MHV68 M2 Manipulation of B cell Signaling Open Access

Rangaswamy, Udaya Shankari (2014)

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Gammaherpesviruses, members of the Herpesviridae family, are found in a wide variety of mammalian species and are characterized by their ability to establish life-long infections in their host. The human gammaherpesviruses Epstein-Barr Virus (EBV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV) are associated with several lymphoid disorders and carcinomas. Murine gammaherpesvirus68 (MHV68) is a well-characterized rodent model of gammaherpesvirus infection that is used to study various features of infection such as acute replication, latency, reactivation, host defense and lymphomagenesis. While latency is established in cell types such as B cells, macrophages, dendritic cells and lung epithelial cells, the long-term primary latent reservoir of MHV68 is memory B cells, similar to EBV. M2 is a MHV68 gene product that is required for efficient establishment of latency and reactivation from latency. M2 expression leads to increased IL-10 levels in primary murine B cells. In the first part, I characterized the signaling mechanism utilized by M2 to induce IL-10. Using an inducible B cell expression system, I demonstrate that M2 induces IRF4 in a manner partially dependent on the NFAT pathway to induce IL-10 expression. I also show that IRF4 is required for efficient establishment of latency and for reactivation from latency. In the second part, I show that Y129 is the critical residue of M2 absolutely required for IL-10 production and IRF4 expression in vitro; and establishment of latency, reactivation and plasma cell differentiation in vivo. Finally, using gene expression data from a microarray analysis, I wanted characterized IL-10 independent genes modulated by M2 and identified Suppressor of Cytokine Signaling-2 (SOCS2) as one such gene. Additionally, I show that M2 interacts and co-localizes with SOCS2. Taken together, this thesis work has identified key molecular players in the host signaling pathway(s) modulated by M2 which will aid in understanding the host evasion strategies utilized by this family of viruses to modulate B cell signaling to their benefit.

Table of Contents

Distribution Agreement
Approval Sheet
Abstract cover page
Cover page
Table of Contents
List of Figures

Chapter I - Introduction
I. Herpesviruses 1
1. An overview 1
2. The Gammaherpesvirus Family 2
3. MHV68 infection of mice - a small animal model to study gammaherpesviruses 3
4. MHV68 M2- a unique latency and reactivation associated gene 5
a) Biochemistry of M2 6
b) In vivo roles of M2 8
II. B cell signaling and differentiation 9
1. An overview 9
2. Signaling molecules of Gammaherpesviruses 10
3. IL-10 - role in B cells 11
4. Plasma cell differentiation -an overview 12
a) IRF4 in plasma cell differentiation 13
b) Plasma cell differentiation associated reactivation in gammaherpesviruses 14
Figures 16
Figure Legends 20

Chapter II -Murine Gammaherpesvirus M2 protein induction of IRF4 via the
NFAT pathway leads to IL-10 expression in B cells

Introduction 21
Materials and methods 25
Results 33
Discussion 49
Figures 55
Figure Legends 69

Chapter III - Differential requirement for tyrosines Y120 and Y129 of MHV68
M2 in plasma cell differentiation and reactivation

Introduction 75
Materials and methods 78
Results 82
Discussion 90
Figures 92
Figure Legends 98

Chapter IV - MHV68 M2 protein increases SOCS2 levels to modulate IFNγ signaling independent of IL-10 induction

Introduction 102
Materials and methods 104
Results 108
Discussion 112
Figures 115
Figure Legends 121

Chapter V - Summary, Future directions and conclusions
Mechanism of M2 induction of IL-10 123
M2 mediated plasma cell differentiation and reactivation from latency 126
M2 mediated changes in gene expression 128
Concluding remarks 130
References 131

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