Identification and characterization of MHV68 and KSHV ORF50 transcripts: A potential role in type I interferon evasion Open Access

Wakeman, Brian Scott (2015)

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Gammaherpesviruses are a lymphtropic subfamily of Herpesviridae, characterized by their ability to establish a lifelong infection within a host. The human gammaherpesviruses are further subdivided into the genera lymphocryptovirus containing Epstein-Barr (EBV or HHV-4), and rhadinovirus containing Kaposi's Sarcoma-Associated Herpesvirus (KSHV or HHV-8). Human gammaherpesviruses demonstrate widespread infectivity resulting in approximately 90% of the world population showing signs of latent EBV infection, and to a lesser extent KSHV infection. While these high infectivity rates are hypothesized to shape the overall global human immune profile, they are usually without major consequences in healthy individuals. This lack of consequences however changes in immunocompromised individuals and those experiencing secondary infections, left untreated EBV and KSHV have been associated with lymphoproliferative disease and lymphomagenesis. Complicating studies of gammaherpesviruses is the highly host specific nature of human infection which results in the use of an animal model utilizing Murine Herpesvirus-68 (MHV68). MHV68 shares large sequence homology with both KSHV and EBV, and one of the most highly conserved genes is that of Orf50, which encodes the protein RTA (Replication and Transcriptional Activator), which acts as a switch between acute and latent infection. The importance of RTA within the viral lifecycle makes it a prime target for drug therapeutics and other potential medical interventions such as vaccines resulting in a critical need for further study and investigation. In this study, we identified three previously unknown MHV68 Orf50 transcripts driven by two previously unknown promoters. We demonstrate that these previously unidentified transcripts retain the ability to drive lytic replication in absence of the previously known transcripts as well as reactivate latently infected virus; however this ability is only observed in the absence of a type I interferon response. Further we identified three previously unknown KSHV transcripts driven by two previously unknown promoters. Unlike MHV68 these KSHV transcripts result in unique isoforms of RTA. These unique isoforms, while maintaining many similar characteristics of known RTA, result in a different viral gene expression profile. These studies further the understanding of the essential lytic protein RTA and help shed light on the complex process regulating the lytic/latent cycle.

Table of Contents

Chapter 1: Introduction...1

1.I. General background

i. Herpesviridae...1

ii. Epstein-Barr Virus (EBV)...5

iii. Kaposi's Sarcoma-Associated Virus (KSHV)...7

iv. Murine Herpesvirus 68 (MHV68)...10

1.II. The Replication and Transcriptional Activator (RTA) protein

i. Function...15

ii. Transcriptional activation by RTA and its regulation...18

1.III. Type I interferon response

i. General background...21

ii. Type I interferon effect...23

iii. Type I interferon effect in gammaherpesviruses...25

1.IV. Figure legends...29

1.V. Figures...30

Chapter 2: Identification of alternative transcripts encoding the essential murine gammaherpesvirus lytic transactivator RTA...32

2.I. Abstract...34

2.II. Introduction...36

2.III. Materials and Methods...39

2.IV. Results...50

2.V. Discussion...66

2.VI. Figure legends...71

2.VII. Figures...77

Chapter 3: Identification of novel KSHV Orf50 transcripts: discovery of new RTA isoforms with variable transactivation potential...87

3.I. Abstract...89

3.II. Introduction...90

3.III. Materials and Methods...93

3.IV. Results...100

3.V. Discussion...110

3.VI. Figure legends...116

3.VII. Figures...120

Chapter 4: Conclusions and Future Directions...127

4.I. The MHV68 exon N3, exon N4, exon N5 and their promoters...128

4.II. Type I interferon sensitivity...133

4.III. The new KSHV transcripts and their promoters...137

4.IV. Summary...139

4.V. Figure legends...140

4.VI. Figures...143

Literature Cited...149

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