Limited nucleotide pools restrict Epstein-Barr virus-mediated B-cell immortalization.

Activation of cellular oncogenes as well as infection with tumor viruses can promote aberrant proliferation and activation of the host DNA damage response. Epstein-Barr virus (EBV) infection of primary human B cells induces a transient period of hyper-proliferation, but many of these infected cells succumb to an ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/Chk2)-mediated senescence-like growth arrest. In this study, we assessed the role of DNA replicative stress and nucleotide pool levels in limiting EBV-infected B-cell outgrowth. We found that EBV triggered activation of the ataxia telangiectasia and Rad3-related (ATR) signaling pathway in the early rapidly proliferating cells, which were also significantly more sensitive to inhibition of the ATR pathway than late attenuated proliferating cells. Through nuclear halo assays, we determined that early EBV-infected cells displayed increased replicative stress and DNA damage relative to late proliferating cells. Finally, we found that early after infection, hyper-proliferating B cells exhibited limited deoxyribonucleotide triphosphate (dNTP) pools compared with late proliferating and EBV-immortalized lymphoblastoid cell lines with a specific loss of purine dNTPs. Importantly, supplementation with exogenous nucleosides before the period of hyper-proliferation markedly enhanced B-cell immortalization by EBV and rescued replicative stress. Together our results suggest that purine dNTP biosynthesis has a critical role in the early stages of EBV-mediated B-cell immortalization.

The DNA damage response in viral-induced cellular transformation.

The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

Effects of the NIK aly mutation on NF-kappaB activation by the Epstein-Barr virus latent infection membrane protein, lymphotoxin beta receptor, and CD40.

Homozygosity for the aly point mutation in NF-kappaB-inducing kinase (NIK) results in alymphoplasia in mice, a phenotype similar to that of homozygosity for deletion of the lymphotoxin beta receptor (LTbetaR). We now find that NF-kappaB activation by Epstein-Barr virus latent membrane protein 1 (LMP1) or by an LMP1 transmembrane domain chimera with the LTbetaR signaling domain in human embryonic kidney 293 cells is selectively inhibited by a wild type dominant negative NIK comprised of amino acids 624-947 (DN-NIK) and not by aly DN-NIK. In contrast, LMP1/CD40 is inhibited by both wild type (wt) and aly DN-NIK. LMP1, an LMP1 transmembrane domain chimera with the LTbetaR signaling domain, and LMP1/CD40 activate NF-kappaB in wt or aly murine embryo fibroblasts. Although wt and aly NIK do not differ in their in vitro binding to tumor necrosis factor receptor-associated factor 1, 2, 3, or 6 or in their in vivo association with tumor necrosis factor receptor-associated factor 2 and differ marginally in their very poor binding to IkappaB kinase beta (IKKbeta), only wt NIK is able to bind to IKKalpha. These data are compatible with a model in which activation of NF-kappaB by LMP1 and LTbetaR is mediated by an interaction of NIK or a NIK-like kinase with IKKalpha that is abrogated by the aly mutation. On the other hand, CD40 mediates NF-kappaB activation through a kinase that interacts with a different component of the IKK complex.