A new gammaherpesvirus lytic gene promoter: identification and epigenetic regulation Öffentlichkeit

Gray, Kathleen Siobhan (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/m039k542n?locale=de
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Abstract

A New Gammaherpersvirus Lytic Gene Promoter: Identification and Epigenetic Regulation
By Kathleen S. Gray

Gammaherpesviruses are characterized by their ability to establish a lifelong infection in cells of lymphoid origin. The human gammaherpesviruses Epstein-Barr (EBV or HHV-4), and to a lesser extent Kaposi's Sarcoma-Associated Herpesvirus (KSHV or HHV-8), are ubiquitous and associated with lymphomagenesis and lymphoproliferative disease in immunocompromised individuals. Although rare given the broad distribution of infection, gammaherpesvirus-associated pathologies have driven aggressive efforts to better understand gammaherpesvirus biology. Narrow host tropism human has necessitated animal models to study gammaherpesvirus infection and is exemplified by the Murine Herpesvirus-68 (MHV68) system.

One of the most conserved functional gammaherpesvirus proteins is that encoded by Orf50, termed Rta (for Replication and Transcriptional Activator), named for its key role in driving both initial lytic replication and reactivation from latent infection.For EBV and KSHV, Rta expression upon reactivation from latency has been well-studied using established latent cell lines, and therefore little is known about the mechanism to initially target Rta for repression in favor of latent infection.

In this study, we identify an additional Rta transcriptional unit conserved among EBV, KSHV, and MHV68 in the existence of a third Rta-coding exon and additional promoter. This promoter drives expression of Rta to sufficiently support lytic replication during both permissive infection and reactivation from latency in certain cell types. We demonstrate that DNA methylation and the de novo DNA methyltransferases (DNMTs) are required to repress Rta transcription during early latency in vivo. Also, we provide evidence of strong selective pressure to methylate this new Rta promoter in infected B cells, even in the absence of DNMT3a and DNMT3b. In summary, we report the effect of de novo DNMT-mediated repression of Rta expression in B cells and demonstrate for the first time a direct requirement for de novo DNA methylation during the establishment of gammaherpesvirus latency.

Table of Contents

TABLE OF CONTENTS

Chapter 1: Introduction...1

1.I. Herpesviruses

i. General background...1
ii. Gammaherpesvirus-associated pathologies: justification for study...3
iii. MHV68 as a model system...4
iv. MHV68 and human gammaherpesviruses: similarities and differences...6

1.II. The gammaherpesvirus Rta protein

i. Gene structure and function in lytic replication...11
ii. Regulation of the Rta promoter...13

1.III. Epigenetic regulation via DNA methylation

i. General background...15
ii. DNA methylation in health and disease...17
iii. Role of DNA methylation in lymphocytes...18

1.IV. Intersection of DNA methylation and gammaherpesviruses

i. Role of DNA methylation in gammaherpesvirus pathogenesis...20
ii. Regulation of EBV latency programs...20
iii. Regulation of EBV latency programs by DNA methylation...23
iv. Regulation of gammaherpesvirus lytic replication by DNA methylation...24
v. The role of de novo DNA methylation in Rta promoter regulation...26

1.V. Figures and Tables...27
1.VI. Figure legends...35

Chapter 2: Alternatively initiated gene 50/RTA transcripts expressed during murine and human gammaherpesvirus reactivation from latency...37

2.I. Abstract...38
2.II. Introduction...40
2.III. Materials and Methods...43
2.IV. Results...53
2.V. Discussion...68
2.IV. Figure legends...77
2.VII. Figures...82

Chapter 3: Deregulated murine gammaherpesvirus gene 50/Rta transcription in vivo in the absence of the de novo methyltransferases DNMT3a and DNMT3b...89

3.I. Abstract...90
3.II. Introduction...92
3.III. Results...96
3.IV. Discussion...103
3.V. Materials and Methods...110
3.IV. Figures...117
3.VII. Figure legends...127

Chapter 4: Conclusions and Future Directions...130

4.I. Rta

i. Rta transcription, splicing, and the function of Exon0...131
ii. Transcription factors regulating the Rta distal promoter...135

4.II. DNA methylation in MHV68 infection

i. Role of de novo DNMTs in B cells...137
ii. De novo methyltransferases and human gammaherpesviruses...141
iii. Role of methylation in regulating gammaherpesvirus Rta: Relevance...144

4.III. Summary...145
4.IV. Figures...146
4.V. Figure legends...155

Literature Cited...158


LIST OF FIGURES and TABLES

Chapter 1:

Figures:

1. A. Kinetics of MHV68 lytic and latent infection
B. Model of MHV68 infection

2. A. Colinearity of gammaherpesvirus genomes
B. Annotation of transposon mutagenesis studies

3. Structure of gammaherpesvirus Rta-encoding transcripts

4. A. Model depicting DNA methylation in heterochromatin formation
B. Key domains of mammalian DNA methyltransferases

5. EBV latent gene transcription programs

Tables:

1. Frequency of MHV68 genome-positive cells
2. MHV68 genes and putative human gammaherpesvirus homologues

Chapter 2:

1. Conservation of G50/BRLF1/Rta coding region among gammaherpesviruses
2. Generation and confirmation of MHV68.G50pKO and MHV68.G50pKO-MR viruses
3. MHV68.G50pKO virus replicates in vitro
4. RACE and RT-PCR analyses identify an additional upstream G50 exon
5. Promoter activity in region immediately 5' to MHV68 E0
6. Quantitative RT-PCR analysis of distal versus proximal promoter-driven transcripts
7. Identification of upstream-initiated transcripts from treated EBV or KSHV latent cell lines
8. Exon 0 extends the EBV and KSHV G50 reading frames
9. G50pKO virus establishes latency in vivo but exhibits severe reactivation defect
10. Bisulfite PCR analysis of CpG methylation in regions containing the proximal and distal G50 promoters
11. G50pKO virus reactivates from peritoneal exudate cells following intraperitoneal infection

Chapter 3:

1. Methyltransferase inhibitor treatment induces MHV68 reactivation
2. In vitro methylation reduces distal Gene 50 promoter activity
3. Rearrangement of Dnmt3a and Dnmt3b alleles in CD19+/CreDnmt3a2loxP/2loxP Dnmt3b2loxP/2loxP splenocytes
4. Evidence of ongoing lytic replication in CD19+/Cre mice during early latency
5. Gene 50 transcripts are elevated in CD19+/Cre mice at day 18
6. Bisulfite PCR analysis of the distal Gene 50 promoter region at day 18
7. Genome frequency at day 42 post-infection
8. Bisulfite PCR analysis of the distal Gene 50 promoter region at day 42 post-infection
9. Bisulfite PCR analysis of the v-cyclin and v-bcl2 promoter region at days 18 and 42 post-infection

Chapter 4:

1. A. Translation of N-terminus of hypothetical MHV68 E0-E2 protein
B. Summary of detected gammaherpesvirus Rta transcripts and translations
C. Results of structural prediction analysis of E0 vs E1-containing proteins
2. Rta can transactivate a methylated Orf57 promoter
3. A. Hypoxia-induced activation of the distal Rta promoter is dependent on a HIF binding site
B. HIF1α activates the distal Rta promoter
C. HIF2α activates the distal Rta promoter
4. Dnmt3a and Dnm3b transcripts are detectable in naïve B cells
5. Analysis of NP-specific antibody response in CD19+/Cre mice versus
littermate controls:
A. ELIspot analysis of secondary antibody response following secondary immunization with NP-CGG
B. NP-specific antibody response following primary immunization
C. NP-specific antibody response following secondary immunization

6. Analysis of antibody response in MHV68-infected CD19+/Cre mice versus littermate controls:
A. MHV68-specific antibody response
B. ELIspot analysis of total IgM and IgG ASCs
7. A. Alternative splicing and promoter usage in transcription of MHV68-LANA B. Bisulfite sequence analysis of LANA latent promoter region

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