PHOSPHATIDYLINOSITOL SIGNALING REGULATES POXVIRAL PATHOGENESIS Open Access

McNulty, Shannon L. (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/m039k5004?locale=en%5D
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Abstract

Smallpox was declared eradicated from nature in 1980, and mass smallpox vaccinations ceased in ~1978. Therefore, the population is extremely sensitive to the accidental release of Smallpox and to naturally occurring poxviruses, such as Monkeypox. Previous work from our lab identified that inhibitors targeting host Abl-family kinases can improve poxviral survival during a lethal (LD75) murine infection. To identify other host-directed antivirals we screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α -/- β -/- ) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α -/- β -/- cells. We extend these observations and demonstrate that Poxviruses regulate not only phosphatidylinositol kinases, but also phosphatidylinositol phosphatases. We found that the phosphoinositide 5'-phosphatase SHIP2 localizes to membranous protrusions formed beneath the virion called, "actin tails." Localization requires phosphotyrosine, Abl- and Src-family tyrosine kinases and N-WASP, but not the Arp2/3 complex nor actin. Cells lacking SHIP2 have normal actin tails, but release more virus. Moreover, viral strains with mutations in release inhibitor A34, release more virus but recruit less SHIP2. Thus, the inhibitory effects of A34 on viral release are mediated by SHIP2. Together, these data suggest that SHIP2 is an intrinsic antiviral factor that regulates dissemination of poxviruses from infected cells. Altogether, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent new therapeutic targets to contain poxviruses.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION.................................................1

1.1 History of Smallpox……………………………………………………………....……3

1.2 Orthopoxviruses Today………………………...…………………………….………7

1.3 Poxviral Structure and Replication……………………………………………..8 1.4 Poxviral Pathogenesis………………………………………………………………..11 1.5 Poxviral Treatment………………………………………………………………......14

1.6 Cellular Phosphatidylinositol Signaling……………………………………..15

1.7 Phosphatidylinositol 3-Kinases……………………………………………….…17 1.8 Phosphatidylinositol 5-Phosphatases……………………………...……...19 1.9 Lipid Signaling in Disease………………………………………………………...20

1.10 Goals of this Dissertation……………………………………………………....23

Literature Cited……..…………………….…………………………………………...…...24

Figure Legends……….……..……………..………………………………………...……..33 Figures 1-2………………………………….……………………………………….......34-35

CHAPTER II: IDENTIFICATION OF SMALL MOLECULES THAT

REDUCE VACCINIA VIRUS REPLICATION AND ACTIN TAIL

FORMATION …..............................................................…..……37

Abstract…………………………………………………………………………………...........38 Introduction…………………………………………………………………………............39

Materials and Methods……………………………………………………………..……...41

Results…………………………………………………………………………………..........…44 Discussion…………………………………………………………………..……………........45

Literature Cited…………………………………………………………..…………....……..50

Figure Legends…………………………………………………………..………….…........53 Tables 1-16….……………………………………………………………..………....…..54-68 Figures 1, 2…………………………………………………………………..……....…...69-70

CHAPTER III: MULTIPLE PHOSPHATIDYLINOSITOL 3-KINASES

REGULATE VACCINIA VIRUS MORPHOGENESIS .…………....…....72

Abstract……………………………………………………………………………....…......…..73 Introduction………………………………………………………………………......……...…74

Materials and Methods………………………………………………………………...…...76

Results……………………………………………………………………………........…....……83 Discussion………………………………………………………………………......………......93 Literature Cited………………………………………………………………....………....…102 Figure Legends…………………………………………………………………........…..….109 Table 1……………………………………………………………………………............…….120

Table S1………………………………………………………………………….............…...121

Figures 1-11……………………………………………………………………..…......122-132 Figures S1-S13………………………………………………………………….........133-145

CHAPTER IV: THE HOST PHOSPHOINOSITIDE 5-PHOSPHATASE

REGULATES DISSEMINATION OF VACCINIA VIRUS ………..…….147

Summary…………………………………………………….....……………………….....…..148 Introduction………………………………………………….....……………………....……..149 Results………………………………………………………….....…………………........…...151

Discussion …………………………………………………….....……………………..........157

Experimental Procedures…………………………….....…………………….………….159 References…………………………………………………….....…………………...….......164 Figure Legends……………………………………………….....……………….………......171 Figures 1-5…………………………………………………….....…………………....…176-180 Figures S1-S4……………………………………………………….....……….....…..181-184 CHAPTER V: ADDITIONAL OBSERVATIONS .…...…………......………186

5.1 Identification of Src- and Abl-family Tyrosine Kinase Substrates

Facilitating Vaccinia Virus Actin Tail Motility and Infectious Virion

Release…………………….................................................................187

5.1.1 Introduction……………………………......……….……………………..........…187

5.1.2 Results………………………………………......………………......……..........…188

5.1.3 Future Directions…………………………......…………………….........………192

5.2 vps34/15

5.2.1 Introduction……………………………………......………………...........………193

5.2.2 Results………………………………………………......…………..............………195

5.2.3 Future Directions………………………………......……………….........………198

5.3 Phosphatidylinositol 5-Kinases

5.3.1 Introduction……………………………………………....………...........…………199

5.3.2 Results……………………………………………………....…….........….....………200

5.3.3 Future Directions………………………………….…......………….........………201

5.4 SHIP2 is not recruited to EPEC pedestals, and does not alter

pedestal morphology………………………………….…….....…………………......…..202 Literature Cited………………………………………….……......………………........……206 Figure Legends……………………………………..….……………......………........…….209 Table 1……………………………………………………………………......…............213-214 Figure 1-14…………………………………………………………….....…........…...215-228 CHAPTER VI: FUTURE DIRECTIONS AND CONCLUSIONS 6.1 Future Directions……………………………………………………………........……..230

6.1.1 Phosphatidylinositol 3-Kinases and Vaccinia Virus...................231

6.1.2 SHIP2 and Vaccinia Virus…………………………………….........……........233

6.2 Conclusions……………………………………………………...…………..........………240

Literature Cited…………………………………………………………………...........………242

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