucleotide pools in limiting EBV-infected B-cell outgrowth.
- To understand the mechanisms behind EBV-mediated B-cell immortalization.
Main Methods:
- Assessed the ataxia telangiectasia and Rad3-related (ATR) signaling pathway activation.
- Performed nuclear halo assays to measure replicative stress and DNA damage.
- Quantified deoxyribonucleotide triphosphate (dNTP) pools in EBV-infected cells.
- Supplemented cells with exogenous nucleosides.
Main Results:
- EBV triggered ATR pathway activation in early proliferating cells, increasing sensitivity to ATR inhibition.
- Early EBV-infected cells showed higher replicative stress and DNA damage than late proliferating cells.
- Early hyper-proliferating B cells had limited purine dNTP pools compared to later stages.
- Exogenous nucleoside supplementation enhanced B-cell immortalization and rescued replicative stress.
Conclusions:
- Purine deoxyribonucleotide triphosphate (dNTP) biosynthesis is critical for early EBV-mediated B-cell immortalization.
- Replicative stress and limited dNTP pools restrict EBV-driven B-cell proliferation.
- Targeting dNTP metabolism could be a strategy to modulate EBV-induced B-cell immortalization.
