Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Paul M. Lieberman

Abstract

Epstein-Barr virus (EBV) is a ubiquitously prevalent human herpesvirus whose persistent latent infection is associated with many lymphomas. The prevalence of this virus can be attributed to its ability to establish a persistent latent infection in host cells, during which only a small number of viral genes are expressed from a highly regulated viral genome. However, it is not completely understood how EBV evades cellular antiviral defenses and regulates chromatin assembly to establish latent infections. The EBV major tegument protein BNRF1 was found to be required for the establishment of latent infections, and contains sequence homology to cellular purine biosynthesis enzymes, yet neither the functional mechanism nor link to purine metabolism is known. We have found that BNRF1 interacts with the cellular transcription co-repressor Daxx, and disrupts the binding between Daxx and its interaction partner, the chromatin remodeler ATRX. By ectopically complimenting BNRF1-null virus with mutant BNRF1, we found that the interaction with Daxx is essential for BNRF1 to support viral gene expression and infection. Furthermore, knockdown studies show that Daxx and ATRX are involved in the maintenance of viral chromatin during latency. With the Daxx-ATRX complex recently found to function as a histone variant H3.3 chaperone, we found that BNRF1 forms a complex with Daxx together with histones H3.3 and H4, suggesting potential modulation of a histone pre-deposition complex. In support of this, we show that BNRF1 can increase the release of histone H3.3 into the cellular free histone pool. We then show in primary infection of B lymphocytes that BNRF1-knockdown virus failed to prevent Daxx-ATRX access to the viral chromatin. This results in enriched H3.3 and decreased active histone H3K4me3 markers on the viral DNA, coinciding with deficient expression of viral latent genes. Our findings demonstrate that EBV BNRF1, by interacting with Daxx and displacing ATRX, prevents repressive histone variant H3.3 chromatin formation on viral DNA. Thus, BNRF1 is essential for the formation of viral chromatin permissive for expressing viral latent genes. This is the first demonstration of a viral tegument FGARAT-homology protein involved in the regulation of chromatin assembly. Most importantly, our findings suggest that active manipulation of a histone pre-deposition complex could turn chromatin-based host antiviral resistances into a mechanism of establishing viral latent chromatin.

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