A Tissue Culture Model of Murine Gammaherpesvirus Replication Reveals Roles for the Viral Cyclin in Virus Replication and Egress. Public

Abstract

The Herpesviridae are a family of clinically relevant viruses that are unique in that they can remain latent in their host cells and may reactivate to cause disease. Gammaherpesviruses, in particular, are often associated with oncogenic conditions that are influenced by the state of the host cell cycle. Passage through the eukaryotic cellular cycle is regulated by cyclins and cyclin dependant kinases (CDKs). Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are two gammaherpesviruses that bring with them a virally encoded homologue to eukaryotic cyclin D (k-cyclin and m-cyclin). In MHV68, m-cyclin can interact with host CDK2 and CDK1/CDC2 this activity can influence lytic replication in the lungs. In-vitro attempts to study this defect have been limited by the lack of available tissue culture models that mimic the growth defect. It is hypothesized that the study of MHV68 replication in a polarized tissue culture cell line would provide a suitable environment to characterize the role of the v-cyclin in virus replication. We report here MHV68 replication in the rat lung cell line RL-65, a spontaneously immortalized, non-transformed polarizable epithelial cell line. These analyses mirror the in-vivo findings that m-cyclin is important in acute virus replication, as well shed new light on its influence on virus egress from infected cells. We also show that the KSHV v-cyclin (k-cyclin), which can functionally interact with different CDK partners (CDK4 and CDK6) than the MHV68 v-cyclin (CDK2 and CDC2), can partially rescue the replication defect observed with a v-cyclin null mutant. In summary, the RL-65 cell line provides an attractive in vitro model that mimics critical aspects of MHV68 replication in the lungs.

Table of Contents

Table of Contents

CHAPTER 1-INTRODUCTION.. 1

1.1 Herpesviridae-Overview.. 2

1.1 a) Morphology and Biological Properties. 2

1.1 b) Alphaherpesvirinae. 3

1.1 c) Betaherpesvirinae. 3

1.2 Gammaherpesvirinae. 4

1.2 a) Taxonomy. 4

1.2 b) Virus Lytic Replication. 4

1.2 c) Virus Latency. 5

1.2 d) Associated Diseases in Humans. 6

1.2 e) Animal Models. 8

1.3 Eukaryotic Cell Cycle & Cyclins. 11

1.3 a) Cell cycle, Cyclin, and CDK Overview.. 11

1.3 b) Cell cycle CKIs. 11

1.3 c) Cyclin and CDK Homologies. 12

1.4 Virus Modulation of Cell Cycle. 14

1.4 a) Virus Modulation of the Cell Cycle-Overview.. 14

1.4 b) Gammaherpesvirus Modulation of the Cell Cycle. 14

1.5 MHV68-Viral cyclin.. 16

1.5 a ) MHV68-cyclin Expression. 16

1.5 b) MHV68-cyclin Biochemical Properties. 16

1.5 c) MHV68-cyclin During Infection. 17

1.6 Epithelial Tissue In the Lungs. 18

1.7 Viral Egress and the Role of Epithelium.. 19

1.7 a) Virus egress. 19

1.7 b) Epithelial Role in Egress. 19

CHAPTER 2-A Tissue Culture Model of Murine Gammaherpesvirus Replication Reveals Roles for the Viral Cyclin in both Virus Replication and Egress from Infected Cells. 21

2.1 Abstract. 22

2.2 Introduction.. 23

2.3 Results and Discussion.. 26

2.4 Materials and Methods. 30

2.5 Conclusions. 34

2.6 Figures. 35

CHAPTER 3-KSHV-cyclin and MHV68-cyclin conserved functions. 42

3.1 Abstract. 43

3.2 Introduction.. 44

3.3 Results and Discussion.. 48

3.4 Materials and Methods. 50

3.5 Conclusions. 55

3.6 Figures. 57

CHAPTER 4-SUMMARY OF WORK AND FUTURE DIRECTIONS. 64

REFERENCES: 70

List of Figures

Figure 1 MHV68 requires v-cyclin for robust growth at early time points in growth arrested RL-65 cells. 35

Figure 2 RL-65 cells form polarized monolayers in transwells. 36

Figure 3. Basolateral shedding of MHV68. 37

Figure 4 MHV68 infection of polarized RL-65 cells leads to piles of infected cells that appear to be extruded from cell monolayer. 39

Figure 5 The vCyclin.stop mutant exhibits a severe defect in egress from growth arrested RL-65 cells. 40

Figure 6 CDK binding is required for efficient MHV68 replication in RL-65 cells. 41

Figure 7 KSHV v-cyclin can partially rescue MHV68 v-cyclin null virus replication defect in growth arrested RL-65 cells. 57

Figure 8 KSHV v-cyclin can partially rescue MHV68 v-cyclin null virus replication defect in lungs following intranasal inoculation. 59

Figure 9 KSHV K-cyclin does not rescue MHV68 v-cyclin null virus reactivation from splenocytes following intranasal inoculation. 61

Figure 10 K-cyclin Virus does not exhibit a preferred Egress Pattern on Polarized RL-65 cells. 63

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, Microbiology
Mot-clé	Viral Cyclin Gammaherpesvirus Epithelium
Committee Chair / Thesis Advisor	Speck, Sam, Emory University
Committee Members	Grakoui, Arash, Emory University Moore, Martin L, Emory University Kalman, Daniel, Emory University Steinhauer, David, Emory University

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