Studies on early cellular responses during Epstein-Barr virus infection

Abstract

The human gamma-herpesvirus Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a broad spectrum of lymphoid and epithelial cell malignancies. A characteristic property of the virus is the capacity to establish a non-productive growthpromoting infection in B-lymphocytes. Although the induction of cell proliferation is a key feature in oncogenesis, it is not sufficient for full malignancy. In the work presented in this thesis my colleagues and I have asked whether the virus might contribute to oncogenesis by triggering additional events that are required for tumor progression. Replicative immortality is dependent on the activation of mechanisms that maintain the integrity of telomeres. Malignant cells achieve this by activating telomerase or a recombination-dependent pathway known as alternative lengthening of telomeres (ALT). We observed multiple signs of telomere dysfunction consistent with the activation of ALT in newly EBV infected Blymphocytes. These include accumulation of telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchange (T-SCE), and low expression of telomere associated proteins such as TRF1, TRF2, POT1, and ATRX, pointing to telomere de-protection as possible cause of telomere damage. The early phase of EBV induced B-cell immortalization is characterized by the accumulation of DNA damage and activation of a DNA damage response (DDR) that limits the efficiency of growth transformation. By comparing the response of B-lymphocytes infected with EBV or stimulated with a potent Bcell mitogen, we found that significant higher levels of damage occur in EBV infected blasts due to stronger and sustained accumulation of reactive oxygen species (ROS). Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but dramatically decreased the efficiency of B-cell transformation, which correlated with selective downregulation of the viral LMP1 and the phosphorylated form of the cellular transcription factor STAT3. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Viral products that are delivered to the infected cells by the incoming virions are likely to play important roles in regulating the cellular response to infection. One of such products, the large tegument protein BPLF1, is a cysteine protease with potent ubiquitin and NEDD8-specific deconjugase activities. We found that targeting of the deneddylase activity of BPLF1 to nucleus of productively infected cells requires processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious viruses. Collectively, the findings summarized in this thesis provide new insights on the capacity of EBV to contribute to tumor initiation and progression by triggering events, such as oxidative stress and ALT, that favor the acquisition of both genomic instability and replicative immortality. Regulation of viral functions by the cellular response to danger signals delivered by incoming virions may further contribute to the remodeling of the host cell environment allowing successful infection.