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Time-course analysis of human primary B lymphocytes infected with EBV
Time-course analysis of human primary B lymphocytes infected with EBV
In vitro infection of human quiescent B cells with Epstein-Barr Virus (EBV) activates them and leads to infinitely proliferating lymphoblastoid B cell lines (LCL), in which the virus establishes a stable latent infection. In this process, the virus triggers dramatic changes in the host cell biology to allow its long-term persistence and guarantee the survival of its host cell. During latency, EBV encoded proteins mimic activating physiological cellular pathways and processes promoting viral success. In my PhD thesis, I wished to investigate the very early steps of EBV infection focusing on changes in the host cell biology. I investigated the early phenotypes of the infected cells, which grow in cell volume and massively induce RNA and proteins synthesis, initially. Within the first two days, the cells show stable metabolic activity and do not proliferate. On day three post infection (p.i), the cells initiate DNA replication and divide followed by a phase of intense cellular proliferation. Starting at day four p.i., the cells show very high metabolic activities characterized by an increased uptake of glucose and enhanced mitochondrial activity. To monitor the obvious alterations in the biology of the EBV-infected B cells, I devised well-controlled and time-resolved RNA expression profiling (RNA-seq) experiments. The analyses of these experiments identified seven different clusters of genes with very specific gene expression patterns in EBV-infected cells compared with uninfected cells. My results document that the virus governs all important cellular processes including proliferation, cell metabolism, various epigenetic mechanisms, and ncRNAs biosynthesis in a very strictly time-controlled manner during infection. The virus delivers its epigenetically naïve genomic DNA to the host cell upon infection, but in latently infected cells the viral DNA is extrachromosomaly maintained and organized identical to cellular chromatin with nucleosomes including mostly repressive histone marks. In my PhD work I wanted to investigate the kinetics of nucleosome assembly on viral DNA as well as the specific positioning of nucleosomes. Within 24 hours after infection, EBV DNA had acquired nucleosomal structures, which accumulated and became more prevalent until day three p.i.. Nucleosome acquisition did not appear to be random, but I found pre-defined locations occupied with nucleosomes very early after infection. In my MNase-seq experiments nucleosome occupancy at certain cellular loci also changes dramatically indicating that dynamic alterations in nucleosome positioning might cause the profound changes in the transcriptome of the virally infected B cells.
EBV, infection, B cells, time-course, RNA-seq, metabolism, MNase-seq
Mrozek-Górska, Paulina
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Mrozek-Górska, Paulina (2018): Time-course analysis of human primary B lymphocytes infected with EBV. Dissertation, LMU München: Faculty of Biology
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Abstract

In vitro infection of human quiescent B cells with Epstein-Barr Virus (EBV) activates them and leads to infinitely proliferating lymphoblastoid B cell lines (LCL), in which the virus establishes a stable latent infection. In this process, the virus triggers dramatic changes in the host cell biology to allow its long-term persistence and guarantee the survival of its host cell. During latency, EBV encoded proteins mimic activating physiological cellular pathways and processes promoting viral success. In my PhD thesis, I wished to investigate the very early steps of EBV infection focusing on changes in the host cell biology. I investigated the early phenotypes of the infected cells, which grow in cell volume and massively induce RNA and proteins synthesis, initially. Within the first two days, the cells show stable metabolic activity and do not proliferate. On day three post infection (p.i), the cells initiate DNA replication and divide followed by a phase of intense cellular proliferation. Starting at day four p.i., the cells show very high metabolic activities characterized by an increased uptake of glucose and enhanced mitochondrial activity. To monitor the obvious alterations in the biology of the EBV-infected B cells, I devised well-controlled and time-resolved RNA expression profiling (RNA-seq) experiments. The analyses of these experiments identified seven different clusters of genes with very specific gene expression patterns in EBV-infected cells compared with uninfected cells. My results document that the virus governs all important cellular processes including proliferation, cell metabolism, various epigenetic mechanisms, and ncRNAs biosynthesis in a very strictly time-controlled manner during infection. The virus delivers its epigenetically naïve genomic DNA to the host cell upon infection, but in latently infected cells the viral DNA is extrachromosomaly maintained and organized identical to cellular chromatin with nucleosomes including mostly repressive histone marks. In my PhD work I wanted to investigate the kinetics of nucleosome assembly on viral DNA as well as the specific positioning of nucleosomes. Within 24 hours after infection, EBV DNA had acquired nucleosomal structures, which accumulated and became more prevalent until day three p.i.. Nucleosome acquisition did not appear to be random, but I found pre-defined locations occupied with nucleosomes very early after infection. In my MNase-seq experiments nucleosome occupancy at certain cellular loci also changes dramatically indicating that dynamic alterations in nucleosome positioning might cause the profound changes in the transcriptome of the virally infected B cells.