Date of Award

8-2-2010

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Dr. I. George Miller

Abstract

Epstein-Barr Virus (EBV) has two states of infection, latent and lytic. During the latent state the viral genome remains stable in cells as episomes and replicates with cellular DNA. During the lytic cycle the viral DNA becomes amplified and packaged in newly formed virions. An unsolved problem is whether newly replicated EBV DNA produced upon lytic cycle activation is associated with histones, and if so, whether these histones are acetylated. This question has biological significance as knowing the chromatin structure of genes is important in determining their function and expression profile. Our hypothesis is that newly synthesized EBV lytic DNA is associated with histones and the histone tails are selectively acetylated. To investigate our hypothesis we performed chromatin immunoprecipitation (ChIP) in HH514-16 cells, a Burkitts Lymphoma cell line, during latent and lytic replication. We used quantitative PCR (qPCR) to detect the relative concentration of DNA among the different samples. We tested three different variables: type of inducing agent, duration of treatment, and different regulatory regions in the genome of Epstein-Barr Virus. We found that in cells induced into the lytic cycle with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), association of newly replicated EBV DNA with acetylated histone 3 (H3) increased ~ 6-10 fold. This increase in association was greatest 72 hrs after treatment. Furthermore, activation of lytic viral replication in HH514-16 cells using a different inducing agent, Azacytidine (AZC), which is known to function as a DNA methyltransferase inhibitor, increased binding of H3 with viral DNA ~8 fold. However, unlike TSA, AZC increased the acetylation state of histones bound to newly synthesized viral DNA only ~ 2 fold. Changing the regulatory region of the EBV genome analyzed in qPCR did not affect our results. Our results suggest that newly replicated viral DNA is associated with histones, a fraction of which are acetylated. The degree of acetylation likely depends on the agent used to induce the lytic cycle. H3 is highly acetylated when an HDACi is used and less acetylated when AZC is used. Our study provides new insight on the epigenetic profile of newly replicated viral DNA during the lytic cycle. It remains to be determined whether histones are packaged together with viral genomes into virions and whether the chromatin state of virion DNA affects gene expression after the virus enters uninfected cells.

Comments

This is an Open Access Thesis.

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