A new gammaherpesvirus lytic gene promoter: identification and epigenetic regulation Pubblico
Gray, Kathleen Siobhan (2009)
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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