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Schmeinck, Anne (2011): Acquisition and loss of chromatin modifications during an Epstein-Barr Virus infection. Dissertation, LMU München: Fakultät für Biologie



Cellular gene regulation depends on fundamental epigenetic mechanisms, but epigenetic modifications also govern the regulation of the life cycle of Epstein-Barr virus (EBV). Promoter usage during latency depends on DNA methylation of the viral genome and CpG-methylation of certain promoters with meZREs is an indispensable prerequisite to switch from the latent to the lytic phase. In my thesis, I wanted to assess the underlying epigenetic principles of EBV’s gene regulation during the establishment of latency and upon lytic reactivation. My results suggested a new classification of viral promoters and phases of gene regulation that depend on the epigenetic state of the viral chromatin. According to this new model, EBV’s infectious cycle consists of an initial abortive lytic, a latent, and a productive lytic phase, and the viral promoters can be classified into default-on, poised-on, and poised-off promoters. Default-on promoters are immediately active upon infection of primary B cells leading to the so-called abortive lytic phase of an EBV infection. Default-on promoters encounter the cell in an environment that supports binding of the basal transcription machinery to activate viral gene transcription. Default-on promoters include the promoters of BALF1, BHRF1, BZLF1, BRLF1, BNLF2a, BCRF1, and Wp. The protein products are indispensible for the permanent establishment of EBV’s genome in latently infected cells supporting growth transformation, immune evasion, and anti-apoptotic cellular pathways. Default-on promoters are epigenetically silenced upon occupancy of EBV’s DNA with nucleosomes very early after infection and a switch to poised-on promoters is initiated. Poised-on promoters embody an epigenetically active state upon infection, but their activation requires an additional, virus-encoded factor to allow initiation of transcription. Wp-induced expression of EBNA1 promotes the switch to the poised-on promoter Cp to sustain long-term EBNA expression. Other poised-on genes including the viral structural proteins are not provided with their cofactor initially. During latency, these promoters are repressed through compaction of chromatin by high nucleosome occupancy, trimethylation of H3K27, and a stable transmission of repressive modifications by Polycomb-mediated long-term silencing. The establishment of a defined DNA methylation pattern on EBV’s DNA further represses poised-on promoters. DNA methylation is a prerequisite for the activation of a third promoter class, termed default-off promoters. Default-off promoters are bound and transactivated by BZLF1 in a methylation-dependent manner. Upon infection of primary B cells, EBV’s DNA is completely unmethylated, impeding an early expression of default-off genes. Only two to three weeks post infection the viral genome has acquired a proper epigenetic configuration that supports transcription of default-off genes. Binding of BZLF1 alone does not suffice to recruit the cellular transcription machinery including RNA polymerase II, but the chromatin requires remodeling, including a loss of nucleosomes and repressive modifications at default-off promoter sites. Default-off genes encode the viral lytic DNA replication machinery. The newly synthesized DNA templates lack epigenetic modifications because lytic DNA amplification is uncoupled from cellular DNA replication, eliminating the epigenetic maintenance mechanisms during the synthesis of viral progeny. As a consequence, default-on promoters and silenced poised-on promoters, which rely on unmethylated, epigenetically naïve templates, become also activated in the onset of the lytic phase. Silenced poised-on promoters require additionally a viral cofactor for their activation. This so-far unknown factor is probably provided upon lytic DNA amplification allowing the transcription of genes encoding for structural proteins that are necessary for the packaging of viral progeny. The released EBV progeny is epigenetically unmodified and ready to infect other cells. In essence, the regulation of EBV’s life cycle by epigenetic mechanisms is a paradigm for viral coevolution with its host. Repressive epigenetic modifications are common cellular defense mechanism to fight invading pathogens. EBV has hijacked this system for the regulation of promoter usage during its own life cycle, which has become a key principle of EBV’s success in infecting and persisting in its host.