Characterizing the Expression and Regulation of MHV68 M1 & Uncovering its Role in Pulmonary Fibrosis Open Access

O'Flaherty, Brigid Moira (2015)

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Coevolution and adaptation to their hosts has led herpesviruses to encode numerous genes which facilitate infection and establishment of lifelong latency. The focus of this dissertation was to evaluate expression and function of MHV68 unique gene M1, which has previously been shown to play a critical role in control of viral reactivation from peritoneal exudate cells through the activation and expansion of INFγ secreting Vβ4+ CD8+ T cells. To gain insights into M1 function, our first aim was to identify the cellular reservoir of M1 expression in vivo. Here we define splenic plasma cells as the predominant source of M1 expression during MHV68 infection. We show that the MHV68 viral replication and transcription activator (Rta), which initiates viral reactivation from latency, synergistically regulates M1 expression with the cellular interferon regulatory factor 4 through protein-protein mediated interaction with the M1 promoter. Furthermore, we identify a novel Rta response element within the M1 promoter, which is conserved in several other MHV68 genes. These findings highlight a mechanism to fine-tune viral gene expression in response to host and viral cues. The second aim of this dissertation was to characterize the role of M1-driven Vβ4+ CD8+ T cell expansion in fibrotic disease. Our lab has shown that M1 expression is required for development of fibrotic disease in IFNγR-/- mice. Here we demonstrate that M1 expression results in heightened levels of inflammation and fibrosis in the lung. Additionally, we find elevated levels of neutrophil and effector CD8+ T cells at 28 days post-infection. We verify the involvement of CD8+ T cells in fibrotic disease through CD8+ T cell depletion which results in protection from fibrosis and lethality. Taken together data raise the possibility that Vβ4+ CD8+ T cells induce fibrosis though cytokine mediated inflammation, which results in altered cellular trafficking and immunopathology. Collectively, these studies provide significant insights into M1 function in MHV68 infection. We link M1 expression with viral reactivation, and provide evidence for the role of Vβ4+ CD8+ T cells as mediators of fibrotic disease.

Table of Contents

Chapter I: Introduction

Herpesviridae. 1

Gammaherpesvirinae. 2

MHV68. 3

M1 Function. 4

M1 Expression. 7

Genetic Requirements. 8

Functional Homologs to M1. 9

Fibrosis. 10

Pulmonary Fibrosis. 10

Role of Infection. 12

Hepatitis C Virus. 12

Torque Teno Virus & Adenovirus. 13

Influenza Virus. 13

Herpesvirus. 14

Cytomegalovirus. 14

Epstein-Barr Virus. 15

Case Reports, Pulmonary Fibrosis in Animals. 16

MHV68 Induced Fibrosis in IFNgR-/- Mice. 16

Atrophy and Fibrosis of the Spleen. 17

Hepatic Fibrosis. 19

Pulmonary Fibrosis. 19

Fidelity of this Model System for IPF. 20

Summary. 21

Figures. 22

Figure Legends. 24

Tables. 26

Chapter II: The Murine Gammaherpesvirus Immediate-Early Rta Synergizes with IRF4, Targeting Expression of the Viral M1 Superantigen to Plasma Cells

Abstract. 28

Introduction. 30

Methods. 33

Results. 40

Discussion. 49

Acknowledgements. 55

Figures. 56

Figure Legends. 67

Tables. 72

Chapter III: CD8+ T cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

Abstract. 74

Introduction. 76

Methods. 79

Results. 84

Discussion. 93

Acknowledgements. 99

Figures. 100

Figure Legends. 114

Tables. 119

Chapter IV: Summary, Discussion, and Future Directions

Characterizing M1 expression in vivo and its transcriptional regulation. 121

Evaluating the role of M1 in MHV68 induced fibrosis. 124

Concluding Remarks. 130

Figures. 132

Figure Legends. 133

References. 134

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