SILENCING SUPPRESSION BY TYPE 1

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Zetang Wu, M.S.

*****

The Ohio State University 2008

Dissertation Committee: Approved by Dr. Deborah S. Parris, Advisor

Dr. Biao Ding

Dr. Kathleen BorisLawrie Advisor Molecular, Cellular, and Developmental Biology Dr. David M. Bisaro Graduate Program

ABSTRACT

It was hypothesized that type1 (HSV1) encodes one or more capable of functioning as a silencing suppressor during productive infection. A transient silencing system was developed that relies on cotransfection of mammalian cells with a plasmid that expresses a silencing target (EGFP) and one that expresses either a perfect EGFPspecific hairpin (dsEGFP) or an imperfect hairpin

(midsEGFP). Cotransfection of target plasmid with a plasmid that expresses perfect or imperfect hairpins specific for lacZ (dsLacZ or midsLacZ, respectively), was used as a control. Results demonstrated that dsEGFP and midsEGFP reduced the EGFP mRNA level by 2.5 and 7fold, respectively, compared with control hairpins, and a 1:1 molar ratio of midsEGFP to target plasmid yielded maximum silencing of EGFP. Silencing by the midsEGFP hairpin decreased the halflife of EGFP mRNA ~2fold compared with the control imperfect hairpin.

Infection of transfected cells with HSV1 wildtype strain KOS increased the steadystate amount of EGFP mRNA, regardless of whether or not the EGFP transcript contained an intron. This increase was due, at least in part, to an increase in the halflife of EGFP mRNA in silenced cells, consistent with silencing suppression. This increased

EGFP mRNA halflife occurred despite the fact that host shutoff functions of HSV1 are

ii known to globally reduce the stability of mRNAs. Indeed with lossoffunction mutations in these host shutoff genes increased the stability of EGFP mRNA in silenced cells to an even greater extent than the wildtype virus. Increased accumulation of target

EGFP mRNA occurred as early as 4 hr after HSV1 infection and did not require viral

DNA synthesis, suggesting that the silencing suppressor activity was an immediateearly gene product or a virion component.

We tested several immediateearly genes and a virion component gene for ability to suppress silencing in an established plant assay and found that only the virion component, US11, could suppress silencing in plants in the absence of other HSV1 genes. Further experiments suggest that US11 could play an important role in silencing suppression in mammalian cells.

Taken together these results demonstrate that infection by HSV1 can suppress established silencing and suggest that this silencing suppression may have evolved as a means to counter a very early host silencing response following virus infection. A prediction of this hypothesis is that replication of HSV1 would be enhanced if the host

silencing response were abrogated. To test this prediction, siRNAs were transfected into

cells to knockdown the RNA slicer activity, argonaut 2 (Ago2), prior to virus infection.

The yields of virus following HSV1 infection of cells treated with siRNA to Ago2,

compared to those treated with RISCfree control RNA duplex, were significantly

increased.

iii

Dedicated to my parents, my wife and daughter

iv

ACKNOWLEDGMENTS

I express my sincere appreciation to my advisor, Dr. Deborah S. Parris for her

guidance, insight, encouragement and constant support. Also, I deeply thank her for her patience throughout the research. Her full support was the key to the success of my work.

I extend my gratitude to my committee members Dr. Biao Ding, Dr. Kathleen

BorisLawrie and Dr. David M. Bisaro for their generosity with their time, valuable

suggestions and enthusiastic support.

I would like to express my sincere gratefulness to all the former and current

members in Dr. Parris’ lab, Dr. Yali Zhu, Dr. Liping Song, Dr. Kelly S. Trego, Dr.

Murari Chaudhuri, and Ms. Houleye Diallo for their help, friendship and encouragement.

Especially, I thank Dr. Yali Zhu for her experienced technical support and stimulating

discussions.

I extend my appreciation to all the former and current members in Dr. Bisaro’ lab,

Dr. Hui Wang, Dr. Kenneth Buckley, Priya Raja, Cody Buchmann, Xiaojuan Yang,

Gireesha Mohannath for generous help and dedicated cooperation. In particular, I would

like to express my sincere thanks to Dr. Hui Wang for his excellent technical support and

useful suggestions, to Dr. Kenneth Buckley for his kind help in revising my candidacy

exam proposal and doing the plant experiment for the project.

v I would also like to express my deepest appreciation to Mrs. Yaoling Shu for helping me do the quantitative realtime PCR.

Finally, I would like to sincerely thank my parents and my family for their continuous love, unconditional support and encouragement. To my parents, Chenan Wu and Zhongyu Tian, I sincerely thank you for lifelong love. To my wife, Shuangling He, and my daughter, Iris Wu, I am sorry for not taking the responsibilities for the family I should have shouldered in the past several years, and I deeply apologize to you for the inconvenience I have brought home over all the challenging time. I know that without your understanding, constant love and encouragement, it would be impossible for me to have completed this work. Thank you all for your support.

vi

VITA

1981 1985………………… B.S, Agronomy, Agronomy Department, Southwest Agriculture University, Chongqing, P.R.C

1985 1988………………… M.S, Biochemistry, The Basic Science Department, Southwest Agriculture University, Chongqing, P.R.C

1988 1998………………… Lecturer/Assistant Professor, The Department of Biology, China West Normal University, Nanchong, Sichuan, P.R.C

1998 2001………………… Research Associate, The Department of Chemical Engineering, The Ohio State University

2001 Present……………… Research Associate, The Department of Molecular , Immunology, and Medical Genetics, The Ohio State University

2003 Present……………… Ph.D student, MCDB, The Ohio State University

PUBLICATIONS

1. Zetang Wu , ShangTian Yang (2003). Butyric acid extractive fermentation by

immobilized clostridium tyrobutyricum in fibroudbed bioreactor. Biotechnol.

Bioeng., 82(1),92102

2. Ying Zhu, Zetang Wu , ShangTian Yang (2002). Butyric acid production from Acid

vii hydrolysate of corn fiber by clostridium tyrobutyricum in a fibroudbed

bioreactor. Process Biochemistry, 38(5), 657 – 666

3. Huang, Yu Liang; Wu, Zetang ; Zhang, Likun; Ming Cheung, Chun; Yang, Shang

Tian (2002). Production of carboxylic acids from hydrolyzed corn meal by

immobilized cell fermentation in a fibrousbed bioreactor. Bioresource Technology,

82(1), 5159

4. Tang Wenqiang, Bai Juan, Wu Shuping, Zetang Wu et. al (1999). Influence of

different pH on the biological activity of calmodulin, an insight into the regulation

mechanism of extracellular calmodulin. Journal of Hebei Normal University, 23(1),

106111

5. Zetang Wu (1998). Studies on the reasons for low activity of SOD of Nanhuang

barley leaves. Journal of Sichuan Teachers College, 19(2), 152155

6. Yang Jin, Zetang Wu (1998). Studies on optimal conditions for measurement of

content by Bradford method. Journal of Sichuan Teachers College, 19(2),

198200

7. Zetang Wu , Hou WanRu (1997). Study on the relation between the influences of

KT, ABA and MDA on SOD activity. SOD conformation and hydrophobicity

changes. Chinese Biochemical Journal, 13(6),716718

viii

8. Hou wanru, Zetang Wu , Xu XinMing (1996). The quantitative measurement standard

for copper sodium chlorophyll. Journal of Sichuan Teachers College, 17(3), 2427

9. Zetang Wu ( 1991). Relationship between superoxide radical and destruction of

chlorophyll during leaf senescence. Plant Physiology Communications, 27(4), 277

279

10. Zetang Wu , Yang Daqi (1991). Studies on the protective effect of ethanol on the

destruction of plasma membrane during plant leave senescence. Journal of Southwest

Agriculture University, 13(2), 204206

11. Zetang Wu , Zhang Gangyuan (1990). Studied on the influence of ABA and 6 benzyl

aminopurine on the destruction of plasma membrane of leaves. Journal of Sichuan

Teachers College, 11(3), 213216

12. Zetang Wu , Zhang GangYaun (1990). Effects of abscisic acid, cytokinin and

malonaldehyde on superoxide dismutase activity. Plant Physiology Communications,

4, 3032

13. Zetang Wu , Yang Daqi (1990). Studies on the relation between enzyme activities

and the destruction of plasma membrane during detached leaf senescence. Journal of

Southwest Agriculture University, 12(4), 371374

ix

FIELDS OF STUDY

Major Field: Molecular, Cellular, and Developmental Biology

x

TABLE OF CONTENTS

Page

Abstract……………………………………………………………………………... ii

Dedication………………………………………………………………………...... iv

Acknowledgments………………………………………………………………… v

Vita…………………………………………………………………………………. vii

List of Tables……………………………………………………………………….. xiv

List of Figures……………………………………………………………………… xv

List of Abbreviations……………………………………………………………….. xvii

CHAPTER 1………………………………………………………………………... 1

INTRODUCTION………………………………………………………………….. 1

1. Background information about herpes simplex virus type 1………………. 1 1.1. HSV1 virion and genetic structures…………………………………. 1 1.2. HSV1 life cycles…………………………………………………….. 4 1.3. Productive cycle of HSV1…………………………………………... 5 1.4. HSV1 encodes mechanisms to alter the cellular environment in 9 favor of viral replication………………………………………………….. 1.5. HSV1 induces interferon response during lytic infection and 10 encodes several mechanisms to antagonize this antiviral defense………. 2. RNA silencing.…………………………………………………………….. 15 3. RNA silencing and viral infection in mammalian cells.…………………... 18 3.1. Mammalian viruses can be targets of siRNAs……………………….. 19 3.2. Mammalian viruses encode virusderived miRNAs and several virus 20 derived miRNAs have been discovered to target viral mRNAs for cleavage…………………………………………………………………… 3.3. RNA silencing might be a general mechanism adopted by 21 mammalian cells to overcome viral infection…………………………….. xi 4. HSV1infection and RNA silencing……………………………………… 23 4.1. HSV1 produces dsRNAs and encodes viral miRNAs during lytic 23 Infection………………………………………………………………….. 4.2. US11 encoded by HSV1 functions similarly to a known silencing 23 suppressor in mammalian cells, NS1 protein………………….. 4.3. Do hosts employ RNAi to antagonize HSV1 infection?…………… 25 CHAPTER 2………………………………………………………………………... 29

MATERIALS AND METHODS…………………………………………………... 29

CHAPTER 3………………………………………………………………………... 50

OPTIMIZATION OF A TRANSIENT SILENCING SYSTEM IN MAMMALIAN CELLS……………………………………………………………. 50

Effect of perfect and imperfect RNA hairpins on target mRNA levels……… 50 Effect of molar ratio of pmidsEGFP to pEGFPC2 on silencing…………….. 52 Effect of midsEGFP on EGFP mRNA degradation rate……………….…….. 53 CHAPTER 4………………………………………………………………………... 62

INFECTION BY HSV1 SUPPRESSSES SILENCING…………………………... 62

Effect of HSV1 infection on steadystate target mRNA levels in 62 silenced and control cells……………………………………………….……. Effect of HSV1 infection on expression of introncontaining EGFP in 63 silenced cells…………………………………………………………………. Effect of infection of silenced and control cells with HSV1 on the halflife 66 of target EGFP mRNA……………………………………………..………… Effect of mutation in viral host shutoff functions or ICP27 on steadystate 67 target levels and the halflife of target mRNA………………………………. Effect of HSV1 infection on miRNAinduced silencing……………………. 70 Effect of knockdown of Ago2 on HSV1 yield of progeny………………... 72 CHAPTER 5………………………………………………………………………... 95

ATTEMPTS TO IDENTIFY A HSV1 SILENCING SUPPRESSOR ……………. 95

Kinetics of HSV1induced silencing suppression…………………………... 95 Expression profiles of HSV1 mRNAs………………………………………. 96 Testing of candidate HSV1 genes for silencing suppression in plants..…….. 97 Effects of infection by a US11 null mutant on silencing suppression….……. 99 CHAPTER 6………………………………………………………………………... 117

DISCUSSION……………………………………………………………………… 117 Mammalian cells mount a RNAi response to inhibit HSV1 replication……. 117 xii Establishment of optimum conditions to achieve maximum silencing……… 118 HSV1 encodes one or more silencing suppressors…………………………. 119 HSV1 suppresses miRNAinduced silencing effectively…………………… 121 Silencing suppressor activity of HSV1 could be an immediateearly gene 121 product or a virion component……………………………………………….. HSV1 US11 can function as a silencing suppressor in plants………………. 122 US11 might be involved in silencing suppression by HSV1 in mammalian 122 cells…………………………………………………………………………... BIBLIOGRAPHY…………………………………………………………………... 125

xiii

LIST OF TABLES

Page Table 1 Description of HSV1 mutants and phenotypes..…………………… 40

Table 2 Sequences of oligos used for expressing RNA hairpins……………. 41

Table 3 Primers used for PCR……………………………………………….. 42

Table 4 PCR amplification…………………………………………………… 43

Table 5 Construction of plasmids……………………………………………. 44

xiv

LIST OF FIGURES

Page Figure 1.1 miRNA and siRNA pathways in mammalian cells………………... 27 Figure 2.1 Organization of EGFP expression plasmids……………………….. 46 Figure 2.2 Structure of RNA hairpin expression plasmids……………………. 48 Figure 3.1 Effects of the perfect and imperfect dsEGFP hairpins on EGFP 54 mRNA levels………………………………………………………. Figure 3.2 Effect of molar ratio of pmidsEGFP and pmidsLac Z to pEGFPC2 57 on steadystate EGFP mRNA levels………………………………. Figure 3.3 Effect of midsEGFP on the halflives of EGFP and βactin 59 mRNAs…………………………………………………………….. Figure 4.1 Effect of HSV1 infection on steady state target and nontarget 74 mRNA levels………………………………………………………. Figure 4.2 Effect of intron within the target EGFP gene on mRNA 77 accumulation in silenced and control cells………………………… Figure 4.3 Effect of HSV1 infection on accumulaton of EGFP mRNA from 79 transcripts with or without intron………………………………….. Figure 4.4 Effects of HSV1 infection on the halflives of target and non 81 target mRNAs……………………………………………………… Figure 4.5 Effects of infection by HSV1 host shutoff mutants on 85 accumulation of target and nontarget mRNAs……………………. Figure 4.6 Effects of loss of host shutoff function on the halflives of target 87 and nontarget mRNAs…………………………………………….. Figure 4.7 Silencing suppression by HSV1 using a miRNAdirected 90 luciferase reporter assay…………………………………………… Figure 4.8 Effect of knockdown of Ago2 on HSV1 yield of progeny……... 93 Figure 5.1 Kinetics of silencing suppression by HSV1………………………. 103 Figure 5.2 Expression profiles of representative immediate early, early and 105 late HSV1 genes…………………………………………………... Figure 5.3 Effects of the HSV1 DNA synthesis inhibitor, PAA, on the ability 107 of HSV1 to suppress silencing……………………………………. Figure 5.4 Silencing suppression assay by HSV1 genes in plants…………… 109 Figure 5.5 Effects of infection by HSV1 US11 null mutant, the rescuant or 112 the wildtype virus on the target EGFP and nontarget β–actin mRNA levels ………………………………………………………

xv Figure 5.6 FIG. 5.6. Effects of infection by HSV1 US11 null mutant, the 114 rescuant or the wildtype virus on the halflives of the target EGFP and β–actin RNAs………………………………………………….

xvi

LIST OF ABBREVIATIONS

Act D Actinomycin D

DMSO Dimethylsulfoxide dsEGFP Perfect EGFP RNA hairpin dsLac Z Perfect Lac Z RNA hairpin

EGFP Enhanced green fluorescence protein hr Hour(s) midsEGP Imperfect EGFP RNA hairpin midsEGP Imperfect Lac Z RNA hairpin m.o.i. Multiplicity of infection

PAA Phosphonoacetic acid

PFU Plaque forming units

xvii

CHAPTER 1

INTRODUCTION

1. Background information about herpes simplex virus type 1

1.1. HSV1 virion and genetic structures

Herpes simplex virus type 1 (HSV1) is an important human pathogen. HSV1 causes cold sores, eye disease and genital lesion in humans. Under rare circumstances,

HSV1 infection results in lifethreatening encephalitis. In addition, HSV1 can cause significant morbility and mortality among immunosuppressed individuals (Reviewed in

Roizman et al., 1996).

Research indicates that 6090% of the adult population in the world is sero positive for HSV1, and there are millions of new cases each year (Carr et al., 2001). It is estimated that the direct medical costs (physician visits and pharmacy claims) of known cases of HSV1 infection in the United States alone are as high as one billion per year— which is considered by most to be a gross underestimate of the actual cost. The direct medical costs of known cases of genital herpes are as high as $208 million in the United

States per year (Arvin, 2002; Rirn, 1995).

1 HSV1 possesses a dsDNA of 152Kbp with 68% G+C content (Boehmer and Villani, 2003). The HSV1 virion has a spherical shape with a diameter of 120 nm.

The outer layer of the virion is the envelope, which consists of altered host membrane and several unique viral glycoproteins (Roizman et al., 1996). Viral glycoproteins are embedded in the envelope and project from the surfaces as short spikes (Roizman et al.,

1974; Stannard et al., 1987). Beneath the envelope is the tegument, an asymmetrically amorphous layer. It is composed of viral enzymes and proteins (Zhou et al., 1999).

Some of the tegument components are involved in taking control of the host cells’ chemical processes and subverting them for viral production while some function to defend against the host immune responses (Coller et al., 2007;Xiong et al., 2007; Bucks et al., 2007; Read and Patterson, 2007; Donnelly et al., 2007; Morrison et al., 1998;

Vittone et al., 2005) . The tegument also contains some proteins with unknown functions.

Nucleocapsids are surrounded by the tegument. The HSV1 nucleocapsid is icosahedral, and consists of 162 capsomers, with a diameter of 100 nm ( Vittone et a.l, 2005, Preston,

1997). The inner layer is the core of the virion, which is composed of a single linear

molecule of dsDNA in the form of a torus (Bucker et al., 1968). The DNA genome is

wrapped by fibrillar spool with the ends anchored to the inner side of the shell

(Reviewed in Roizman et al., 1996).

The HSV1 genome is divided into two unique regions, termed unique long

region (U L) and unique short region (U S) (Hayward et al., 1975, Morese et al., 1978). The two unique regions are bounded by internal inverted repeats while the genome termini are

2 bounded by regions of ~ 400 bp direct repeats called the “a” sequence, which is in an

inverted orientation relative to the junction between U L and U S (Roizman et al., 2001).

The U L and U S regions of the genome have the capacity to invert with respect to

each other at a high frequency, generating four equimolar isomers of viral DNA

(Roizman et al., 2001). In the genome, there are three origins, with two in U S and one in

UL (Mocarski, ES et al., 1982, Vlazy et al., 1982). Experiments have indicated that the three origins have an equal efficiency to initiate viral DNA replication in tissue culture

(Knopf et al., 1986, Quinn et al., 1985). In infected cells, the HSV1 genome has been discovered to exist in at least three different forms: linear, circular and concatemeric

(Deshmane et al., 1995; Garber et al., 1993; Poffenberger et al., 1985). In virions, the genome is in linear form. However, the two ends of the genome join together resulting in an active endless form for DNA replication within several hours after infection. Viral

DNA replication has been thought to be initiated by an origindependent mechanism at the early stages of infection and switches to a rolling cycle mode at the late stages of infection (Strang and Stow, 2005). Rolling cycle replication of the genome results in formation of headtotail concatemers, which are cleaved into a monomeric unit and packaged into the virions for the second round of infection (Deiss et al., 1986).

The HSV1 genes, like those of their eukaryotic hosts, are not arranged in operons, and in most cases have individual promoters. However, unlike eukaryotic genes, very few HSV1 genes contain introns (Roizman et al., 2001). The HSV1 genome encodes at least 85 different products (Sacks et al., 1985). Less than half of the encoded proteins are essential for the virus to grow in tissue culture (Schranz et al., 1989; Weber

3 et al., 1987). However, the other “dispensable” genes function to modulate the cellular environment for virus production, promote celltocell spread or defend against the host immune system, and many have been proven to be required for in vivo infection (Chou et al., 1990; SubakSharpe and Dargan, 1998).

1.2. HSV1 life cycles

HSV1 alternates between two life cycles in vivo, lytic infection and latency

(Decman et al., 2005; Preston, 2000). During lytic infection, the virus replicates and infects adjacent cells. In humans, lytic infection usually lasts for 510 days depending on the site of infection. Subsequently, the virus is cleared from the infected tissue due to the host’s adaptive immune defenses (Kaufman et al., 1998). During lytic infection, a fraction of viruses gains access to neuronal termini, which innervate the epithelial cells at the site of lytic replication. The virus is transported by retrograde flow to the cell bodies of neurons in a sensory ganglion such as the trigeminal ganglion (TG) or dorsal root ganglion (DRG). In sensory ganglia, the virus replicates briefly and subsequently establishes latency, a quiescent state (Metcalf et al., 1987; Pereira FA, 1996). Virus replication is not necessary for latency to be established. However, brief replication of the virus increases the pool for establishment of latency. A variety of external stimuli or physiological stresses have been shown to activate the latent viruses leading to productive replication. Activated viruses from latency are transported by anterograde transport to the initially infected tissues or adjacent organs, where new rounds of lytic infection can occur and cause diseases in humans (Carr et al., 2001).

4 1.3. Productive cycle of HSV1

The lytic replication cycle can be divided into several steps: attachment, entry, uncoating, macromolecular synthesis, assembly of viral nucleocapsids, maturation of viruses, and egress from the infected cells (Roizman et al., 2001).

Entry of HSV1 into cells and injection of the genome DNA into the nucleus of infected cells

Entry of the virus into cells is achieved through interaction of viral glycoprotein(s) with cellular receptors (Roizman et al., 1996; Roizman et al., 2001). The interaction directly results in fusion of the virion envelope with the cell membrane. At least five HSV1 glycoproteins, which are gB, gC,gD, gH, gL respectively, have been demonstrated to be involved in viral entry into cells (Reske et al., 2007; Aguilar et al.,

2007). During attachment, gC interacts with heparan sulfate proteoglycan on the cell membrane surface (kwon et al., 2006). This interaction is labile until other glycoproteins, such as gB and gD, begin to participate in the entry process. gB also harbors a site for interaction with other glycosoaminoglycans (Bender et al., 2005) while gD provides a stable attachment to cellular receptors such as the herpesvirus entry mediator (HVEM)

(Tsvitov et al., 2007). Binding to cellular receptors causes a conformational change of gD, which facilitates the interaction of gD with the gH/gL heterodimer (Galdiero et al.,

2004; Cairns et al., 2005). Fusion domains of the gH/gL complex and gB allows for the virus to penetrate cells in a pHindependent manner (Roizman et al., 1996). What cellular receptors, other than HVEM, are required for HSV1 entry is unknown. However, it is widely accepted that HSV1 might utilize different receptors on different cell types,

5 which is very common among all large, complex DNA viruses. Alternatively, the virus could use common cell surface molecules rather than specific proteins as receptors for attachment (Reske et al., 2007). Endocytosis is not required for the entry of HSV1 into the host. However, it may serve as an alternative route for penetration (Nicola et al.,

2003).

Fusion of the viral glycoproteins with the cell membrane results into release of the capsid into the cytoplasm. Next, the capsid is transported along microtubules to the nucleus (Sodeik et al., 1997), where it binds to the nuclear pore complexes (NPCs) and

the viral genome is injected into the nucleus via the nuclear pore (Ojala et al, 2000;

Batterson et al., 1983; Tognon et al., 1981). The viral genome is then circularized in the

nucleus (Strang et al., 2005). Release of the genome into the nucleus is accompanied by a

tegument protein, VP16, which functions to enhance expression of immediate early genes

through interactions with cellular factors (Mcknight et al., 1987).

Viral gene expression

In the nucleus, the genome is expressed in a highly coordinated manner (Honess

et al., 1974, Roizman and Knipe, 2001). Viral proteins are classified into three distinct

categories based on their expression orderimmediate early genes, early genes and late

genes.

Immediate early genes are transcribed by the cellular machinery without prior

viral gene expression. A tegument protein, VP 16 (or αTIF), also plays an important role in this process (Roizman and Knipe, 2001). Oct1, one member of Oct gene family, is the key cellular protein for initiating expression of HSV1 immediate early genes. This

6 protein contains a DNAbinding structure known as the POU domain, which is shared by the Oct gene family. Oct1 binds to an octamer sequence, TAATGARAT (R is a purine nucleotide). HSV1 immediate early genes have at least one copy of such an octamer sequence in their promoters (Herr et al., 1995). The binding of Oct1 to TAATGARAT in the immediate early gene promoters results in recruitment of HSV1 VP16 and a cellular protein, HCF (host cellular factor) to form a complex (O’Hare, 1993). The formation of this threecomponent complex brings the Cterminal domain of VP16, a strong transcriptionactivating element, into proximity with the preinitiation complex to initiate transcription. Other members of the Oct protein family can bind to

TAATGARAT in the promoters of HSV1 immediate early genes. However, their binding prevents expression of viral genes (Turner et al., 1996). A main function of HSV

1 immediate early genes is to transactivate early and/or late genes.

Among early gene products are the enzymes and accessory proteins for viral DNA synthesis. Early gene expression requires the prior expression of immediate early genes.

However, accumulation of early gene products leads to reduced expression of IE genes and initiation of genome replication (Roizman and Knipe, 2001). Proteins encoded by late genes include tegument and capsid proteins, and glycoproteins. They are actually divided into two subgroups, late (γ 1) and true late (γ 2) genes depending on whether their synthesis is strictly dependent upon viral DNA replication. γ 1 genes are expressed prior to

DNA replication. However, viral DNA replication enhances their expression. γ 2 gene products are not expressed in the absence of viral DNA replication (Roizman et al.,

1996).

7 HSV1 DNA replication and cleavage/packaging

The linear viral DNA is circularized once the viral DNA is released into the nucleus of the infected cells. Viral DNA replication begins shortly after early genes are expressed and is detectable as early as 3 hours and continues up to fifteen hours postinfection (Roizman et al., 1963, Roizman et al., 1964; Igarashi et al., 1993). It has been hypothesized that at an early stage of viral infection, DNA synthesis initiates at origins with the circularized genome as template and proceeds in a theta mechanism

(Garber et al., 1993). At a late stage of infection, DNA synthesis switches to a rolling circle mode to generate headtotail concatemers. Finally, the headtotail concatemers are cleaved into unitlength genome and packaged into performed caspids (Boeher and

Lehman, 1997).

Viral envelopment and egress

Once genome are assembled into , the viral particles attach to patches of modified inner lamellae of the nuclear membranes and migrate to the inner nuclear membrane, where capsids become enveloped as they bud into the perinuclear space (Mettenleiter, 2002). Then, the viral particles fuse with the outer nuclear membrane, releasing viral nucleocapsids into the cytoplasm, where the naked nucleocapsids undergo tegumentation in the cytoplasmic compartment. The tegumented viral particles obtain reenvelopment by moving into the Golgi apparatus, and Golgi apparatus guides the viral particles for egress (Skepper et al., 2001; Schwartz and

Roizman, 2001).

8 1.4. HSV1 encodes mechanisms to alter the cellular environment in favor of viral

replication

Host protein synthesis is rapidly shut off during HSV1 lytic infection, thereby

allowing viral mRNAs to dominate the host translation machinery (Roizman and Pellett,

2001). The host protein synthesis shutoff is attributed to the complementary functions of

two viral genes, ICP27 and virion host shutoff protein U L41 (vhs).

ICP27 is an immediate early multifunctional protein (Zhou et al., 2002; McCarthy

et al., 1989; Smith et al., 2005). HSV1 ICP27 binds RNAs and shuttles between the

nucleus and the cytoplasm, allowing it to function in both places. ICP27 is a trans

activator, essential for expression for a subset of HSV1 early and late gene expression

(McCarthy et al., 1989). ICP27 interacts with a key component of the cellular

spliceosome, SAP145 (Spliceosomeassociated protein). This association prevents the

formation of an active spliceosome, thereby reducing or abrogating splicing of host pre

mRNAs (Bryant et al., 2001). Unspliced transcripts are sequestered in the nucleus, which promotes turnover. ICP27 also interacts with the cellular mRNA transport factors

Aly/Ref and/or TAP1, thereby facilitating the transport of the predominant intronless

viral mRNAs from the nucleus into the cytoplasm (Chen et al., 2005; Chen et al., 2002).

Recently, it was shown that ICP27 is directly involved in promoting translation by binding components of the translation initiation machinery to promote viral gene

translation (Perkins et al., 2003; FontaineRodriguez et al., 2004).

The virion host shutoff protein (vhs) is a HSV1 late gene. However, because it is

a virion component, it can function early after infection (Kwong et al., 1987; Pak et al.,

9 1995; Smibert et al., 1992; Fenwick M et al. 1988, Fenwick et al., 1978). Virion host shutoff protein is a nuclease (Feng et al., 2002). It has been shown to interact with the cellular translation initiation factor eIF4H and the related factor eIF4B (Feng et al., 2001;

Feng et al., 2005). It has been proposed that this interaction is essential for vhs to function. Viral host shutoff protein first endonucleolytically cleaves sequences at the 5’ ends of mRNAs, followed by a 5’ to 3’ exonucleolytic degradation (PerezParada et al.,

2004). It has been reported that vhs degrades cellular housekeeping, heatshock and viral mRNAs without selectivity (Pak et al., 1995; Smibert et al., 1992; Schek, 1985, Strom et al., 1987). However, some reports suggest that vhs may not degrade the cellular rRNAs

(Schek, 1985, Strom et al., 1987). The combined activities of ICP27 and vhs lead to an efficient shutoff of host protein synthesis, which exclusively promotes viral gene expression.

1.5. HSV1 induces interferon response during lytic infection and encodes several

mechanisms to antagonize this antiviral defense

Upon viral infection, mammalian hosts initiate an interferon response in response to HSV1 replication. In turn, the virus adopts several mechanisms to antagonize this host’s restriction.

Interferon response

Interferons are a large family of cytokines first discovered for their ability to interfere with viral replication (Goodbourn et al., 2000). Interferons (IFNs) are divided into two major classes, type 1 and type II, based on their functions, (Joklik, 1990). Only type I interferons, which consists of IFN α and IFN β, has been demonstrated to be

10 involved in innate antiviral defense (Goodbourn et al., 2000). Once mammalian cells are exposed to viruses, virusencoded ligands interact with tolllikereceptors on cell surface

(Rasmussen et al., 2007; KurtJones et al., 2005). Interactions of viral ligands with the receptors activate the receptors. Activated receptors initiate a signaling cascade that culminates in upregulation of type I IFN α and IFN β expression. IFN β expression can

also be stimulated by dsRNAs generated during viral replication of RNA and DNA

viruses. Once IFN α and IFN β are produced, they are secreted from the infected cells.

Secreted interferons bind to their cognate receptors on the plasma membrane of the

adjacent cells. The interaction between IFNs and the receptor activates JAK and STAT by initiating phosphorylation events. Activated JAK and STAT translocate to the nucleus,

where they bind to the interferonstimulating response elements (ISRE) within the promoter of interferonstimulating genes (ISGs), thereby promoting the expression of

ISGs (deVeer et al., 2001; Le Page et al., 2000; Stark et al., 1998). Among all ISGs, the

most important are dsRNAdependent protein kinase R (PKR) (Gale et al., 1998), 25A

synthetase (OAS) and RNAase L (Silverman, 1997a; Silverman et al., 1997b). PKR,

which is activated by dsRNAs or PACT (Patel et al., 1998; Peter et al., 2001; Li et al.,

2006), phosphorylates the eukaryotic initiation factor eIF2 α of translation machinery

resulting in cessation of protein synthesis, thereby inhibiting viral replication. OAS

catalyzes the formation of 25A (Silverman, 1997a), therefore activating RNase L.

Activated RNase L cleaves cellular and viral mRNAs, thereby limiting viral replication

(Silverman et al., 1997b). Among all the three key antiviral components in the interferon

response, PKR plays the most critical role in viral defenses since inactivation of the

11 translation machinery by PKR will ultimately lead to apoptosis, a process that can clear viruses and completely prevent viruses from further infection (Mossman, 2002).

PKR also plays an important role in limiting viral replication in virusinfected cells. dsRNAs, which are generated during viral replication for all viruses including RNA and DNA viruses (Schneider and Mohr, 2003), activate PKR. Activated PKR would deplete active eIF2 α, thus inhibiting cellular and synthesis, and blocking

viral replication.

Structurally, PKR is composed of two Nterminal dsRNA binding motifs

(dsRBMs), one central dimerization motif and one Cterminal catalytic domain

(Puthenveetil et al., 2006). Under a noninductive condition, the N and Cterminal

regions interact with each other resulting in an inactive conformation with ATP binding

site embedded within the molecule (Li et al., 2006). PKR can be activated by dsRNAs or

ssRNAs with extensive secondary structures or PACT (Patel et al., 1998; Peters et al.,

2001; Mohr, 2004). dsRNAs bind to PKR through interaction between dsRNAs and the

two dsRBMs of PKR while activation of PKR by PACT is achieved by interaction of

PACT domain III with the central dimerization region of PKR (Peters et al., 2001).

Activation of PKR by PACT does not require PACT domains I and II. However, the

domains I and II of PACT enhance the interaction between the domain III of PACT and

the central region of PKR. Binding of dsRNAs or PACT to PKR disrupts the interaction between the N and Cterminals of PKR. Disruption of N and Cterminal interaction of

PKR facilitates exposure of the ATP binding site and the formation of the PKR

homodimer for activation (Li et al., 2006a and 2006b). TRBP (The TAR RNAbinding

12 Protein) is a dsRNA binding protein and a component of RISC in RNAi pathway (St

Johnston et al., 1992; Kok et al., 2007). TRBP also enhances HIV replication via association with TAR (Dorin et al., 2003). TRBP and PACT belong to the same family of

RNA binding proteins, and both possess three dsRBMs (dsRNA binding motifs).

Moreover, the dsRBMs of TRBP shares a high degree of homology with those of PACT

(Gupta et al., 2003; St Johnston et al., 1992). However, TRBP and PACT have opposite effects on PKR activity. TRBP inhibits PKR activity through interaction between the first two dsRBDs of TRBP and the Nterminal dsRBDs of PKR (Cosentino et al., 1995, Daher et al., 2001; Gupta et al., 2003).

Most pathogenic mammalian viruses encode antagonists to block the host cells’ interferon response (Gale et al., 1998). Interestingly, almost all antagonists encoded by viruses act on PKR and target it for inactivation (Chang et al., 1992, Tan et al., 1998; Roy et al., 1990, Liu et al., 1995). This also indirectly reflects the importance of PKR in anti viral defense. PKR antagonists encoded by viruses include noncoded RNAs and various proteins with no similarity in sequence and structures (Mathews, 1990; Clemens et al.,

1994; Cosentino et al., 1995). They function to inhibit PKR by diverse mechanisms ranging from modulating PKR expression to interfering with binding of substrates to

PKR. For example, VA RNAs, encoded by Adenovirus negatively regulate PKR activity by preventing binding of activator dsRNAs to PKR (Mathews, 1990). virus E3L and reovirus ơ prevent activation of PKR by sequestering dsRNAs (Chang et al., 1992).

13 HSV1 encodes different proteins to block the host interferon response at each stage of its replication during lytic infection

Mammalian cells mount an innate interferon response to counter viral infection

(Goodbourn et al., 2000; Joklik, 1990). However, successful mammalian viruses encode mechanisms to overcome the host interferon response (Tan et al., 1998). Research has demonstrated that several proteins encoded by HSV1 cocoordinately block the host interferon response throughout HSV1 lytic infection (Peters et al., 2002; Mossman et al.,

2000; Mulvey et al., 2004). The host interferon response is initiated upon viral infection, but this response can immediately be overcome due to the function of an immediate early product ICP0 (Eidson et al., 2002). HSV1 ICP0 is a multifunctional protein, produced early after HSV1 infection, which transactivates viral gene expression, and possesses an ubiquitin ligase activity (Boutell et al., 2002; Van Sant et al., 2001). An ICP0 mutant is very sensitive to IFN treatment (Mossman et al., 2000). IFNs themselves are not harmful to viruses. The key for IFN response is the IFN initiated downstream cascade (Gale and katze, 1998). Expression of ISGs is stimulated by promyelocytic leukemia (PML) bodies.

ICP0 functions as an ubiquitin ligase to dismantle PML body structure, thereby preventing the production of IRGs and modulating the cellular environment for viral replication (Boutell et al., 2002; Van Sant et al., 2001).

At the early late stage of HSV1 infection, one γ1 gene (early late genes) product,

γ134.5, blocks the host shutoff of protein synthesis (Mulvey et al., 2004). γ134.5 binds to

the catalytic subunit of protein phosphatase 1α (PP1α) and forms γ134.5PP1α complex,

which constitutes an active PP1α (He et al., 1997). Activated PP1α dephosphorylates the

14 inactivated eIF2α due to phosphorylation by PKR and prevents the block to cellular protein synthesis. At a very late stage of HSV1 infection, US11 is synthesized and works

with γ134.5 to block the shutoff of protein translation. Mutation of US11 results in a 10 fold reduction in the production of viral late proteins in some cells, thereby reducing the formation of viral progeny (Mulvey et al., 2004). US11 binds to PKR and prevents the dimerization of PKR, which is required for its activation. Thus, US11 preserves viral translation rates (Peters et al., 2002).

2. RNA silencing

RNA interfence (RNAi) is a phenomenon in which small RNAs (2125 nt) inhibit gene expression. RNAi was first discovered in transgenic plants as cosuppression of the exogenous transgenes and its homologous endogenous counterparts (Napoli et al., 1990;

Matzke et al., 1989). Subsequent study in C. elegans indicated that the effector molecules

in gene silencing were 2125 nt small RNAs (Guo et al., 1995; Tabara et al., 1998). Since

then, additional lines of evidence have demonstrated that RNAi is a highly conserved

mechanism to regulate gene expression in nearly allliving organisms (Li and Ding,

2006). For the purpose of this dissertation, discussion will be limited to two categories of

interfering RNAs, small interfering RNA (siRNA) and microRNA (miRNA).

siRNAs and miRNAs are 2125 nt long. However, they are different from each

other in their biogenesis and functions. miRNAs are generated from singlestranded RNA

hairpin precursors while siRNAs are derived from dsRNAs (Baulcomble, 2004). miRNAs

can initiate gene silencing either by inhibiting translation or by inducing cleavage of their

targets depending on the level of their sequence complementarity to their targets

15 (Hutvagner and Zamore, 20002; Llave et al., 2000; Rhoades et al., 2002). Generally speaking, miRNAs induce translation inhibition in mammalian cells because of low sequence complementarities to their targets while miRNAs in plants initiate cleavage of their targets due to high sequence complementarities to their targets (Llave et al., 2000;

Rhoades et al., 2002). siRNAs have perfect homologies to their targets (Fig. 1.1). Thus, siRNAs usually initiate gene silencing through cleavage of their target mRNAs. In some species, siRNAs induce methylation of their cognate DNA and/or lead to formation of heterochromatin (Li and Ding, 2006; Wang et al., 2005; Vaucheret, 2007).

Nevertheless, siRNAs and miRNAs share many features in common. In addition to their similar size, siRNAs and miRNAs are the end products derived from cleavage of their precursor RNAs by a RNAase IIIlike enzyme, Dicer. They have a similar chemical structure. That is they are small RNAs with 2 nt 3’ overhangs, 5’phosphate and 3’ hydroxyl groups. Both function through a RNAinduced silencing complex (RISC)

(JonesRhoades et al., 2006; Tang, 2005). Moreover, siRNAs and miRNAs are functionally interchangeable. siRNAs can inhibit gene expression by repressing translation when imperfect target sequences are introduced into the 3’ UTRs of their target mRNAs. Likewise, miRNAs can function like siRNAs to cleave mRNAs when perfectly matched target sequences are engineered into the open reading frame (ORF) of

the target mRNAs (Doench et al., 2003; Zeng et al., 2003).

There are some characteristic features in RNAi pathways that differ among

species. A good example of these differences is that siRNAs and miRNAs in plants are

methylated by a RNAspecific methyl transferase, HEN1. HEN1catalyzed methylation

16 of siRNAs and miRNAs are essential for robust gene silencing in plants in that methylation of siRNAs and miRNAs prevents siRNAs from oligouridylation, which promotes their instability (Boutet et al., 2003; Li et al., 2005;Yu et al., 2005; Park et al.,

2002; Xie et al., 2003). RNA dependent RNA polymerases (RDRP) are required for robust RNAi pathways in plants and C. elegans in that primary siRNAs can initiate mRNA cleavage, but are not sufficient to induce gene silencing. Secondary siRNAs, which are derived from dsRNAs amplified with the cleaved mRNA fragments as templates by RDRP, initiate additional rounds of cleavage on the targets, thereby ensuring effective silencing of target genes (Xie et al., 2004; Herr et al., 2005; Dalmay et al., 2000; Mourrain et al., 2000; Sijen et al., 2001; Smardon et al., 2000; Vaistij et al.,

2002). However, no RDRPs have been identified to date in mammals.

Another unique characteristic of RNAi in C.elegans and plants is that signals of

RNAi can be transferred to adjacent cells or can be spread throughout the whole

organism when exogenous genes are introduced into plants and worms or plants and

worms are infected by viruses (Voinnet, 2005; Hamilton et al., 2002; Himber et al., 2003; palauqui et al., 1997). It has been demonstrated that spread of RNAi signals into adjacent

cells or establishment of systemic silencing in plants and worms also needs the function

of RDRPs (Dunoyer et al., 2005; Himber et al., 2003; Schwach et al., 2005).

miRNAs are processed from primary miRNA precursors solely by Dicers in plants while miRNAs in animals are generated by a twostep process (Fig. 1.1). Primary

miRNAs are first cleaved into 6070 nt premiRNAs by a processing center known as

DroshaParsa complex. Subsequently, premiRNAs are transported in the cytoplasm into

17 by exportin5 (Brownawell and Macara, 2002). In the cytoplasm, premiRNAs are processed into mature miRNAs by Dicer (Park et al., 2002; Reinhart et al; Papp et al.,

2003; Kurihara and Watanable, 2004, Hiraguri et al., 2005).

Nevertheless, siRNAs and miRNAs, collectively function to control gene expression (Agrawal et al., 2003; Li and Ding, 2006; Wang et al., 2005; Vaucheret,

2007). Research has demonstrated that interfering RNAs are the key components in regulation of development among plants, insects, worms and animals (Baulcombe, 2004;

Brodersen and Voinnet, 2006; Ding et al., 2004; Wienholds and Plasterk, 2005).

Misregulation of miRNA expression results in developmental defects in animals and plants (Vaucheret et al., 2004; Lee et al., 1993; Brennecke et al., 2003; Lecellier et al.,

2005, Wienholds and Plasterk, 2005). Inappropriate timing of miRNA expression or failure to timely express certain miRNAs leads to diseases including cancers in humans

(Calin et al., 2004; Michael et al., 2003; Takamizawa et al., 2004). Work has also shown that RNAi plays a critical role in viral defense in plants, and might be involved in defenses against viral infection in animals (Anandalakshmi et al., 1998; Bayne et al.,

2005; Bisaro, 2006; Lu and Cullen, 2004, Vanitharani et al., 2005).

3. RNA silencing and viral infection in mammalian cells

The role of RNAi in antiviral defenses in plants and in insects has been well

documented (Voinnet et al., 2003; Reed et al., 2003; Li et al., 2004). Almost all pathogenic viruses in plants encode silencing suppressors, which are essential for

effective viral replication (Li and Ding, 2006; Silhavy et al., 2002; Vargason et al., 2003).

A wellknown innate antiviral defense of mammalian cells is the interferon response

18 (Goodbourn et al., 2000; Honda et al., 2005). To date no naturally occurring siRNA pathway has been identified in mammalian cells. These features have led to an extensive debate about whether mammalian cells use a RNAi response to overcome viral infection.

However, an increasing number of lines of evidence suggest mammalian cells can employ RNAi as an antivial defense strategy.

3.1. Mammalian viruses can be targets of siRNAs

Introduction of viralspecific siRNAs prior to infection can greatly restrict viral replication in mammalian cells. Furthermore, siRNAmediated restriction of viral replication has been observed in cells infected with major classes of viruses including

DNA, RNA and (Omoto et al., 2004; Qin et al., 2003; Bhuyan et al., 2004;

McCaffrey et al., 2003; Ge et al., 2003; Wilson et al., 2005; Dave et al., 2006). siRNAs that target HIV Tat or Rev reduced HIV replication 5fold (Coburn and Cullen, 2002).

Introduction of siRNAs specific to NS5b resulted in nondetectable level of NS5 protein and a 16fold reduction in accumulation of HCV replicon RNAs (Wilson et al., 2005;

Randal et al., 2003). Expression of siRNAs directed against HBV led to a 13fold reduction in HBV RNAs and the genomic DNA was undetectable (McCaffrey et al.,

2003). Introduction of siRNAs that target vaccinia virus E3L decreased the yield of progeny 50fold (Dave et al., 2006). Application of siRNA specific for HSV1 gD results in smaller plaque size, similar to that observed with gD null mutant (Bhuyan et al., 2004). siRNAs raised against nucleocasid protein (NP) or RNA transcriptase (PA) of influenza virus abrogate not only accumulation of target mRNAs but virion RNA as well (Ge et al.,

2003).

19 3.2. Mammalian viruses encode virusderived miRNAs and several virusderived miRNAs have been discovered to target viral mRNAs for cleavage

Virusderived miRNAs have been identified in several mammalian viruses. For example, five Epstein Barr virus (EBV) viral miRNAs have been reported (Pfeffer et al.,

2005). Kaposi sarcomaassociated herpesvirus (KSHV), human cytomegalovirus, and herpesvirus 8 have been identified to encode eleven, nine, and nine miRNAs, respectively

(Pfeffer et al., 2004; Cai et al., 2005; Samols et al., 2005). With time, it is expected that more and more virusencoded miRNAs will be discovered. Among all virusderived miRNAs, several have been demonstrated to be used by viruses in favor of viral replication (Gupta et al., 2006; Sullivan et al., 2005). SV40 encodes miRNAs during its infection, which possesses perfect complementarity to large T antigen. Expression of these miRNAs reduces the expression of T antigen but doesn’t affect the yield of infectious virus relative to that generated by a mutant with mismatches introduced in the predicted target region. However, wildtype SV40 infected cells are less sensitive to lysis

induced by cytotoxic T cells, and the wildtype virus triggers less cytokine production in

cells compared with the mutant (Sullivan et al., 2005). These results demonstrate that

SV40 might use miRNAs encoded from its genome to inhibit the expression of antigens,

in order to modulate the host CTL response favorable for the virus. Depletion of

svaRNAs, which are siRNAs generated from VA RNAs during adenovirus replication, by

antisense RNAs affects adenovirus production. This suggests that adenovirus svaRNAs play an important role in viral replication. These results also demonstrate that adenovirus

might use virus–encoded siRNAs to modulate either expression of viral genes or

20 expression of cellular genes in favor of viral replication (Aparicio et al., 2006). Latency associated transcripts (LATs) are the only gene expressed to a detectable level during

HSV1 latency. Research has discovered that HSV1 miRNAs encoded from LAT gene can prevent host cells from undergoing apoptosis by inhibiting the expression of pre apoptotic genes, TGFβ and SMAD3 (Gupta et al., 2006). However, several virus encoded miRNAs have been demonstrated to function as inducers for cleavage of their cognate viral mRNAs, thereby resulting in inhibition of viral replication. vsiRNA#1 , vsiRNA# 2 and –derived miRNAs, which are encoded from HIV genome and reach detectable levels during HIV infection, target the HIV genome and induce cleavage of the mRNA. Thus, Env expression is repressed and HIV replication is inhibited

(Bennasser et al., 2005). Virusencoded miRNAs are also produced from EBV during latency and lytic infection. Among five EBVencoded miRNAs, miRBART2 has a perfect a match with 3’ UTR of the viral DNA polymerase (), and is abundantly expressed during lytic infection. Cotransfection of a plasmid expressing miRBART2 with a plasmid expressing EBV pol led to the cleavage of the pol mRNA. These results suggest that EBVencoded miRBART2 could inhibit viral replication by silencing pol during lytic infecton (Pfeffer et al., 2004).

3.3. RNA silencing might be a general mechanism adopted by mammalian cells to overcome viral infection

Whether mammalian cells mount an RNAi response to viral infection is very controversial. However, an increasing number of lines of evidence indicate that mammalian cells might use RNA silencing as a general mechanism to defend viral

21 infection. First of all, it has been known that mammalian cells encode hundreds of miRNAs and several viruses have also been discovered to encode viral miRNAs

(Wienholds et al., 2005; Pfeffer et al., 2005; Walter et al., 2006; Cai et al., 2006; Cai et al., 2005; Cui et al., 2006). The functions of most cellular and virusencoded miRNAs are unknown (Wienholds et al., 2005). However, the possibility can’t be ruled out that mammalian cells might employ cellular or viralencoded miRNAs to attack viruses.

Moreover, recent research has demonstrated that mammalian cells can use either cellular miRNAs or virusencoded siRNAs to target PFV1, EBV, SV40 and HIV respectively

(Lecellier et al., 2005; Pfeffer et al., 2004; Omoto et al., 2004; Bennasser et al., 2005).

Thirdly, several viruses have been identified to encode silencing suppressors (Lu and

Cullen, 2004; Andersson et al., 2005; Sullivan et al., 2005; Lecellier et al., 2005;

Bennasser et al., 2005; Bennasser et al., 2006; Haasnoot et al., 2007; Triboulet et al.,

2007). For example, adenovirus encoded VA RNAs can block silencing pathway by preventing transport of premiRNAs into the cytoplasm and inhibiting Dicer activity (Lu

and Cullen, 2004; Andersson et al., 2005). PFV encodes a silencing suppressor, Tas, to

counter the restriction induced by miR32 by an unknown mechanism. When cells are

infected by HIV, the host uses virusencoded miRNAs to silence viral genes. In response

to the hostinduced gene silencing, HIV encodes Tat protein to interfere with Dicer

activity and prevent the production of mature miRNAs. Deletion or mutation resulting in

TAT or Tas compromised activity impacts PFV and HIV replication greatly (Bennasser

et al., 2005; Lecellier et al., 2005).

22 4. HSV1infection and RNA silencing

4.1. HSV1 produces dsRNAs and encodes viral miRNAs during lytic infection

HSV1 produces various dsRNAs by symmetric transcription of both strands of viral DNA during lytic infection (Jacquemont and Roizman, 1975a; Jacquemont and

Roizman, 1975b; Kozak and Roizman, 1975). dsRNAs can elicit a host IFN response.

However, HSV1 encodes multiple functions to block or abrogate the host IFN response. dsRNAs also can activate the host RNAi pathway (Li and Ding, 2006), although there is no evidence that dsRNAs generated during HSV1 lytic infection are involved in initiation of RNAi pathway.

Computational screening of the HSV1 genome suggests that it can encode 13 miRNA precursors and 24 miRNAs. Among the predicted miRNA precursors and miRNAs, several premiRNAs and one mature miRNA have been experimentally identified during lytic infection (Cui et al., 2006) even though their functions need to be elucidated.

4.2. US11 encoded by HSV1 functions similarly to a known silencing suppressor in mammalian cells, influenza NS1 protein

US11 is a RNA binding protein encoded by HSV1. US11 is a γ2 gene, expressed

very late during HSV1 lytic infection (Roller and Roizman, 1992). However, US11 can

function early during HSV1 infection since it is incorporated into virions (Roller and

Roizman, 1992). US11 is not essential for the virus to grow in tissue culture and in

animals (Roizman and Knipe, 2001). US11 consists of two major domains: the N

terminal transactivation motif and Cterminal dsRNAbinding domain (Maclean et al.,

23 1987; Roller et al., 1996). The Cterminal domain, which is comprised of tandem RXP repeats, is involved in multiple functions of US11 (Peters et al., 2002; Roller et al., 1996;

Bryant et al., 2005; Diefenbach et al., 2002; Giraud et al., 2004; Koshizuka et al., 2001).

However, US11 is a multifunctional protein. For example, US11 can bind to single and double stranded RNAs. US11 can bind to singlestranded RNAs shorter than 46 nt with high affinity (Bryant et al., 2005). It has been hypothesized that short RNA binding by

US11 is important for its regulation of viral gene expression (Atrill et al., 2002; Roller and Roizman, 1991). US11 can also bind to longer than 39 nt dsRNAs without sequence preference. However, binding of US11 to dsRNAs is less efficient compared with single stranded RNA (Khoo et al., 2002). Another function of US11 is that US11 binds to DNA via its Nterminal transactivation motif, and promotes gene expression (Palu et al., 2001;

SchaererUthurralt et al., 1998). US11 also has been demonstrated to play an important role in blocking activation of PKR (Peters et al., 2002), and preventing HIPK2induced cell growth arrest (Giraud et al., 2004). In addition, US11 promotes anterograde transport of unenveloped HSV1 nucleocapsids to axons (Diefenbach et al., 2002).

HSV1 US11 doesn’t share homology in sequence with influenza NS1 (Chien et al., 1997; Chien et al., 2004; Wang et al., 1999; Liu et al., 1997; Qian et al., 1995;

Poppers et al., 2000; Roller et al., 1996). However, US11 shares extensive similarities in functions with NS1. NS1 is a nonstructural protein encoded by influenza virus. It is essential for efficient viral growth and antagonizes host interferon response (Dauber et al., 2004). Also, it has been demonstrated to function as a silencing suppressor in plants, insects, worms and mammalian cells (Bucher et al., 2004; Li et al., 2004; Haasnoot et al.,

24 2007). US11 and NS1 are both dsRNAbinding proteins (Chien et al., 1997; Roller et al.,

1996). They both can bind to PKR and block activation of PKR by PACT or dsRNAs

(Khoo et al., 2002; Li et al., 2006; Peters et al., 2002). Moreover, US11 and NS1 both block the activation of PKR by PACT without disturbing the interaction between PACT

and PKR. Also, they both directly interact with PACT (Peters et al., 2002; Li et al.,

2006), one known component of holoRISC of RNA silencing pathways in mammalian

cells (Lee et al., 2006; Kok et al., 2007). Based on similarities between NS1 and US11, it

is reasonable to predict that HSV1 US11 might be a silencing suppressor encoded by

HSV1 and function in suppressing silencing by a similar mechanism to NS1.

4.3. Do hosts employ RNAi to antagonize HSV1 infection?

Whether HSV1 infection initiates the host RNAi response and whether HSV1

encodes silencing suppressor(s) are unknown. HSV1 encodes miRNAs and generates

dsRNAs during lytic infection. Also, HSV1 encodes a protein with functions similar to a

known silencing suppressor in mammalian cells. Therefore, we hypothesized that HSV1

elicits the host RNAi pathway during its lytic infection, and in turn HSV1 encodes

silencing suppressors to antagonize the host RNAi response for its full replication. In

addition, we further hypothesized that US11 is a silencing suppressor encoded by HSV1.

To test our hypothesis, a transient silencing system was generated and optimized, and the

effect of HSV1 on silencing was analyzed. Also, mutant assays and experiments with

wellcharacterized silencing suppressor assay system in plants were conducted to

determine the possible function of US11 in suppressing silencing. To address whether

HSV1 is the target of RNAi during lytic infection, experiments was conducted to

25 analyze the effect of knockdown of Argonaute2, the slicer of RNAinducedsilencing complex (RISC) by siRNAs on the yield of viral progeny.

26

FIG. 1.1. miRNA and siRNA pathways in mammalian cells. Primary transcripts of miRNAs are first cleaved into 6070 nt premiRNAs by a processing center known as DroshaParsa complex in the nucleus. In the cytoplasm, premiRNAs are processed into mature miRNAs by Dicer. Exogenously introduced RNA duplexes are cleaved into 2125 nt RNA duplexes by Dicer. The guide strand in the RNA duplexes is incorporated into RISC, and direct RISC to target its cognate mRNA for cleavage. A: miRNA pathway. B: siRNA pathway.

27

miRNA siRNA

~70 nt Primary transcript Dicer Dicer Drosha (?) 21-25 nt 21-25 nt Pre-miRNA-like RISC (RNP) hairpin RISC (RNP)

Nucleus Cytoplasm

Translation repression (?) mRNA cleavage mRNA cleavage

A: miRNA pathway B: siRNA pathway

FIG. 1.1. miRNA and siRNA pathways in mammalian cells

28

CHAPTER 2

MATERIALS AND METHODS

Cells and viruses . Baby hamster kidney (BHK), african green monkey kidney

(Vero), and HEp2 cells were maintained in Dulbecco’s modified Eagle’s essential

medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS, Atlanta Biologics,

Atlanta, GA), 100 units/ml penicillin, 100 g/ml streptomycin sulfate, 0.03% glutamine,

and 0.075% sodium bicarbonate for closed containers or 0.225% sodium bicarbonate for

open containers in a 5% CO 2 atmosphere. Human embryonic kidney (HEK) 293T cells

were grown in similar media but supplemented with 10% FBS. A derivative of Vero cells

that expresses ICP27 under the control of its own promoter (22 cells. Smith et al., 1992),

were maintained in 10% FBScontaining DMEM supplemented with 500 g/ml G418.

HSV1 wildtype strains KOS and 17syn+ and several mutant viruses were used in this study. The phenotypes of mutant viruses are listed in Table 1.

Virus amplification. For stock preparation, viruses were propagated in Vero or

22 cells as appropriate using an input multiplicity of infection (m.o.i.) of ≈0.01 plaque

o forming Units (PFU) / cell. Cells were incubated at 34 C in a 5% CO 2 atmosphere and harvested when at least 90% of cells showed cytopathic effects. Harvested cells in

29 medium were disrupted by sonication, clarified by lowspeed centrifugation, and aliquots were stored at 80 o C. In experiments in which virus was used to infect BHK cells, stocks were prepared by propagation in BHK cells as detailed above. Virus titers were determined by plaque assay as previously described (Parris et al., 1978).

Plasmids . The enhanced green fluorescence protein (EGFP) expression plasmid, pEGFPC2, was purchased from Clontech (Mountain View, CA). In this construct (Fig.

2.1A), EGFP is expressed under the control of the human cytomegalovirus immediate early promoter (pCMV IE). Plasmid pintronEGFP (Fig 2.1B), in which the primary transcript that encodes EGFP contains a short intron (136 nt) 5’ of the open reading frame (ORF), was derived from pEGFPC2. The chimeric intron contains the 5’donor splice site from the first intron of the human βglobin gene, and the branch together with

3’acceptor sequence from an intron preceding an immunoglobulin gene heavy chain variable region (Bothwell et al., 1981; Senapathy et al., 1990; Huang et al., 1990a; Huang et al., 1990b). The intron was derived from plasmid pRLCMV from Promega (Madson,

WI). Plasmid pRLCMV was cut by SnaBI and NheI. The resulting 560 bp fragment, which consists of a 300 bp 3’ end portion of the pCMV IE and the chimeric intron, was used to replace the 415 bp SnaBINheI fragment of pEGFPC2.

Plasmid pUClinker, which was constructed to facilitate cloning the human U6 promoter (pHu6) into pUC19, was generated by replacing the multiple cloning site

(MCS) of pUC19 with an EcoR1BamH1Xba1 linker. The linker upper oligo is 5’

PAATTCGCGGATCCCGCTAGT3’, and the linker lower oligo is: 5’PCTAGA

CTAGCGGGATCCGCG3’. Plasmid pUCHu6, which possesses the Hu6 promoter in

30 the pUC19 backbone, was generated by ligating a 301 bp polymerase chain reaction

(PCR) product into the EcoRI and BamHI sites of pUClinker. The Hu6 promoter fragments corresponding to nucleotides 64355 of the human U6 gene (GenBank accession number M14486), was amplified from HEp2 cell DNA by PCR using the upper and lower primers shown in Table 3 to generate EcoRI and BamHI sites at 5’ and 3’ ends, respectively. The PCR conditions used for the amplification are detailed in Table 4.

Plasmids pmidsEGFP and pmidsLac Z, express imperfect hairpins of EGFP and

Lac Z sequences, respectively, whereas plasmids pdsEGFP and pdsLac Z, express perfect

hairpins of EGFP and Lac Z sequences, respectively. Plasmids were constructed by sub

cloning a 7880 bp sequence containing the appropriate inverted repeat into the BamHI

and XbaI sites downstream of the Hu6 promoter in pUCHu6. The imperfect inverted

repeat DNA sequences were designed by utilizing the RNAioligoretriever program

(http://katahdin.cshl.org:9331/RNAi/html/rnai.html), and the perfect inverted repeat DNA

sequences were designed using Invitrogen website BLOCKIT RNAi designer

(https://rnaidesigner.invitrogen.com/rnaiexpress/). The imperfect hairpins were

transcribed using the pol III Hu6 promoter to yield a 28 nt sequence that is completely

complementary to a portion of the target mRNA, a 9 nt loop sequence, and a 28 nt sense

strand sequence in which several mismatches were introduced (Fig. 2.2A). For pmidsEGFP, the 28 nt antisense strand sequence was completely complementary to nucleotides 121148 in the EGFP ORF. For pmidsLac Z, the 28 nt antisense strand sequence was complementary to nucleotides 5885 in the Lac Z ORF. The perfect inverted hairpin transcripts have the sensestrand sequences at the 5’ end of the loop and

31 the antisense stand sequences are located at the 3’ end of the transcripts (Fig. 2.2B). In pdsEGFP, the 21 nt antisense sequence is complementary to nucleotides 122141 in the

EGFP ORF, and in pdsLac Z, the 19 nt antisense sequence is complementary to nucleotides 294312 in the Lac Z ORF. To each pUCHu6 RNA construct, a BamHI sticky end sequence was added at the 5’ end of the repeat, and a sequence containing 5 T residues and a XbaI sticky overhang was added to 3’ end of the repeats. The pentadT sequence serves to terminate pHu6 driven transcription. The sequences of the upper and lower oligos used for constructing RNA hairpin expression plasmids are shown in Table

2.

Plasmid pcDNAUS11, which expresses HSV1 US11 under the control of the pCMV IE in mammalian cells, was constructed as follows. A 676 bp HSV1 US11 containing fragment, which consists of US11 ORF together with 102 bp upstream and

127 bp downstream of the US11 ORF, was amplified by PCR using HSV1 infected Vero cell DNA as template. The primer sequences and the PCR amplification parameters are shown in Tables 3 and 4, respectively. The 676 bp US11containing fragment was sub cloned into the TA cloning vector pCR®2.1 (Invitrogen, Carlsbad, CA) to create plasmid

TAUS11m. The 719 bp US11 containing fragment was released from TAUS11m by

EcoRI cleavage and cloned into the EcoRI site of pcDNA3.1 () (Invitrogen, Carlsbad,

CA).

Plasmids pKJBGFP, pKJBdsGFP, pKJBGUS (Kenneth Buckley, unpublished), express modified green fluorescence protein (GFP), double stranded GFP (dsGFP), and

βglucuronidase (GUS), respectively, in a plant binary vector under the control of the

32 enhanced 35S promoter. These plasmids were generated by excising the HindIIIXbaI gene fragments from pRTL2based GFP, βglucuronidase (GUS), dsGFP expression

constructs (kind gifts from James Carrington, Oregon State University. Johansen and

Carrington, 2001) and inserting them into the HindIII and XbaI sites of pKJB5033, a plant binary vector (a derivative of pBI121, Clontech, Mountain View, CA; Kenneth

Buckley, unpublished). Plasmid pKJBdsGFP contains the fulllength ORF of GFP, an

120 bp intron from the Arabidopsis RTM1 Col0 (Chrisholm et al., 2000), and the entire

GFP coding region in the antisense orientation. Plasmid pBI19, which expresses

Cymbidium ringspot virus -encoded 19kDa protein under the control of the 35S promoter in the binary vector pBIB61, was obtained from David Baulcombe (The

Sainsbury Laboratory, John Innes Centre, The United Kingdom) (Voinnet et al., 2003)

To make pBIUS11 for expression of HSV1 US11 in a plant binary vector, the

460 bp fulllength US11containing fragment was amplified by PCR using TAUS11m as described above as template with an upper primer containing a BspHI recognition site and a lower primer possessing an XbaI recognition site at the 5’ end, respectively. The primer sequences and the PCR amplification parameters are shown in Tables 3 and 4,

respectively. The PCR product was ligated into the BspHI and XbaI sites of pUC18T

CAT (Kenneth Buckley, unpublished). The BspHIXbaI US11 gene fragment was

transferred to pRTL2CAT and then to the binary vector, pBICAT (Enami et al., 1994)

as a HindIIIXba1 fragment.

Plasmids pBIICP0, pBIICP4 and pBIICP27 (Kenneth Buckley, unpublished),

which express HSV1 ICP0, ICP4 and ICP27, respectively, in a plant binary vector under

33 the control of the enhanced 35S promoter, were generated by excising the HindIII XbaI gene fragments from pRTL2based ICP0, ICP4 and ICP27 expression constructs

(Kenneth Buckley, unpublished) and inserting them into the HindIII and XbaI sites of pBICAT, a plant binary vector. The cloning strategies for these constructs are summarized in Table 5.

DNA isolation. Uninfected and infected cell DNA was isolated using the

DNAeasy Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s

instructions. DNA concentrations were determined based on spectrophotometric readings

(1 OD corresponds to 50 g/ml of DNA).

Polymerase chain reaction. Polymerase chain reactions (PCR) were conducted

in a total volume of 100 l using the hot start method (94 oC, 10 min prior to addition of

Taq polymerase (Invitrogen, Carlsbad, CA). All PCR reactions except for those with

US11 contained 20 mM TrisHCl pH 8.4, 50 mM KCl, 250 M dNTP, 2.5 M upper primer, 2.5 M lower primer, 1.5 mM MgCl 2, 100 ng template DNA, and 2.5 units of Taq polymerase. The PCR reaction for UScontaining sequence was as described above but

also contained 5% dimethyl sulfoxide (DMSO). The PCR primers and amplification parameters for various DNA fragments are given in Tables 3 and 4, respectively.

Quantitative realtime PCR. Reverse transcription was conducted using the

Superscript II kit from Invitrogen (Carlsbad, CA). The reaction was

carried out in a total volume of 20 l containing 2 g of total RNA, 25 mM Tris pH 8.3,

37.5 mM KCl, 1.5 mM MgCl 2, 0.1 M primer, 10 M DTT, 500 M dNTPs, and 200 units of reverse transcriptase. A primer specific to Ago2 (the primer sequence is listed

34 in Table 3) was used for the cDNA synthesis of Ago2 while random hexamers were employed for the cDNA synthesis of 18S rRNA, which was used as the loading control.

The RNA and primer were denatured at 70 oC for 10 minutes. For the cDNA synthesis of

Ago2, mixtures containing the reverse transcription buffer, DTT, dNTPs, primer, and the denatured RNAs were incubated at 42 oC for 2 minutes. Two hundred units of reverse

transcriptase were added, and the reactions were carried out at 42 oC for an additional 30 minutes. For the cDNA synthesis of 18S rRNA, all components for reverse transcription were incubated at 48 oC for 90 minutes. Real time PCR (RTPCT) reactions were

conducted in a total volume of 20 l using Sybergreen master mix from Roche

(Pleasanton, CA ) according to the manufacturer’s instructions . Ago2 RTPCR was

carried out using primers at a final concentration of 0.063 M under the following

conditions: 95 oC, 10 min; 40 cycles of 95 oC, 30s / 65 oC, 20s / 72 oC, 30s. 18S rRNA RT

PCR was conducted using primers at a final concentration of 0.252 M as follows: 95 oC,

10 min; 40 cycles of 95 oC, 15s / 56 oC, 9s / 72 oC, 20s. Ago2 and 18S rRNA PCR products of known concentrations were utilized as templates to make the standard curves.

DNA fragments used for making standard curves were amplified from HEK 293T cell genomic DNA by PCR. The primer sequences and PCR amplification conditions are shown in Table 3 and 4, respectively.

DNA digestion by restriction enzymes and DNA recovery from gel. DNA

digestions by restriction enzymes were conducted by standard procedures according to

the manufacturer’s instructions. Recovery of DNA from a gel was carried out using the

QIAEX kit from Qiagen (Valencia, CA). DNA was eluted in 3050 l of 10 mM Tris

35 HCl pH 8.5. Purified DNA was separated on a 1% agarose gel and quantified using the precision marker from BioRad (Hercules, CA) as standards.

Mammalian cell transfection. 2.5x10 5 BHK cells or 1.4x10 6 HEK 293T were seeded into 35 mm dishes or 7.5x10 5 BHK cells were seeded into 60 mm dishes in antibioticfree medium. The following morning, transfection was carried out in antibiotic and FBSfree DMEM supplemented with 0.1 mM nonessential amino acids using lipofectamine 2000 from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. For plasmid DNA transfection in 35 mm dishes, 700 ng of total DNA was diluted into a total volume of 85 l by OptiMem (Invitrogen, Carlsbad, CA) and was mixed with 85 l of 1:20 diluted lipofectamine 2000 in OptiMem. For 60 mm dishes, 2.5

g of total DNA was diluted into a total volume of 250 l by OptiMem and was mixed

with 250 l of 1:20 diluted lipofectamine 2000. For RNA transfection, 200 pmol RNA

duplex was diluted into 85 l by OptiMem and was mixed with 85 l of 1:20 diluted

lipofectamine 2000, and the resulting mixture was used for transfecting 35 mm dishes.

The transfection medium was replaced with DMEM containing 7.5% FBS for BHK cells

or 10% FBS for HEK 293T cells at 4 hr posttransfection. To silence Ago2 in 293T

cells, RNA duplex specific to Ago2 with upper oligo: 5’GCACGGAAGUC

CAUCUGAAUU3’, and lower oligo: 5’pUUCAGAUGGACUUCCGUGCUU3’, was

utilized while nontarget siControl RNA duplex from Dharmacon (Chicago, IL) was used

as the silencing specificity control.

Total RNA isolation. Total RNA from mammalian cells was isolated using the

RNAeasy plus kit from Qiagen (Valencia, CA) according to the manufacturer’s

36 instructions. Plant leaf total RNA was isolated using TRIzol from Invitrogen (Carlsbad,

CA) according to the manufacturer’s instructions.

Luciferase activity assay. Firefly and renilla luciferase activities were assayed using the Dual Glo Luciferase Assay System (Promega, Madson, WI) according to the manufacturer’ instructions.

Probe preparation for northern blot. 32 P radioactively labeled DNA probes were used for all northern blots except for GFP mRNA detection in plants. DNA probes were made with Rediprime II random priming labeling system from GE Healthcare

(Piscataway, NJ) based on the manufacturer’s instructions. All the templates for making

DNA probes were produced by PCR (the primer sequences and the PCR amplification parameters are given in Tables 3 and 4, respectively). Probes containing 1.23x10 7 DPMs were used for northern blots.

Antisense GFP RNA was used as the probe for GFP mRNA detection in plants.

GFP antisense RNA was generated by in vitro transcription using the StripEZ RNA kit from Ambion (Austin, TX) according to the manufacturer’s instructions. The synthesized

o RNA was cleaved into RNA fragments of 70100 nt long by 185 M Na 2CO 3 at 60 C for

3 hr.

Northern blot. Total RNA (510 g per lane) was separated on 1% argarose gel containing 6.7% formaldehyde in MOPS buffer (20 mM MOPS, 5 mM sodium acetate,

10 mM EDTA pH 7). RNAs were visualized following ethidium bromide (EtBr) staining with a UV transluminator (BioRad, Hercules, CA). Gels were equilibrated in 10xSSC

(1xSSC contains 0.15 M NaCl and 0.015 M sodium citrate), and RNAs were transferred

37 to neutral nylon membranes (GE Healthcare, Piscataway, NJ) by ascending diffusion using paper towels. Membranes were baked at 80 oC for 2 hr to fix RNAs. Pre hybridization was conducted in hybridization buffer containing 50% formamide, 5xSSC,

0.1% SDS, 250 g/ml of sheared single stranded salmon sperm DNA, 20 mM K 2HPO 4

KH 2PO 4 buffer pH 7.4, 5xDenhardts (1xDenhardts consists of 0.02% Polyvinyl pyrrolidine, 0.02% bovine serum albumin and 0.02% ficoll400) at 42 oC for 4 hr.

Hybridization was carried out in the same buffer as prehybridization at 40 oC overnight

(≈14 hr). Blots were washed twice in 2xSSC containing 0.1% SDS at RT for 30 minutes, then washed twice with 0.5xSSC containing 0.1% SDS at 50 oC for 30 minutes. Blots were briefly equilibrated in 2xSSC and exposed to a storage phoshpor screen. For detection of βactin mRNA, the previously hybridized probes were removed by boiling the membranes in distilled water for 1 minute prior to blotting. Subsequent hybridization, and wash steps were conducted using the same conditions described above. Messager

RNA levels were normalized to 18S rRNA, based on relative quantities observed in EtBr stained gels prior to blotting.

mRNA halflife assay. A stock solution of actinomycin D (Act D; Sigma, st.

Louis, MO) dissolved in DMSO (5 mg/ml) was used to supplement medium to obtain a final Act D concentration of 5 g/ml. Medium containing Act D was added to uninfected cells 48 hr posttransfection or to infected cells 6 hr postinfection. At various times after

Act D treatment, cells were harvested and total RNA was isolated. Messager RNA levels were determined by northern blot analysis and were normalized to 18S rRNA. Halflife curves were estimated by plotting time (in hr) after Act D treatment versus relative

38 mRNA amount, and the data were fit to an exponential decay function. Halflives were calculated according to the following equations:

kt Equation 1: E i=E 0e

Equation 2: t 1/2 =0.693/k

where E i is the proportion of remaining mRNA amount at time i compared to that at time 0, t is time (in hr), k is the decay constant and t 1/2 is the halflife of a given mRNA.

Plant silencing suppression assay. Suppression of gene silencing was assayed in a two or threecomponent system using agrobacterium infiltration of three to four week old WT or GFP transgenic line 16c N. benthamiana plants (Ruiz et al., 1998). Overnight cultures of agrobacteria were diluted to an OD 600 of 1.0. In a 2component assay, equal amounts of two agrobacterium cultures, one expressing GFP and the other expressing the gene of interest under the control of the 35S promoter, were mixed, and GFP transgenic line 16c N. benthamiana plants were infiltrated by the mixture. In a 3component system, equal amounts of three agrobacterium, which express target GFP, dsGFP and the test gene each under the control of the 35S promoter, were mixed, and WT N. Benthamiana plants were coinfiltrated with the agrobacterium mixture. Three leaves per plant were infiltrated from the underside of the leaf, and at both sides of the midvein with 10 l of the culture mixture, and four plants were inoculated with a given mixture of agrobacteria.

After 310 days, GFP expression was monitored by assessing green fluorescence under a

UV light lamp and by measuring GFP mRNA levels by northern blot analysis.

Statistical method. Data analysis with the studentT test was conducted.

39 Gene Isogenetic Protein properties Mutant Growth Source of the mutant HSV1 mutant inactivated wildtype strain and function(s) phenotype host Replicates Tegument normally in component; dsRNA tissue culture; binding protein; mild replication David Leib 1711 US11 17syn+ binds and inhibits phenotype in Vero PKR; expressed late mice; (Washington University) cooperates with (Mulvey et al, 2003) γ34.5 to lower replication in mice

Only partially Rescuant contains rescues David Leib 1711R US11 rescuant 17syn+ US11 gene (not fully phenotype (D. Vero (Washington University)

characterized) Leib, personal comm.).

4 Tegument protein; Replicates 0 RNase that normally in Sullivan Read (University

nonspecifically tissue culture; of MissouriKansas City) vhs1 vhs KOS degrades host and Host protein Vero (Read and Frenkel, 1983) viral mRNA; synthesis responsible for early inhibition is host shutoff abrogated ICP27 expressed IE; Fails to interferes with replicate in 27lac10 ICP27 KOS splicing and export tissue culture; Rozanne SandriGoldin of introncontaining inhibition of 22 cells (U. California, Irvine) genes; shuttles early and late (Smith et al, 1992) between nucleus and viral gene cytoplasm expression; host gene expression occurs late

TABLE 1. Description of HSV1 mutants and phenotypes 40

Name of Transcript Oligo Adapter at 5’ end Sequence a inverted repeat (IR) Upper BamHI 5’-PGATC CGGCGATGCCACCTACGGCAAGTTCAAGAGACTTG IRdsEGFP dsEGFP CCGTAGGTGGCATCGCCTTTTTT-3’ Lower XbaI 5’-PCTAG AAAAAAGGCGATGCCACCTACGGCAAGTCTCTTGA ACTTGCCGTAGGTGGCATCGCCG-3’ Upper BamHI 5’-PGATC CTGCAGATGAACTTCAGGGTCAGCTTGCCGAAGCT IRmidsEGFP midsEGFP TGGGCAGGCTGATCCTGAGGTTCATCTGCATCATTTTTTT-3’ Lower XbaI 5’-PCTAG AAAAAAATGATGCAGATGAACCTCAGGATCAGCCT GCCCAAGCTTCGGCAAGCTGACCCTGAAGTTCATCTGCAG-3’ Upper BamHI 5’-PGATC CGGTTGTTACTCGCTCACATTTGAGAAAATGTGAG IRdsLac Z dsLac Z CGAGTAACAACCTTTTT-3’ Lower XbaI 5’-PCTAG AAAAAGGTTGTTACTCGCTCACATTTTCTCAAATG

4

1 TGAGCGAGTAACAACCG-3’ Upper BamHI 5’-PGATCCCAAGGCGATTAAGTTGGGTAACGCCAGGGAAGCT IRmidsLac Z midsLac Z TGCTTGGCGTTATCCGACTTAGTCGCCTTGCAGTTTTTTT-3’ Lower XbaI 5’-PCTAG AAAAAAATGATGCAGATGAACCTCAGGATCAGCCT GCCCAAGCTTCGGCAAGCTGACCCTGAAGTTCATCTGCAG-3’

a Underlined is the sticky end sequence of BamHI or XbaI

TABLE 2. Sequences of oligos used for expressing RNA hairpins

41

PCR product Primer a Restriction site at 5’ end Sequence b Hu6 promoter U EcoRI 5’-TCTTTGGAATTC AAGGTCGGGCAGGAAGGCCTA-3’ L BamHI 5’-CGCGGATCC TAGTATATGTGCTGCCGAAGCGAGGAC-3’ US11m c U 5’-AGTGCGTGTGGGTTGGGCTTC-3’ L 5’-CGTGACCGACAGTCCCCGTAATC-3’ US11p d U BspHI 5’-CGTCATGA GCCAGACCCAACCCCCGG-3’ L XbaI 5’- GTCTAGA CTATACAGACCCGCGAGCCG-3’ βActin U 5’-TCGCAATGGAAGAAGAAATC-3’ L 5’-GGGGCCTCGGTCAGAAGCAC-3’ Ago2 U 5’-GTGGAGCTGGAGGTCACG-3’ L 5’-CAGGGCCTGGATCGTCTC-3’ UL42 U 5’-GCCCTGCCAGGTGGTCC-3’ L 5’-GCCCGTGATCGCCAACTC-3’ EGFP U 5’-CGCTACCCCGACCACA-3’ 5’-TGATGCCGTTCTTCTGCT-3’ 4 L 2 ICP27 U 5’-GGCCCCGGAGCGAAAG-3’ L 5’-TGCGGCCCGAGGATTG-3’ 18S rRNA U 5’-TCAAGAACGAAAGTCGGAGG-3’ L 5’-GGACATCTAAGGGCATCACA-3’

a U: upper primer. L: lower primer. b Underlined is the restriction recognition site. c for construction of TAUS11m. d for construction of pBIUS11

TABLE 3. The primers used for PCR

42

Product name Application Template Restriction Restriction Product Amplification a

site at 5’ site at 3’ length

end end (bp)

Hu6 promoter Construction of HEp2 cell DNA EcoR1 BamHI 25 cycles of 94 oC, 2 min / 60 o C, 30s o pUCHu6 303 / 70 C, 1min. o o US11m Construction of TA HSV1 infected No No 676 25 cycles of 94 C, 2 min / 70 C, 1 US11 Vero cell DNA min US11p Construction of pBI Plasmid TA BspHI XbaI 460 25 cycles of 94 oC, 2 min / 65 o C, 30s US11 US11 / 70 oC,1min.

Template for making pcDNAβ –Actin 25 cycles of 94 oC, 2 min / 65 o C, 30s βactin probe b (Bill Lafuse, No No 328 / 70 oC,1min.

unpublished)

Template for making 25 cycles of 94 oC, 2 min / 65 o C, 30s

Ago2 RTPCR standard 293T cell DNA No No 150 / 70 oC, 1min.

curve UL42 Template for making HSV1 infected No No 313 25 cycles of 94 oC, 2 min / 55 o C, 30s 4 o 3 probe Vero cell DNA / 70 C, 1min. o o EGFP Template for making Plasmid No No 267 40 cycles of 94 C, 2 min / 70 C, probe pEGFPC2 1min. ICP27 Template for making HSV1 infected No No 301 25 cycles of 94 oC, 2 min / 65 o C, 30s probe Vero cell DNA / 70 oC, 1min. 18S rRNA Template for making 293T cell DNA No No 400 25 cycles of 94 oC, 2 min / 56 o C, 30s RTPCR standard / 72 oC, 30s. curve

a all PCRs were conducted with a hot start (94 oC, 10min). b for northern blot.

TABLE 4. PCR amplification

43

Plasmid name Property Parental Cloning strategy plasmid pintronEGFP Expresses intron pEGFPC2 560 bp fragment with 130 bp chimeric intron at 3’ end cleaved from plasmid containing EGFP pRL, and cloned into the SnaBI and NheI sites of pEGFPC2 pUClinker Intermediate construct pUC19 pUC19 MCS replaced by EcoR1BamH1Xba1 linker pUCHu6 Possesses human U6 pUClinker 303 bp Hu6 promoter containing PCR product cloned into the EcoR1 and promoter in pUC19 BamH1 sites of pUClinker pmidsEGFP Expresses imperfect EGFP pUCHu6 Synthesized oligos ligated into the BamH1 and XbaI sites of pUCHu6 RNA hairpin (midsEGFP) pdsEGFP Expresses perfect EGFP pUCHu6 Synthesized oligos ligated into the BamH1 and XbaI sites of pUCHu6 RNA hairpin(dsEGFP) pmidsLac Z Expresses imperfect Lac Z pUCHu6 Synthesized oligos ligated into the BamH1 and XbaI sites of pUCHu6

4 RNA hairpin (midsLac Z) 4 pdsLac Z Expresses perfect Lac Z pUCHu6 Synthesized oligos ligated into the BamH1 and XbaI sites of pUCHu6 RNA hairpin (dsLac Z) TAUS11 Intermediate construct pCR®2.1 676 bp US11 containing PCR product amplified from HSV1 infected Vero cell DNA, cloned into A overhang in pCR®2.1 pcDNAUS11 Expresses US11 in pcDNA3.1() 719 bp US11 containing EcoR1 fragment cleaved from TAUS11, cloned into the mammalian cells EcoR1 sites of pcDNA3.1() PBIUS11 Expresses US11 in in a PBICAT Fulllength US11 cleaved from pRTL2US11 (Kenneth Buckley, unpublished), plant binary vector and cloned into the HindIII and Xba1 sites of pBICAT

TABLE 5. Construction of plasmids

Continued

44

TABLE 5 Continued

Plasmid name Property Parental Cloning strategy plasmid pKJBGFP (Kenneth Buckley, unpublished) Expresses GFP in a plant pKJB5033 GFPcontaining fragment cleaved from binary vector Kenneth pRTL2GFP, and cloned into HindIII and Buckley, XbaI of pKJB5033, a plant binary vector unpublished) pKJBdsGFP (Kenneth Buckley, unpublished) Expresses dsGFP in a plant pKJB5033 dsGFPcontaining fragment cleaved from binary vector pRTL2dsGFP, and cloned into HindIII and XbaI of pKJB5033 pKJBGUS (Kenneth Buckley, unpublished) Expresses pKJB5033 GUScontaining fragment cleaved from

4 pRTL2GUS, and cloned into HindIII and

5 βglucuronidase (GUS) in a plant binary vector XbaI of pKJB5033 pBIICP0 Expresses ICP0 in a plant pBICAT Fulllength ICP0 cleaved from pRTL2 (Kenneth Buckley, unpublished) binary vector ICP0 (Kenneth Buckley, unpublished), and cloned into a HindIIIXba1 sites of pBI CAT PBIICP4 Expresses ICP0 in a plant pBICAT Fulllength ICP4 cleaved from pRTLICP4 (Kenneth Buckley, unpublished) binarty vector (Kenneth Buckley, unpublished) and cloned into the HindIII and Xba1 sites of pBICAT pBIICP27 (Kenneth Buckley, unpublished) Expresses ICP27 in a plant pBICAT Fulllength ICP27 cleaved from pSelect binarty vector ICP27 (Kenneth Buckley, unpublished), and cloned into the HindIII and Xba1 sites of pBICAT

45

FIG. 2.1. Organization of EGFP expression plasmids. Plasmids pEGFPC2 (A) and pintronEGFP (B) are constructs from which transcripts encoding EGFP without or with an intron, respectively, are expressed under the control of the pCMV IE. The chemeric intron in pintronEGFP was subcloned using the SnaBINheI fragment from plasmid pRL CMV (Promega) to replace the SnaBINheI fragment from pEGFPC2 as described in the text.

46

pUC ori pCMV IE SnaB I HSV TK Nhe I poly A EGFP pEGFP C2 4.7kb

SV40 poly A Kan/Neo R

pSV40 pBacteria

FIG. 2.1A. Plasmid pEGFPC2

pUC ori pCMV IE SnaB I HSV TK Chimeric intron poly A Nhe I pintronEGFP 5.1kb EGFP

Kan/Neo R SV40 poly A pSV40 pBacteria

FIG. 2.1B. Plasmid pintronEGFP

FIG. 2.1. Organization of EGFP expression plasmids

47

FIG. 2.2. Structure of RNA hairpin expression plasmids . Imperfect (A) or perfect (B) RNA hairpins were expressed from an inverted repeat under the control of the pol III Hu6 promoter. For imperfect hairpins, the repeats consist of a 28nt antisense strand sequence, a 9nt loop, and a 28nt sensestrand sequence. The antisense portion in the inverted repeat is fully complementary to a portion close to the 5’ end of the target mRNA while several mismatches were introduced into the sense strand sequence. For perfect hairpins, the repeats contain a 19 or 21nt sense strand sequence, followed by 9nt loop. The 3’ portion of the repeats is 19 or 21nt antisense strand sequence.

48

R Anti-sense Amp pHu6RNAi Hu6 Transcription 27nt Hu6 RNA 9nt spacer promoter

28 nt 28nt sense Anti-sense sense (imperfect IR) Inverted repeat Primary transcript Transcription Stop (AAAAA)

FIG. 2.2A. Imperfect dsRNA expression plasmid

Transcription Stop (TTTTT)

R Sense Amp pHu6RNAi Hu6 Transcription 27nt Hu6 RNA 9nt spacer promoter

sense anti - sense Anti-sense (19 or 21 nt ) (19 or 21 nt) Inverted repeat Transcription Primary transcript Stop (AAAAA)

FIG. 2.2A. Perfect dsRNA expression plasmid

FIG. 2.2. Structure of RNA hairpin expression plasmids

49

CHAPTER 3

OPTIMIZATION OF A TRANSIENT SILENCING SYSTEM IN MAMMALIAN CELLS

Effect of perfect and imperfect RNA hairpins on target mRNA levels

We hypothesized that HSV1 elicits the host’s RNAi response, and in turn encodes a silencing suppressor(s) to overcome this antiviral effect for optimum replication. To test our hypothesis, an effective silencing system needed to be developed and optimized. Enhanced green fluorescence protein (EGFP), expressed from the plasmid, pEGFPC2, under the control of the cytomegalovirus immediate early pol II promoter (pCMV IE), was chosen as the target of RNA silencing (Fig. 2A). Imperfect

(midsEGFP) or perfect (dsEGFP) EGFP hairpin was expressed from plasmids pmidsEGFP or pdsEGFP, respectively, under the control of the pol III human U6 promoter to induce silencing. The midsLac Z and dsLac Z, which were expressed from the pmidsLac Z and pdsLac Z, respectively, were utilized as controls for target specificity. In the construct pmidsEGFP or pmidsLac Z, the predicted guide strand in the hairpin had a sequence completely complementary to a region close to the 5’ end of the target mRNA while several mismatches (G:U) were introduced into the passenger strand.

50 Therefore, the mature forms of the RNA duplexes from midsEGFP or midsLac Z were expected to function like siRNAs to effect cleavage of the mRNA target.

To test the effects of midsEGFP and dsEGFP on EGFP expression, BHK cells were cotransfected with pEGFPC2 and pmidsEGFP or pEGFPC2 and pdsEGFP for expression of the target EGFP gene and silencing inducer. As controls, BHK cells were cotransfected with pEGFPC2 and pmidsLac Z or pEGFPC2 and pdsLac Z. At 52 hr posttransfection, the steadystate EGFP mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. For quantification, the steadystate EGFP mRNA levels in cells receiving the target and silencing inducer were set to 1. The steady state EGFP mRNA level in cells receiving pEGFPC2 and pmidsEGFP (180 ± 10 arbitrary units) was much lower than that in cells cotransfected with pEGFPC2 and pmidsLac Z (1250 ± 30 arbitrary units) (Fig. 3.1A). After normalization to 18S rRNA levels, it was found that midsEGFP resulted in a 7fold reduction in the steadystate

EGFP mRNA level compared to that obtained with the control midsLac (Fig. 3.1C).

Expression of dsEGFP also reduced the steadystate EGFP mRNA level (450 ± 10 arbitrary units) compared with that in cells that expressed the control perfect hairpin dsLac Z (980 ± 20 arbitrary units) (Fig. 3.1B). However, coexpression of pdsEGFP with pEGFPC2 led to a 2.2fold reduction in EGFP mRNA level relative to that in cells co

transfected with pEGFPC2 and pdsLac Z (Fig. 3.1D). These results suggest that the

imperfect hairpin, midsEGFP, was more effective in silencing EGFP than the perfect

hairpin, dsEGFP. Therefore, midsEGFP was selected as the silencing inducer used for the

remainder of experiments.

51 Effect of molar ratio of pmidsEGFP to pEGFPC2 on silencing

To further optimize the transient silencing system, the amount of pmidsEGFP required to achieve maximum silencing of EGFP was determined. The molar ratios of hairpin to target plasmids were changed by adjusting the amount of pmidsEGFP or pmidsLac Z while keeping the amount of the target expression plasmid pEGFPC2 constant (420 or 500 ng for 35 or 60 mm dishes, respectively). Hu6pUC19 DNA was added as filler to keep the total DNA transfected constant (0.7 or 2.5 g for 35 or 60 mm dishes, respectively). Steadystate EGFP mRNA levels 52 hr after transfection were determined by northern blot analysis, and were normalized to 18S rRNA. The steady state EGFP mRNA level in cells receiving pmidsLac Z and pEGFPC2 at a molar ratio of

25 : 1 was set to 1. Results indicated that varying the molar ratio of pmidsLac Z to pEGFPC2 ranging from 2 5: 1 to 6:1 did not significantly alter the steadystate EGFP

mRNA levels (Fig. 3.2), suggesting midsLac Z has no effect on EGFP expression. A 1:1

molar ratio of pmidsEGFP to pEGFPC2 resulted in a ~7fold reduction in the steady

state EGFP mRNA level compared with a 1:1 molar ratio of pmidsLac Z to pEGFPC2,

consistent with previous findings described in Fig. 3.1C. Increased steadystate EGFP

mRNA levels were observed when the molar ratio of pmidsEGFP to pEGFPC2 was

reduced to below 1:1. The midsEGFP lost the ability to silence EGFP when the molar

ratio of pmidsEGFP to pEGFPC2 dropped to 2 5: 1, resulting in a steadystate EGFP

mRNA level comparable to that in control cells receiving pmidsLac Z and pEGFPC2

(Fig. 3.2). However, increasing the molar ratio of pmidsEGFP to pEGFPC2 to greater

than 1:1 did not reduce the steadystate EGFP mRNA levels further. Therefore, a 1:1

molar ratio of pmidsEGFP to pEGFPC2 was selected to achieve silencing of EGFP.

52 Effect of midsEGFP on EGFP mRNA degradation rate

The characteristic feature of siRNA pathways is that siRNAs initiate cleavage of their perfectly complementary target mRNAs. To confirm that the reduced steadystate

EGFP mRNA level in silenced cells was associated with an increased rate of mRNA degradation, experiments were conducted to measure the halflife of EGFP mRNA in silenced vs. control cells. Cells were cotransfected with pEGFPC2 and pmidsEGFP or with pEGFPC2 and pmidsLac Z with a molar ratio of 1:1. Fortytwo hr post transfection, Act D was added into medium to a final concentration of 5 g/ml in order to block additional transcription. Total RNA was isolated at various times after the addition of Act D. EGFP and βactin mRNA levels were determined by northern blot analysis

(Fig. 3.3A and B), and were normalized to 18S rRNA levels. Halflife curves for EGFP

and βactin mRNAs were made by plotting the relative EGFP or βactin mRNA amount

at various times after Act D treatment vs. time, and the data were fitted to an exponential

decay function as described in equation 1 in Chapter 2. The halflife of mRNA was

calculated according to equation 2 given in Chapter 2. The halflife curves for βactin mRNA in silenced and control cells were indistinguishable, and represented a halflife of

21.7 and 21.9 hr, respectively (Fig. 3.3C). These results indicate that midsEGFP had no significant effect on the degradation rate of a nontarget mRNA. However, it was observed that the halflife of EGFP mRNA in silenced cells was 2.8 hr, 2fold less than that observed in control cells (5.5 hr) (Fig. 3.3D). These results demonstrate that increased degradation rate of EGFP mRNA contributed to the reduced level of EGFP mRNA in those cells.

53

FIG. 3.1. Effects of the perfect and imperfect dsEGFP hairpins on EGFP mRNA levels. BHK cells were cotransfected with pEGFPC2 and either pmidsEGFP or pdsEGFP for expression of the imperfect (midsEGFP) or perfect (dsEGFP) hairpin, respectively, for silencing. In controls, BHK cells were cotransfected with pEGFPC2 and either the midsLac Z expression plasmid (pmidsLac Z) or the dsLac Z expression plasmid (pdsLac Z). Steadystate EGFP mRNA levels present at 52 hr posttransfection were determined by northern blot analysis, and were normalized to 18S rRNA levels and reported as arbitary units (Au) A: Steadystate EGFP mRNA levels in cells receiving pEGFPC2 and a plasmid expressing an imperfect hairpin. B: Steadystate EGFP mRNA levels in cells receiving pEGFPC2 and a plasmid expressing a perfect hairpin. C: Quantification of the northern blot shown in A.The relative steadystate EGFP mRNA amount in silenced cells was set at 1. D: Quantification of the northern blot shown in B. The relative steadystate EGFP mRNA amount in silenced cells was set at 1.

54

EGFP+ midsEGFP EGFP+midsLac Z

1 2 3 4 5 6 7 8 9 10

EGFP mRNA

18S rRNA

180±10 1250±35 Normalized Levels (Au) FIG. 3.1A. EGFP mRNA northern blot (midsEGFP as the silencing inducer)

EGFP+dsEGFP EGFP+dsLac Z

1 2 3 4 5 6 EGFP mRNA

18S rRNA

450±10 980±20 Normalized Levels (Au)

FIG. 3.1B. EGFP mRNA northern blot (dsEGFP as the silencing inducer)

Continued

55

FIG. 3.1 continued

8

6

4

2

Relative EGFP mRNA amount 0 Silenced control FIG. 3.1C. Quantification of the northern blot shown in A

8

6

4

2

Relative EGFPRelative mRNAamount 0 Silenced Control FIG. 3.1D. Quantification of the northern blot shown in B

FIG. 3.1. Effects of the perfect and imperfect dsEGFP hairpins on expression of EGFP

56

FIG. 3.2. Effect of molar ratio of pmidsEGFP and pmidsLac Z to pEGFPC2 on steadystate EGFP mRNA levels. The amounts of total DNA and pEGFPC2 were kept constant while the amount of pmidsEGFP (silenced cells) or pmidsLac Z (control cells) was altered. Total DNA was kept constant by the addition of Hu6pUC19 DNA. Steady state EGFP mRNA levels present were determined by northern blot analysis at 52 hr after transfection, and were normalized to 18S rRNA levels. The relative steadystate EGFP mRNA level in cells receiving pEGFPC2 and pmidsLac Z with a molar ratio of 2 5: 1 was set at 1.

57

1.0

0.8 Control 0.6 Silenced

0.4

0.2

Relative EGFP mRNA amount 0.0 2-5 2-4 2-3 2-2 2-1 20 21 22 22.59 Molar ratio of dsRNA to target plasmid

FIG. 3.2: Effect of molar ratio of pmidsEGFP and pmidsLac Z to pEGFPC2 on steadystate EGFP mRNA levels

58

FIG. 3.3. Effect of midsEGFP on the halflives of EGFP and βββactin mRNAs. At 42hr posttransfection, Act D (5 g/ml) was applied to silenced and control cells, which were cotransfected with pEGFPC2 and pmidsEGFP (silenced) or cotransfected with pEGFP C2 and pmidsLac Z (control) with a molar ratio of 1:1. Total RNA was isolated at various times after the addition of Act D. Northern blot analysis for EGFP and βactin mRNAs was conducted, and relative EGFP and βactin mRNA amounts were quantified by normalizing to18S rRNA levels. Halflife curves for EGFP and βactin mRNAs were made by fitting the data to an exponential decay function (equation 1 described in Chapter 2) to estimate halflife. A: Northern blot analysis of EGFP and βactin mRNA levels in silenced cells after Act D treatment. B: Northern blot analysis of EGFP and β actin mRNA levels in control cells after Act D treatment. C: Halflife curves for βactin mRNA in silenced and control cells. D: Halflife curves for EGFP mRNA in silenced and control cells

59

Time (hr) after Act D

Treatment 0 1 2 3 4.5 6 9 12 18 24

EGFP mRNA

βactin mRNA

18S RNA

FIG. 3.3A. mRNA levels in silenced cells

Time (hr) after Act D Treatment 0 2 4.5 6 9 12 18 24

EGFP mRNA

βactin mRNA

18S RNA

FIG. 3.3B. mRNA levels in control cells

Continued

60

FIG. 3.3 Continued

110 100 Silenced t 1/2 = 21.7 hr

90 Control t 1/2 = 21.9 hr 80 70 -actin mRNA-actin amount β β β β 60 50

Relative 40 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 3.3C. Halflife curves for βββ–actin mRNA

100 Silenced t 1/2 = 2.8 hr

80 Control t 1/2 = 5.5 hr

60

40

20

Relative EGFPRelative mRNAamount 0 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 3.3D. Halflife curves for EGFP mRNA

FIG. 3.3. Effect of midsEGFP on the halflives of EGFP and βββactin mRNAs

61

CHAPTER 4

INFECTION BY HSV1 SUPPRESSES RNA SILENCING

Effect of HSV1 infection on steadystate target mRNA levels in silenced and control cells

To address whether HSV1 encodes one or more silencing suppressors, we

compared the steadystate target EGFP mRNA levels in HSV1infected silenced and

control cells with those in similarly transfected mockinfected cells. At 42 hr after

transfection, silenced and control cells, which received the target EGFP expression plasmid pEGFPC2 and either the midsEGFP expression plasmid pmidsEGFP or the

control hairpin expression plasmid pmidsLac Z, respectively, were mockinfected or

infected with HSV1 virus (strain KOS) at a multiplicity of infection (m.o.i.) of 5 plaque

forming units (PFU)/cell. Cells were harvested 10 hr postinfection, and total RNA was

isolated. EGFP and βactin mRNA levels were determined by northern blot analysis and

were normalized to 18S rRNA. Northern blotting indicated that the steadystate βactin

mRNA levels in mockinfected cells, whether or not silenced, were similar (Fig. 4.1A).

However, infection of silenced cells with HSV1 led to a much lower level of βactin

mRNA compared with mock infection of silenced cells (Fig. 4.1A). A much lower 62 steadystate βactin mRNA level was also observed in HSV1infected control cells compared to that in mockinfected control cells (Fig. 4.1A). Quantitative analysis showed that HSV1 infection reduced βactin mRNA levels both in silenced and control cells by

~ 2.5fold compared with mock infection (Fig. 4.1B). In contrast, HSV1 infection enhanced the steadystate EGFP mRNA level in control cells by 25% compared with mock infection (Fig. 4.1C). However, the EGFP mRNA level in HSV1infected silenced cells was fivetimes that in mockinfected silenced cells (Fig. 4.1A and C). The lowlevel enhancement of EGFP mRNA level observed in control cells infected with HSV1 may have reflected a nonspecific effect of HSV1 on gene expression. Alternatively, it is possible that overexpression of EGFP resulted in a low level of specific silencing that was suppressed by HSV1 infection. Nevertheless, the much larger enhancement of

EGFP mRNA level in silenced cells as a result of infection suggests that HSV1 can specifically suppress silencing of the target EGFP.

Effect of HSV1 infection on expression of introncontaining EGFP in silenced cells

Intronless mRNAs are poorly exported into the cytoplasm compared with intron

containing mRNAs in mammalian cells (Chen et al., 2002). HSV1 infection facilitates

the transport of intronless mRNAs from the nucleus into the cytoplasm (Chen et al.,

2005; Chen et al., 2002). The EGFP, expressed from pEGFPC2 does not contain an

intron. Therefore, the enhancement of the target EGFP mRNA level in silenced cells by

HSV1 infection (see Fig. 4.1C) might have been due to the more effective export of

intronless EGFP mRNA out of the nucleus. To address whether enhancement of the

target mRNA level in silenced cells by HSV1 is attributed to the lack of an intron in the

EGFP transcript, plasmid pintronEGFP, which expresses introncontaining EGFP

63 (intronEGFP), was constructed from pEGFPC2 by cloning a 136 bp chimeric intron upstream of the AUG of the EGFP ORF in pEGFPC2 (Fig. 2.1B). To analyze the effect of midsEGFP on accumulation of introncontaining vs. nonspliced EGFP transcripts,

BHK cells were cotransfected with pintronEGFP or pEGFPC2 together with a hairpin

expression plasmid. For silenced cells, the hairpin expression plasmid was pmidsEGFP

and for the control cells, it was pmidsLac Z. Total RNA was isolated 52 hr post

transfection, and the steadystate EGFP mRNA levels were determined by northern blot

analysis and normalized to 18S rRNA levels. For comparison purposes, the steadystate

EGFP mRNA level in cells receiving the intronless EGFP expression plasmid pEGFPC2

and pmidsEGFP was set at 1. The steadystate EGFP mRNA level in cells receiving pEGFPC2 and pmidsEGFP was 5.7fold lower than that in cells cotransfected with pEGFPC2 and pmidsLac Z (Fig. 4.2), similar to that in silenced cells reported in Fig.

3.1C. Surprisingly, the steadystate EGFP mRNA level in cells receiving pintronEGFP and pmidsLac Z was 1.8fold lower compared to that in cells cotransfected with pEGFP

C2 and pmidsLac Z (Fig. 4.2), suggesting that transcripts for intronEGFP did not accumulate to the same extent as those transcripts without intron. Cotransfection of pmidsEGFP with pintronEGFP resulted in a 6.7fold reduction in EGFP mRNA level compared with cotransfection of pmidsLac Z (Fig. 4.2), indicating that silencing of intronEGFP was effective despite an overall reduction in the mRNA level in control cells.

To analyze the effect of HSV1 infection on expression of the introncontaining

EGFP gene, BHK cells were cotransfected with pintronEGFP and a plasmid expressing the midsEGFP or midsLac Z. BHK were also cotranfected with pEGFPC2 (no intron)

64 together with pmidsEGFP or pmidsLac Z. At 42hr posttransfection, cells were either mockinfected or infected with HSV1 KOS (m.o.i. of 5 PFU/cell). Cells were harvested

10 hr after infection. Total RNA was isolated and the steadystate EGFP mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA levels. For comparison purposes, the steadystate EGFP mRNA level in mockinfected silenced cells

(i.e. those receiving pEGFPC2 and pmidsEGFP) was set at 1. HSV1 infection increased the steadystate EGFP mRNA level in control cells receiving the intronless target gene

(pEGFPC2) and pmidsLac Z by 24% compared with mock infection (Fig. 4.3). Similar enhancement of the EGFP mRNA level in the control cells cotransfected with pintronEGFPC2 and pmidsLac Z by HSV1 infection (32%) was also observed compared with mockinfection. HSV1 infection resulted in a 4.8fold increase in the steadystate EGFP mRNA level in silenced cells receiving pEGFPC2 and pmidsEGFP compared with mock infection, which was comparable to that observed in the previous experiments (see Fig. 4.1C). The steadystate EGFP mRNA level in HSV1 infected silenced cells cotransfected with pintronEGFP and pmidsEGFP was 5.0fold higher than that in the mockinfected silenced cells. These results indicate that the presence or absence of an intron did not influence the ability of HSV1 to enhance the target mRNA level in silenced cells. Moreover, the presence of an intron did not alter the ability of

HSV1 to produce lowlevel enhancement of EGFP accumulation in control cells.

65 Effect of HSV1 infection of silenced and control cells on the halflife of target EGFP mRNA

As indicated in Fig. 4.1C, HSV1 infection increased the steadystate target mRNA level in silenced cells. The mechanism by which that increase was achieved could have been due to increased expression of target gene, increased mRNA stability, or both.

True silencing suppression by HSV1 predicts that at least some of the increased mRNA level is due to increased mRNA stability. Therefore, the effect of HSV1 infection on the halflife of the target EGFP mRNA was determined. BHK cells were cotransfected with the target EGFP plasmid together with a plasmid expressing either the silencing inducer midsEGFP or the control hairpin midsLacZ. Fortytwo hr after transfection, silenced and control cells were either mockinfected or infected with HSV1 KOS at a m.o.i. of 5

PFU/cell. Viral genes were allowed to be expressed for 6 hr before Act D (5 g/ml) was applied to cells. Cells were harvested at various times after Act D treatment and the remaining levels of EGFP and βactin mRNAs were determined by northern blot analysis

(Fig 4.4A and B). The amounts of EGFP and βactin mRNAs were normalized to 18S rRNA, and the relative amounts of EGFP and βactin mRNAs at the time when Act D was applied (time 0) were set as 100%. The data were fit to an exponential decay function (equation 1) as described in Chapter 2, and the halflives of mRNAs were calculated using equation 2 given in Chapter 2. The halflife of βactin mRNA in HSV1 infected silenced cells (15.5 hr), was not significantly different from that in HSV1 infected control cells (15.4 hr). However, these halflives were shorter than that in mock infected cells (~21 hr) (Fig. 4.4C and D), consistent with an increased degradation rate for cellular mRNAs following HSV1 infection (Zhou et al., 2002; PerezParada et al.,

66 2004). In control cells, HSV1 infection decreased the halflife of EGFP mRNA (4.1 hr) compared to that in mockinfected cells (5.4 hr) (Fig. 4.4E and F). By contrast, in silenced cells, HSV1 infection increased the halflife of EGFP mRNA (4.5 hr) compared to that in mockinfected cells (2.7 hr) (Fig. 4.4E and F). In fact, the halflives of EGFP mRNA were comparable in silenced and control cells following HSV1 infection (4.5 vs.

4.1 hr, respectively). These results indicate that HSV1 infection completely reversed silencing, and suggest that HSV1 encodes at least one silencing suppressor. However, the halflife of EGFP mRNA in HSV1 infected silenced cells was significantly and reproductively shorter that that in mockinfected control cells (Fig. 4.4E and F).

Effect of mutation in viral host shutoff functions or ICP27 on the steadystate target mRNA level and the halflife of the target mRNA

As demonstrated in Fig. 4.4E and F, HSV1 increased the degradation rate of the

EGFP mRNA in control cells compared to that observed in mockinfected control cells

(Fig. 4E and F). It was predicted that the shorter halflife of EGFP mRNA in HSV1 infected control cells was attributed to one or more known functions of HSV1 that result in virusspecific host shutoff. Most of the host shutoff is due to the functions of ICP27 and the virion host shutoff protein (vhs) encoded by the virus (Bryant et al., 2001; Pak et al., 1995; Smibert et al., 1992; Schek, 1985). To test this prediction, we examined viral mutants with a defect in ICP27 or vhs for their respective effects on steadystate target

EGFP mRNA levels and the halflife of EGFP mRNA in both silenced and control cells.

Cells were cotransfected as before with target and hairpin expression plasmids. Forty two hr posttransfection, silenced and control cells were either mockinfected or infected with wildtype (KOS), vhs mutant (vhs1) or ICP27 mutant (27lac10) at a m.o.i. of 5

67 PFU/cell. Cells were harvested 10 hr postinfection, and total RNA was isolated. Steady state EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA levels. For comparison purposes, the steadystate EGFP and βactin mRNA levels in mockinfected silenced cells were each set at 1. The steady state βactin mRNA levels in mockinfected silenced and control cells were not significantly different from each other (Fig. 4.5A). Infection of either control or silenced cells with wildtype virus reduced the steadystate βactin mRNA levels by ~2.5 fold compared with mockinfection (Fig. 4.5A). However, the steadystate βactin mRNA levels in vhs mutantinfected silenced or control cells or in ICP27 mutantinfected silenced or control cells were comparable to or slightly greater than that observed in mock–infected control cells (Fig.4.5A). Those results are consistent with previously reported effects of these mutations on cellular mRNA accumulation (Kwong et al., 1987;

Pak et al., 1995; Smibert et al., 1992).

However, the effects of these mutations on EGFP mRNA levels were strikingly different. In control cells infected with the wildtype virus (KOS), vhs or ICP27 mutant, the steadystate EGFP mRNA was 0.5, 0.7 and 2fold higher than that observed with mock infection (Fig. 4.5B). In silenced cells, infection by the wildtype virus enhanced the steadystate EGFP mRNA level to 5fold that observed with mock infection (Fig.

4.5B). However, infection of silenced cells by the vhs or ICP27 mutant resulted in a 7 fold or 10.3fold increase in the steadystate EGFP mRNA levels, respectively, compared with mock infection (Fig. 4.5B). Thus, the loss of host shutoff function actually further enhances the accumulation of EGFP mRNA following infection.

68 The effect of loss of vhs or ICP27 function on the halflife of the target EGFP mRNA was next determined to assess the possible cause of increased EGFP mRNA accumulation. At 42 hr after transfection, silenced and control cells were either mock infected or infected with the vhs1 or ICP27 mutant at a m.o.i. of 5 PFU/cell. Viral genes were expressed for 6 hr and Act D (5 g/ml) was applied to cells. Total RNA was isolated at various times after Act D treatment. The amounts of EGFP and βactin mRNAs were determined by northern blot analysis, and were normalized to 18S RNA levels. The halflife curves for EGFP and βactin mRNAs were plotted as described above. The halflives of βactin mRNA in mockinfected silenced and control cells, 20.6 and 21.6 hr, respectively (Fig 4.6A and B), were not significantly different from each other. Infection by the vhs mutant did not significantly alter the halflife of βactin mRNA in either silenced or control cells compared with mock infection (Fig. 4.6A and

B), consistent with the phenotype previously reported for this virus (Sorenson, et al.,

1991). Infection by the ICP27 mutant resulted in similar halflives of βactin mRNAs in silenced and control cells (20.5 versus 20.8 hr, respectively), and were comparable to that in mockinfected control or silenced cells (Fig. 4.6A and B). The halflives of EGFP mRNA in mockinfected silenced and control cells were 2.8 and 5.5 hr, respectively (Fig.

4.6C and D). In silenced cells, infection by the vhs mutant resulted in the halflife of

EGFP mRNA (5.6 hr) similar to that observed in mock infected control cells (5.5 hr), but substantially longer that that observed in mockinfected silenced cells (2.8 hr) (Fig. 4.6C and D). Likewise, infection of silenced cells by the ICP27 mutant increased the halflife of EGFP mRNA (5.3 hr) compared to that in mockinfected silenced cells. Moreover, the halflife of EGFP mRNA in ICP27 mutant infected silenced cells (5.3 hr) was virtually

69 the same as that in mockinfected control cells (5.5 hr) (Fig. 4.6Cand D). These results demonstrate that somewhat a shorter halflife of EGFP mRNA observed in silenced and control cells infected with wildtype virus compared to that observed in mockinfected control cells is due to the host shutoff functions of vhs and ICP27. Moreover, these results further support the hypothesis that HSV1 infection can completely reverse silencing.

Effect of HSV1 infection on miRNAinduced silencing

As described in Fig. 4.4, HSV1 infection suppressed siRNAinduced silencing.

Whether HSV1 suppresses miRNAinduced silencing is not known. Therefore, we determined the effect of HSV1 infection on miRNAinduced silencing using a surrogate reporter assay described elsewhere (Zeng et al., 2003). In the assay, firefly luciferase

(Fluc), which was expressed from a pCMV IEdriven expression construct, pCMVLuc miR30 (P), and contains 8 tandem target sequences of human miR30 in the 3’ UTR, was used as the target (Fig. 4.7A). Human miR30, which was expressed from plasmid pH1 miR30 under the control of the pol III H1 promoter, was utilized as the silencing inducer while miR21 expressed from plasmid pH1miR21 was exploited as the silencing specificity control. Renilla luciferase (Rluc), which was expressed from plasmid pRL under the control of pCMV IE, was used as another silencing specificity control, and as an internal transfection control. BHK cells were cotransfected with 30 ng pCMVLuc miR30 (P), 2 ng pRL, and 30 ng pH1miR30. In controls, BHK cells were cotransfected with 30 ng pCMVLucmiR30 (P), 2 ng pRL, and 30 ng pH1miR21. To monitor whether or not there is any promoter competition for expression of Fluc and Rluc, BHK cells were also transfected with 2 ng pRL or 30 ng pCMVLucmiR30 (P) or co

70 transfected with both. At 42 hr posttransfection, cells were either mockinfected or infected with HSV1 (KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested 10 hr after infection, lysates were prepared, and the activities of Rluc and Fluc were assayed. The activities of Rluc in cells receiving pRL alone and in cells cotransfected with pRL and pCMVLucmiR30 (P) were 21400 and 20600 Arb units (Fig. 4.7B). Moreover, the Fluc activities in cells transfected with pCMVLucmiR30 (P) alone and in cells cotransfected with pCMVLucmiR30 (P) and pRL were not significantly different from each other

(5000 versus 5500 Arb units) (Fig. 4.7C). These results suggest that under the conditions used, there was no promoter competition for expression of Fluc and Rluc.

The activities of Rluc in cells cotransfected with pRL, pCMVLucmiR30 (P)

and pH1miR30, and in cells cotransfected with pRL, pCMVLucmiR30 (P) and pH1

miR21 were 50000 and 51000 Arb units, with both not significantly different from that

in cells receiving pRL and pCMVLucmiR30 (P), suggesting that miR30 and miR21 did

not have a general effect on expression of RLuc. The relative Fluc activity (Fluc activity /

RLuc activity) in cells receiving pRL, pCMVLucmiR30 (P) and pH1miR30 was 23.5

fold lower than that in cells receiving pRL, pCMVLucmiR30 (P) and pH1miR21,

indicating miR30 silenced Fluc effectively (Fig. 4.7C). HSV1 infection increased the

activity of Rluc in cells cotransfected with pRL and pCMVLucmiR30 (P) by 2.5fold

compared with mock infection. Also, a similarfold enhancement of RLuc activities

following HSV1 infection was observed regardless of the presence of other plasmids

(Fig. 4.7B). HSV1 infection also increased the relative Fluc activity compared with

mock infection in cells that were not specifically silenced, and the level of this

enhancement (≈2.2) was similar to that observed for Rluc (Fig. 4.7B). These results

71 indicate that HSV1 has a general effect on expression of transfected reporter genes Fluc and Rluc. However, HSVI infection increased the relative activity of Fluc in cells under specific silencing condition by 11fold compared with mock infection (Fig. 4.5C). This higher level of enhancement indicate that HSV1 infection can specifically suppress miRNAinduced silencing in addition to its general ability to enhance reporter expression.

Effect of knockdown of Ago2 on HSV1 yield of progeny

Fig. 4.4E and F indicate that HSV1 encodes or induces a silencing suppressor. To determine the biological significance of this silencing suppression, the effect of knock down of Argonaute 2 (Ago2) on HSV1 yield of progeny was analyzed. Ago2 possesses the slicer activity responsible for mRNA cleavage by RISC. If the host mounts a silencing response as a natural antiviral strategy, it was expected that knockdown of

Ago2 would increase the yield of progeny following HSV1 infection. To test this prediction, we first tested an RNA duplex specific to Ago2 for its ability to reduce Ago

2 mRNA level (The sequences were obtained from a publication. Meister et al., 2004;

Chapter 2). HEK 293T cells were transfected with the Ago2 specific RNA duplex or a

RISCfree RNA control duplex. At 48 hr posttransfection, Ago2 mRNA levels were

determined by quantitative realtime PCR. It was found that the copy number of Ago2

mRNA in cells receiving Ago2 specific RNA duplex was 4.1 ± 0.5x10 3 copies per g

RNA, which was 9.6fold lower (a 91% reduction) than that in cells receiving the control

RNA duplex (the copy number of Ago2 mRNA was 4.44 ± 0.61x10 4 copies per g

RNA). These results indicate that Ago2 was effectively silenced by the Ago2 specific

RNA duplex. To assess the effect of knockdown of Ago2 on HSV1 yield of progeny,

72 cells that received either the control or Ago2 specific RNA duplex were infected by

HSV1 at a m.o.i. of 1 or 10 PFU/cell at 48 hr posttransfection. Cells were harvested 18 hr after infection, and the yield of virus progeny was determined by plaque assay. Knock down of Ago2 resulted in the yield of progeny of 155 ± 20 PFU/cell, which was 2.5fold higher than that in cells receiving the control RNA duplex (56 ± 3 PFU/cell) when a m.o.i. of 1 PFU/cell was used. Similarly, at an input m.o.i of 10 PFU/cell, the yield of the virus progeny in cells receiving the Ago2 specific RNA duplex (1290 ± 145 PFU/cell) was enhanced by 2.7fold compared to that in cells receiving the control RNA duplex

(480 ± 30 PFU/cell). These results indicate that HSV1 replication is impeded by the presence of a potent silencing system despite its ability to suppress silencing following

infection. These results further suggest that lytic replication of HSV1 in 293T cells

elicits a host silencing response.

73

FIG. 4.1. Effect of HSV1 infection on steady state target and nontarget mRNA levels. Fortytwo hr after transfection, silenced and control cells were either mock infected or infected with HSV1 virus (KOS) at a m.o.i. of 5 PFU/cell. EGFP and βactin mRNA levels present at 10 hr p.i. were determined by northern blot analysis, and were normalized to 18S rRNA. The relative βactin and EGFP mRNA amounts in mock infected silenced cells were set at 1 for comparison. A: Northern blot analysis of EGFP and βactin mRNAs. B: Quantification of βactin mRNA levels shown in A . C: Quantification of EGFP mRNA levels shown in A.

74

Silenced cells Control cells

Mock KOS Mock KOS 1 2 3 1 2 3 1 2 3 1 2 3

EGFP mRNA 190 ± 20 930 ± 30 1270± 5 1580 ± 75 Normalized levels (Au)

βββ− Actin mRNA

850 ± 30 340 ± 15 780± 20 310 ± 15 Normalized levels (Au)

18S rRNA

FIG. 4.1A. EGFP and β–actin mRNA levels in cells

Continued

75 FIG. 4.1 Continued

Mock infected 1.0 KOS infected

0.8

0.6

0.4 -actin amount mRNA β β β β 0.2

Relative Relative 0.0 Silenced Control

FIG. 4.1B. Quantification of βββactin mRNA leve ls shown in A

10

8 Mock infected KOS infected 6

4

2

amount EGFP mRNA Relative 0 Silenced Control

FIG. 4.1C. Quantification of EGFP mRNA levels shown in A

FIG. 4.1. Effect of HSV1 infection on steadystate target and nontarget mRNA levels

76

FIG. 4.2. Effect of intron within the target EGFP gene on accumulation of EGFP mRNA in silenced and control cells. BHK cells were cotransfected with pintronEGFP or pEGFPC2 and a plasmid expressing either midsEGFP or midsLac Z. Cells were harvested 52 hr posttransfection, and total RNA was isolated. EGFP mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The relative amount of EGFP mRNA in cells receiving pEGFPC2 and pmidsEGFP was set at 1. The experiments were conducted in triplicates.

77

7 5.7 6 Silenced cells Control cells 5

4 6.7

3

2

1

RelativeEGFP mRNAamount 0 n n ro ro nt nt i + I No

FIG. 4.2. Effect of intron within the target EGFP gene on accumulation of EGFP mRNA in silenced and control cells.

78

FIG. 4.3. Effect of HSV1 infection on mRNA accumulation from EGFP transcripts with or without intron . BHK cells were cotransfected with pintronEGFP or pEGFPC2 and a plasmid expressing either midsEGFP or midsLac Z. At 42 hr posttransfection, cells were either mock infected or infected with HSV1 virus (KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested 10 hr posttransfection, and total RNA was isolated. EGFP mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The relative amount of EGFP mRNA in cells receiving pEGFPC2 and pmidsEGFP was set at 1.

79

8 1.2 Silenced, mock 7 Silenced, infected Control, mock 6 Control, infected 4.8 5 1.3 4 3 5.1 2 1

Relative EGFP mRNA amount EGFP mRNA Relative 0 n n o o r r t t n n i i o + N

FIG. 4.3. Effect of HSV1 infection on mRNA accumulation from EGFP transcripts with or without intron

80

FIG. 4.4. Effect of HSV1 infection on the halflives of target and nontarget mRNAs. At 42hr after transfection, silenced and control cells, were either mockinfected or infected with HSV1 virus (KOS) at a m.o.i. of 5 PFU/cell. Viral genes were allowed to be expressed for 6 hr. Act D (5 g/ml) was applied to cells, and cells were harvested at various times after Act D treatment. EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The amounts of EGFP and β actin mRNAs at the time when Act D was applied (time 0) were set to 100%, and half life curves were plotted based on an exponential decay function described in Chapter 2. A: Northern blot analysis of EGFP and β–actin mRNA levels in HSV1 infected silenced cells after Act D treatment. B: Northern analysis of EGFP and β–actin mRNA levels in HSV1 infected control cells after Act D treatment. C: Halflife curves for β–actin mRNA in mockinfected silenced and control cells. D: Halflife curves for β–actin mRNA in HSV1infected silenced and control cells. E: Halflife curves for EGFP mRNA in mock infected silenced and control cells. F: Halflife curves for EGFP mRNA in HSV1 infected silenced and control cells.

81

Time (hr) after Act D

Treatment 0 2 4 6 9 12 18 24

EGFP mRNA

βactin mRNA

18S rRNA mRNA

FIG. 4.4A. mRNA levels in HSV1infected silenced cells

Time (hr) after Act D Treatment 0 2 4 6 9 12 18 24

EGFP mRNA

βactin mRNA

18S rRNA mRNA

FIG. 4.4B. mRNA levels in HSV1infected control cells Fig. 4.4B

Continued

82 FIG. 4.4 Continued

110

100 Silenced t 1/2 = 20.4 hr 90 Control t 1/2 = 21.2 hr 80 70 60 -actinmRNA amount β β β β 50 40

Relative 30 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.4C. Halflife curves for βactin mRNA in mockinfected cells

100 Silenced t 1/2 = 15.5 hr Control t = 15.4 hr 80 1/2

60 -actin mRNA-actin amount β β β β 40

Relative Relative 20 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.4D. Halflife curves for βactin mRNA in

HSV1 infected cells

Continued

83 FIG. 4.4 Continued

100

Silenced t 1/2 = 2.7 hr 80 Control t 1/2 = 5.4 hr 60

40

20

Relative EGFPRelative amountmRNA 0 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.4E. Halflife curves for EGFP mRNA in mockinfected cells

100

80 Silenced t 1/2 = 4.5 hr Control t 1/2 = 4.1 hr 60

40

20

Relatvie EGFPRelatvie mRNA amount 0 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.4F. Halflife curves for EGFP mRNA in HSV1infected cells

FIG. 4.4. Effect of HSV1 infection on the halflives of target and nontarget mRNAs

84

FIG. 4.5. Effects of infection by HSV1 host shutoff mutants on accumulation of target and nontarget mRNAs. Silenced and control cells were either mockinfected or infected with HSV1 KOS, vhs or ICP27 mutant at a m.o.i. of 5 PFU/cell 42 hr post transfection. Total RNA was isolated 10 hr after infection. Steadystate EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The relative amounts of EGFP and βactin mRNA in mock infected silenced cells were set to 1. A: Steadystate βactin mRNA levels in silenced and control cells. B Steadystate EGFP mRNA levels in silenced and control cells. .

85

12

10 Silenced cells Control cells 8 6

-actin mRNA level mRNA -actin β β β β 4

2 Relative Relative 0 k S 1 0 oc O s- c1 M K vh la 27

FIG. 4.5A. Steadystate βactin mRNA levels

12 Fig. 4.5B

10 Silenced cells Control cells 8

6

4

2 RelativeEGFP mRNA level 0 k S 1 0 oc O s- c1 M K vh la 27 FIG. 4.5B. Steadystate EGFP mRNA levels

FIG. 4.5. Effects of infection by HSV1 host shutoff mutants on accumulation of target and nontarget mRNAs

86

FIG. 4.6. Effect of loss of host shutoff function on the halflives of target and non target mRNAs. At 42hr after transfection, silenced and control cells were either mock infected or infected with vhs or ICP27 mutant at a m.o.i. of 5 PFU/cell. Viral genes were allowed to be expressed for 6 hr. Act D (5 g/ml) was applied to cells, and cells were harvested at various times after Act D treatment. EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The amounts of EGFP and βactin mRNAs at the time when Act D was applied (time 0) were set as 100% and halflife curves were plotted based on an exponential decay function. A: Halflife curves for β–actin mRNA in vhs or ICP27 mutantinfected silenced cells. B: Halflife curves for β–actin mRNA in vhs or ICP27 mutantinfected control cells. C: Halflife curves for EGFP mRNA in vhs or ICP27 mutantinfected silenced cells. D: Halflife curves for EGFP mRNA in vhs or ICP27 mutantinfected control cells.

87

100

90 Mock t 1/2 = 20.6 hr 80 ICP27 t 1/2 = 20.5 hr vhs-1 t 1/2 = 21.1 hr 70

-actinmRNA amount 60 β β β β

50

Relative 40 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.6A. Halflife curves for β–actin mRNA in vhs or ICP27 mutantinfected silenced cells

100

90 Mock t 1/2 = 21.6 hr ICP27 t 1/2 = 20.6 hr 80 vhs-1 t 1/2 = 20.5 hr 70

-actin mRNA-actin amount 60 β β β β

50

Relative 40 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.6B. Halflife curves for β–actin mRNA in vhs or ICP27 mutantinfected control cells

Continued

88 FIG. 4.6 Continued

100 Mock t 1/2 = 2.8 hr 80 ICP27 t 1/2 = 5.3 hr vsh-1 t 1/2 = 5.6 hr 60

40

20

Relative EGFPRelative mRNA amount 0 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.6C. Halflife curves for EGFP mRNA in vhs or ICP27 mutantinfected silenced cells

100 Mock t 1/2 = 5.5 hr 80 ICP27 t 1/2 = 5.7 hr vhs-1 = 5.4 hr 60 t1/2

40

20

RelativeEGFP mRNA amount 0 0 4 8 12 16 20 24 Time (hr) after Act D treatment

FIG. 4.6D. Halflife curves for EGFP mRNA in vhs or ICP27 mutantinfected control cells

FIG. 4.6. Effect of loss of host shutoff function on the halflives of target and nontarget mRNAs

89

FIG. 4.7. Silencing suppression by HSV1 using a luciferase reporter assay. Firefly luciferase (Fluc), which was expressed from plasmid pCMVLucmiR30 (P) under the control of pCMV IE and contains 8 tandem target sequences of human miR30 in the 3’ UTR, was used as the silencing target. Human miR30 (the silencing inducer), and miR21 (the control), were expressed from the plasmid pH1miR30 or pH1miR21 under the control of a pol III H1 promoter. Renilla luciferase (Rluc), expressed from plasmid pRL under the control of the pCMV IE, was used as the target specificity and internal transfection control. BHK cells were cotransfected with plasmids as indicated. At 42 hr posttransfection, cells were either mockinfected or infected with HSV1 KOS at a m.o.i. of 5 PFU/cell. Cells were harvested 10 hr after infection. Lysates were made, and the activities of Fluc and Rluc were assayed. Experiments were conducted in triplicates. A: Schematic representation of the Fluc construct that contains the target sequences of miR 30 in the 3’ UTR and the predicted sizes of fulllength and cleaved mRNA. B: Effect of HSV1 on the activity of Rluc in silenced and control cells. C: Effect of HSV1 on the activity of Fluc in silenced and control cells.

90

2.3 kb

1.7 kb

Firefly luciferase

miR30 target sites

FIG. 4.7A. Schematic representation of the Fluc construct and the predicted sizes of fulllength and cleaved mRNA

Continued

91 FIG. 4.7 Continued

10 5 Mock Infected

10 4 c c 0 1 Renilla luciferase activity (Arb activity Renilla luciferase units) lu lu -3 -2 R F iR iR c+ m m lu c+ c+ R lu lu +F +F uc uc Rl Rl

FIG. 4.7B. Rluc activity in cells

10 5 Mock Infected

10 4

10 3 Relative firefly luciferase Relative firefly (Fluc/Rluc) activity 10 2 c 0 1 uc lu -3 -2 Fl R iR iR c+ m m lu c+ c+ F lu lu +R +R uc uc Fl Fl

FIG. 4.7C. Fluc activity in cells

FIG. 4.7. Silencing suppression by HSV1 using a luciferase reporter assay

92

FIG. 4.8. Effect of knockdown of Ago2 on HSV1 yield of progeny. HEK 293T cells were transfected with Ago2specific RNA duplex or a RISCfree RNA control duplex. Fortyeight hr posttransfection, silenced and control cells were infected with HSV1 KOS at a m.o.i of 1 or 10 PFU/cell. Cells were harvested 18hr after infection, and the virus yield of progeny was determined by plaque assay.

93

240 Control RNA duplex Ago-2 specific RNA duplex 200

160

120

80

Progeny yield (PFU/cell) 40

FIG. 4.8A. Effect of knockdown of Ago2 on HSV1 yield of progeny (imput m.o.i. of 1 PFU/cell)

2000 Control RNA duplex Ago-2 specific RNA duplex 1600

1200

800

Progenyyield(PFU/cell) 400

FIG. 4.8B. Effect of knockdown of Ago2 on HSV1 yield of progeny (imput m.o.i. of 10 PFU/cell)

FIG. 4.8. Effect of knockdown of Ago2 on HSV1 yield of progeny

94

CHAPTER 5

ATTEMPTS TO INDENTIFY A HSV1 SILENCING SUPPRESSOR

Kinetics of HSV1induced silencing suppression

To begin to identify the silencing suppressor(s) encoded by HSV1, the kinetics of silencing suppression activity were examined. Fortytwo hr posttransfection, silenced cells, which received the target expression plasmid pEGFPC2 and a midsEGFP expression plasmid pmidsEGFP, were either mockinfected or infected with HSV1

(strain KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested at various times after infection, total RNA was isolated, and levels of EGFP and βactin mRNAs were determined by northern blot analysis. The steadystate βactin mRNA levels in mock infected cells remained relatively constant over a 24 hr time period (Fig. 5.1A). By contrast, the steadystate βactin mRNA levels in HSV1 wildtype virusinfected cells decreased with time after HSV1 infection (Fig. 5.1A), consistent with previous findings

(see Fig. 4.4C and D). However, HSV1 infection increased the steadystate EGFP mRNA levels in silenced cells as early a 4 hr postinfection, whereas the steadystate

EGFP mRNA levels in mockinfected silenced cells decreased continuously following infection (Fig. 5.1B).

95 Expression profiles of HSV1 mRNAs

The timing for silencing suppression was compared to the expression profiles of

HSV1 genes known to be classified as immediateearly, early, or late. BHK cells were infected with HSV1 (KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested, and total

RNA was isolated. The steadystate ICP27 (immediate early), UL42 (early), US11 (true late) mRNA levels were examined by northern blot analysis, and were normalized to 18S rRNA. The maximum mRNA level for each gene was set as 100%. ICP27 mRNA was detectable by 4 hr postinfection, and the levels rapidly increased until 8 hr postinfection and remained relatively constant through 12 hr postinfection. However, the ICP27 mRNA levels dramatically deceased thereafter with no ICP27 mRNA detectable at 24hr postinfection (Fig. 5.2). UL42 mRNA was first detected at 6 hr postinfection and increased through 10 hr postinfection. The maximum UL42 mRNA level was observed

12 hr after infection, but gradually decreased thereafter. The mRNA of US11, a true late gene, was detected at 8 hr postinfection, and continued to accumulate through 24 hr postinfection. These expression profiles are consistent with those previously published

(Honess and Roizman, 1974).

To determine whether silencing suppression is dependent on viral DNA replication, the effect of phosphonoacetic acid (PAA) was examined. PAA inhibits HSV

1 DNA synthesis by binding to the DNA polymerase and preventing extension of the primer/template (Elliott et al., 1980). Expression of HSV1 true late genes (γ 2) requires

DNA replication. Therefore, PAA treatment not only blocks viral DNA replication, but

also prevents expression of γ 2 genes. BHK cells were cotransfected with the target

EGFPexpression plasmid, pEGFPC2, and a midsEGFPexpression plasmid,

96 pmidsEGFP. Cells were exposed to PAA (400 g/ml) or to PAAfree media 41 hr post transfection. One hr later, PAAtreated and PAAfree cells were either mockinfected or infected with HSV1 KOS (m.o.i. of 5 PFU/cell). Cells were collected at various times after infection, total RNA was isolated, and northern blot analysis was conducted as described preciously. In the mockinfected cells, regardless of the presence or absence of

PAA, the steadystate βactin mRNA levels remained relatively constant throughout the time course of the experiment (Fig. 5.3A). However, it was found that PAA treatment caused a slight increase in βactin mRNA levels in mockinfected cells. HSV1 infection resulted in a rapid reduction in βactin mRNA levels regardless of the presence of PAA

(Fig.5.3A). The steady state EGFP mRNA levels in mockinfected silenced cells decreased in the presence or absence of PAA. Following infection, the kinetics of EGFP mRNA accumulation was similar in the presence vs absence of PAA. However, PAA treatment did decrease EGFP mRNA levels in silenced cells. These results indicate that silencing suppression by HSV1 does not require viral DNA synthesis or de novo expression of HSV1 true late genes.

Testing of candidate HSV1 genes for silencing suppression in plants

The kinetics for silencing suppression by HSV1 was consistent with that for an immediate early or virion protein (see Fig. 5.1B and Fig. 5.2). Therefore, the silencing suppressor activities of HSV1 immediate early genes, ICP0, ICP4 and ICP27, and a virion component gene US11, were determined in well defined silencing suppression assay systems in plants (the work was performed by Kenneth Buckley in Dr. David

Bisaro’s lab at the Ohio State University). In the plant systems, all genes were cloned into binary plasmids and expressed under the control of the 35S promoter. The plasmid

97 constructs were transformed into Agrobacterium tumefacients for delivery into plants . In

the assays with the twocomponent system, GFP transgenic line 16c N. benthamiana plants were coninfiltrated with two plasmids one of which expressed the target GFP gene and the other expressed the test gene. In the threecomponent silencing suppression assay system (Fig. 5.4A), N. benthamiana plants were coinfiltrated by three plasmids one expressing GFP, one expressing the silencing inducer dsGFP and the third expressing the test gene. GFP fluorescence was monitored under UV light and GFP mRNA levels were assessed by northern blot analysis 58 days after coinfiltration. In the three component system, no green fluorescence was observed at the infiltration spots in the plants receiving the control plasmid, which expressed βglucuronidase (GUS). Instead, the spots appeared red, due to the inherent fluorescence of chlorophyll under UV light

(5.4B). Coinfiltration of p19, a known potent silencing suppressor in plants encoded by

Cymbidium ringspot virus (Lakatos et al., 2004), resulted in very bright green

fluorescence at the coinfiltration spot. Coinfiltration of HSV1 US11 resulted in GFP

fluorescence, though not intense as observed with p19 (Fig. 5.4B).

In the twocomponent system, weak GFP fluorescence was observed at the co

infiltration spot in plants receiving the control plasmid (Fig. 5.4B), indicating that GFP in

the twocomponent system was not silenced as effectively as in the threecomponent

system. Coinfiltration of p19 resulted in extremely bright green fluorescence. As

observed in the threecomponent system, coexpression of US11 significantly enhanced

GFP fluorescence compared with coinfiltraton of the control plasmid (Fig. 5.4C).

However, coinfiltration of HSV1 ICP0, ICP4 or ICP27 both in the three and two

98 component systems had no significant effects on GFP fluorescence compared with the control plasmid (data not shown there).

The suppression of GFP silencing by p19 and US11 was also confirmed by northern blotting. In the twocomponent assay system, p19 coinfiltration led to remarkable accumulation of GFP mRNA (5fold RNA less loading than other samples) compared with the control (Fig. 5.4D). The GFP mRNA level in US11 coinfiltrated plants was lower than that in p19 coinfiltrated plants, but coinfiltration of HSV1 US11 resulted in a much higher GFP mRNA level compared with GUS coinfiltration. Co infiltration of HSV1 ICP0, ICP4 or ICP27 did not affect GFP mRNA levels significantly compared with the control (Fig. 5.4D). Similar effects of p19 and HSV1 US11 on GFP mRNA levels were also observed in threecomponent system assays (data not shown there). These results suggest US11 can function as a silencing suppressor in plants without the need of expression of other HSV1 genes.

Effects of infection by a US11 null mutant on silencing suppression

We predicted that US11 could suppress silencing in the context of HSV1 replication. Therefore, we determined the effects of infection by a US11 null mutant, a rescued virus, and the wildtype virus on accumulation of the target EGFP mRNA and the halflife of EGFP mRNA in silenced cells. Because no US11 null mutant in the KOS background was available, we tested one constructed in the 17syn+ background. BHK cells were cotransfected with the EGFPexpression plasmid pEGFPC2 and a plasmid expressing midsEGFP or midsLac Z. At 42hr posttransfection, cells were either mock infected or infected by a US11 null mutant (1711), the recuant (1711R) (1711 and 1711R were obtained from David leib at the University of Washington) or the isogenic wildtype

99 virus (17syn+) at a m.o.i. of 5 PFU/cell. Cells were harvested 10 hr after infection, and total RNA was isolated. The steadystate EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. US11 mutant, the recuant, and the wildtype virus reduced the steady state βactin mRNA levels in cells silenced with midsEGFP 3.6, 3 and 3.4fold, respectively, compared with mock infection

(Fig. 5.5A). Similar reductions in the steadystate βactin mRNA levels in control cells infected with either of the three viruses were also observed compared with mock infection. Infection by the US11 mutant, the US11 rescuant, or the wild type (17syn+) resulted in 3.7, 2.6, 3fold decrease in the steadystate βactin mRNA level in control cells compared with mock infection (Fig. 5.5A). These results suggest that HSV1

17syn+ like HSV1 KOS affects accumulation of βactin mRNA (Fig. 4.1A). However, infection by the US11 mutant, the rescuant, or the wildtype HSV1 17 syn+ had no significant effect on the steadystate EGFP mRNA level in control cells compared with mock infection (Fig. 5.5B). The steadystate EGFP mRNA level in control cells infected with the US11 mutant, the US11 rescuant or the wildtype virus were 90, 110 and 120% relative to that in mockinfected control cells. In contrast, the wildtype virus increased the steadystate EGFP mRNA level in silenced cells by 1.5fold compared with mock infection. Infection by US11 mutant enhanced the steadystate EGFP mRNA level in silenced cells by only 25% compared with mock infection. The US11 rescuant increased the steadystate EGFP mRNA level in silenced cells by 78%. This enhancement was significantly less than that by the wildtype infection (p < 0.01), but significantly greater than that observed with the US11 mutant (Fig. 5.5B) ((p < 0.01).

100 To further evaluate the function of US11 in silencing suppression in mammalian cells, we examined the effects of infection by these viruses on the halflife of EGFP mRNA in silenced cells. BHK cells were cotransfected with pEGFPC2 and a plasmid expressing either midsEGFP or midsLac Z. At 42 hr posttransfection, cells were either mockinfected or infected with viruses at a m.o.i. of 5 PFU/cell. Act D (5g/ml) was applied to cells 6 hr postinfection. Cells were harvested at various times after the addition of Act D, and total RNA was isolated. Steadystate EGFP and βactin mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA.

Halflive curves for EGFP and βactin mRNAs were plotted as described in Chapter 2.

Infection by US11 mutant, the rescuant or the wildtype virus reduced the halflife of β actin mRNA in control cells by ≈ 3fold compared with mock infection (the halflife of β actin mRNA in mockinfected control cells was 19.7 hr, Fig. 5.6A). Similar results were obtained in silenced cells by comparing infection by the US11 null mutant, the rescuant or the wildtype virus with mockinfection (Fig. 5.6B). Infection by US11 mutant, the rescuant or 17syn+ reduced the halflife of βactin mRNA in silenced cells from 20 hr to

6.1, 6.6, or 6.8 hr, respectively. These results indicate that infection by HSV1 17syn+ virus reduces the stability of βactin mRNA more than the KOS wildtype strain (Fig.

4.4). Infection of control cells by US11 mutant, the rescuant or the wildtype virus resulted in a comparable halflife of EGFP mRNA (≈ 4 hr). These halflives of EGFP mRNA were shorter than the halflife observed in mockinfected control cells (5.5 hr) as found for the KOS wildtype virus (Fig. 4.4C). Surprisingly, all the halflives of EGFP mRNA in the US11 mutant, the rescuant and the wildtype infected silenced cells were shorter than that in mockinfected silenced cells (2.6 hr) (Fig. 5.6D). However, the half

101 life of EGFP mRNA in the wildtypeinfected silenced cells (1.8 hr) was significantly longer than that in US11 mutantinfected silenced cells (1.2 hr. Fig. 5.6D). In contrast, no significant difference in the halflife of EGFP mRNA in the US11 mutant and the rescuantinfected silenced cells was observed.

These results indicated that in the absence of US11, HSV1 17syn+ almost lost the ability to enhance the target mRNA level in silenced cells, and a further reduction in the halflife of the target mRNA in the mutantinfected silenced cells was observed compared to that observed in silenced cells infectd with 17syn+ wt virus.

102

FIG. 5.1. Kinetics of silencing suppression by HSV1. Silenced cells were either mock infected or infected with HSV1 (KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested at various times after infection, total RNA was isolated, and steadystate βactin (A) and EGFP (B) mRNA levels were determined by northern blot analysis. The steadystate β actin and EGFP mRNA levels at time 0 (the time cells were infected) were set as 100%.

103

100

HSV-1 infected Mock infected 50 -actin mRNA-actinamount β β β β

Relative Relative 0 0 4 8 12 16 20 24 Time (hr) after infection

FIG. 5.1A. βactin mRNA levels in mock and HSV1 infected silenced cells

250

200 HSV-1 infected Mock infected 150

100

50

Relative EGFP mRNA amount mRNA EGFP Relative 0 0 4 8 12 16 20 24 Time (hr) after infection

FIG. 5.1B. EGFP mRNA levels in mock and

HSV1 infected silenced cells

FIG. 5.1. Kinetics of silencing suppression by HSV1

104

FIG. 5.2. Expression profiles of representative immediate early, early and late HSV1 genes. BHK cells were infected with HSV1 (KOS) at a m.o.i. of 5 PFU/cell. Cells were harvested at various times after infection, and total RNA was isolated. Steady state ICP27 (immediate early), UL42 (early) and US11 (true late) mRNA levels were determined by northern blot analysis using probes generated from PCR products described in Chapter 2, and were normalized to 18S rRNA. The maximum accumulation level for each gene was set as 100%.

105

120 ICP27 100 UL42 US11 80

60

40

Relative mRNA level level mRNA Relative 20

0 0 4 8 12 16 20 24 Time (hr) after infection

FIG. 5.2. Expression profiles of representative immediate early, early and late HSV1 genes

106

FIG. 5.3. Effect of the HSV1 DNA synthesis inhibitor, PAA, on the ability of HSV1 to suppress silencing. At 41 hr posttransfection, silenced cells, which received plasmids expressing EGFP and midsEGFP, were exposed to 400 g/ml phosphonoacetic acid (PAA). One hr later, PAAtreated and PAAfree cells were either mockinfected or infected with HSV1 KOS (a m.o.i. of 5 PFU/cell). Cells were harvested at various times after infection, and total RNA was isolated. Steadystate βactin (A) and EGFP (B) mRNA levels were determined by northern blot analysis, and were normalized to 18S rRNA. The steady state EGFP and βactin mRNA levels at time 0 (the time when cells were infected) were set as 100%.

107 140

120

100 Mock, no PAA 80 Mock + PAA 60 Infected, no PAA Infected + PAA -actin -actin mRNA amount β β β β 40

20

Relative 0 0 4 8 12 16 20 24 Time (hr) after infection

FIG. 5.3A. βactin mRNA levels in PAAfree, PAAtreated mock or HSV1 infected silenced cells

Mock, no PAA 200 Mock + PAA Infected, no PAA 150 Infected + PAA

100

50

Relative EGFP mRNA amount mRNA EGFP Relative 0 0 4 8 12 16 20 24 Time (hr) after infection

FIG. 5.3B. EGFP mRNA levels in PAAfree, PAAtreated mock or HSV1 infected silenced cells

FIG. 5.3: Effect of the HSV1 DNA synthesis inhibitor, PAA, on the ability of HSV1 to suppress silencing.

108

FIG. 5.4. Silencing suppression assay by HSV1 genes in plants. In the plant silencing suppression assay systems, all genes were cloned into binary plasmids and under the control of 35S promoter and all constructs were introduced into agrobaterium. In the assays with the twocomponent system, GFP transgenic line 16c N. benthamiana plants were coninfiltrated with two plasmids expressing the target GFP and a test gene. In the three component silencing suppression assays, N. benthamiana plants were coinfiltrated with three plasmids, one expressing the target GFP, and one expressing the silencing inducer dsGFP, and the third one expressing a test gene. GFP fluorescence was monitored under UV light, and GFP mRNA levels were determined by northern blot analysis 58 d after coinfiltration. In the assays, p19, a known silencing suppressor in plants, was used as the positive control. Glucuronidase (GUS) was used as the negative control. Three leaves per plant were coinfiltrated and four plants were used for one gene assay. A: Schematic representation of the threecomponent silencing suppression assay system. B: GFP fluorescence with the threecomponent assay. C: GFP fluorescence with the twocomponent assay D: GFP northern blot with the twocomponent assays.

109

Component 1: 35S-GFP GFP

Component 2: 35S - GFP GFP dsGFP Component 3: test Protein, RNAi, inhibitor construct

Constr ucts transformed into Agrobacterium The three - tumefacients for delivery component to N. benthamiana cells by silencing assay agroinfiltration

• UV Inspection

• GFP mRNA blot

FIG. 5.4A. Threecomponent silencing suppression assay system

Continued

110

FIG. 5.4 Continued

FIG. 5.4B. GFP fluorescence in 3component assay

FIG. 5.4C. GFP fluorescence in 2component assay

FIG.5.4D. GFP northern blot with 2component system

FIG. 5.4. Silencing suppression assay by HSV1 genes in plants

111

FIG. 5.5. Effects of infection by HSV1 US11 null mutant, the rescuant or the wild type virus on the target EGFP and nontarget β–actin mRNA levels. BHK cells were cotransfected with an EGFP expression plasmid and a plasmid expressing either midsEGFP or midsLac Z. At 42 hr posttransfection, cells were either mockinfected or infected with the US11 null mutant, the rescuant virus or the isogenic wildtype virus, 17syn+ at a m.o.i. of 5 PFU/cell. Viral genes were allowed to be expressed for 6 hr. Cells were harvested, and total RNA was isolated. The steadystate β –actin (A) and EGFP (B) mRNA levels were determined by northern blot analysis, and normalized to 18S rRNA. The steady state EGFP and β–actin mRNA levels in mockinfected silenced cells were set at 1.

112

3.0

2.5 Silenced cells Control cells 2.0

1.5 -actin mRNAlevel β β β β 1.0

0.5

Relative 0.0 k + 1 R c n 71 1 Mo sy 1 71 17 1

FIG. 5.5A. βactin mRNA levels levels

10 Silenced cells Control cells 8

6

4

2 RelativeEGFP mRNA level 0 k + 1 R c n 71 1 Mo sy 1 71 17 1

FIG. 5.5B. EGFP mRNA levels

FIG. 5.5. Effects of infection by HSV1 US11 null mutant, the rescuant or the wildtype virus on the target EGFP and nontarget β–actin mRNA levels

113

FIG. 5.6. Effects of infection by HSV1 US11 null mutant, the rescuant or the wild type virus on the halflives of the target EGFP and β–actin mRNAs. BHK cells were cotransfected with an EGFP expression plasmid and a plasmid expressing either midsEGFP (silenced) or midsLac Z (control). At 42 hr posttransfection, cells were either mockinfected or infected at a m.o.i. of 5 PFU/cell. Viral genes were allowed to be expressed for 6 hr. Cells were exposed to 5 g/ml Act D, and were harvested at various times after Act D treatment. Total RNA was isolated. The steadystate β–actin and EGFP mRNA levels were determined by northern blot analysis, and normalized to 18S rRNA. The steady state EGFP and β–actin mRNA levels at time 0 (the time when Act D was applied) were set as 100%, and halflives for EGFP and β –actin mRNAs were plotted as described in Chapter 2. A: Effects of infection by the US11 null mutant, the rescuant or the wildtype virus on the halflife of β–actin mRNA in control cells. B: Effects of infection by US11 null mutant, the rescuant or the wildtype virus on the halflife of β– actin mRNA in silenced cells. C: Effects of infection by the US11 null mutant, the rescuant or the wildtype virus on the halflife of EGFP mRNA in control cells. D: Effects of infection by the US11 null mutant, the rescuant or the wildtype virus on the halflife of EGFP mRNA in silenced cells.

114

Mock t = 19.7 hr 120 1/2 US11 t =6 hr 100 1/2 US11R t 1/2 = 6.4 hr 80 17syn+ t 1/2 = 6.3 hr 60

-actin mRNA-actin amount 40 β β β β 20

Relative 0 0 4 8 12 16 20 Time (hr) after Act D treatment

FIG. 5.6A. Halflife curves for βactin mRNA in

control cells

120 Mock t 1/2 = 20 hr US11 t 1/2 = 6.1 hr 100 US11R t 1/2 = 6.6 hr 17syn+ t = 6.8 hr 80 1/2

60

-actin mRNA -actin amount 40 β β β β 20

Relative 0 0 4 8 12 16 20 Time (hr) after Act D treatment

FIG. 5.6B. Halflife curves for βactin mRNA in silenced cells

Continued

115

FIG. 5.6 Continued

100 Mock t 1/2 = 5.5 hr 80 US11 t 1/2 = 4.0 hr US11R t = 4.2 hr 60 1/2 17syn+ t 1/2 = 4.1 hr 40

20

Relative mRNAEGFP amount 0 0 5 10 15 20 Time (hr) after Act D treatment

FIG. 5.6C. Halflife curves for EGFP mRNA in control cells

100 Mock t = 2.6 hr 80 1/2 US11 t 1/2 = 1.2 hr 60 US11R t 1/2 = 1.3 hr

17syn+ t 1/2 = 1.8 hr 40

20

Relative mRNAEGFP amount 0 0 5 10 15 20 Time (hr) after Act D treatment

FIG. 5.6D. Halflife curves for EGFP mRNA in silenced cells

FIG. 5.6: Effect of infection by US11 null mutant, the rescuant or the wild type on the halflife of the target EGFP mRNA in silenced cells

116

CHAPTER 6

DISCUSSION

RNA interference (RNAi) is a mechanism by which small RNAs (2125 nt)

inhibit gene expression in a sequencespecific manner. Collectively, interfering RNAs

function to regulate development and growth among almost all organisms (Baulcombe,

2004; Brodersen and Voinnet, 2006; Ding et al., 2004; Wienholds and Plasterk, 2005). It

has been demonstrated that RNAi plays a critical role in antiviral defense in plants,

insects and animals (Anandalakshmi et al., 1998; Bayne et al., 2005; Bisaro, 2006; Lu

and Cullen, 2004, Vanitharani et al., 2005). Moreover, it has been found that several

mammalian viruses are the targets of the host’s RNAi response and in turn encode

silencing suppressors to antagonize this host antiviral effect for optimal viral replication

(Lecellier et al., 2005; Pfeffer et al., 2004; Omoto et al., 2004; Bennasser et al., 2005).

Therefore, it is increasingly accepted that RNAi is involved in the antiviral defenses in

mammalian cells. However, whether or not HSV1 infection elicits the host’s RNAi

response and whether HSV1 encodes silencing suppressor(s) are not clear.

Mammalian cells mount a RNAi response to inhibit HSV1 replication

Numerous doublestranded RNAs are generated through symmetric transcription of both strands of viral DNA during lytic infection (Jacquemont and Roizman, 1975a;

Jacquemont and Roizman, 1975b; Kozak and Roizman, 1975), which could activate the 117 host RNAi pathway. In addition, computational screening of the HSV1 genome suggests that HSV1 could encode 13 miRNA precursors and 24 miRNAs, and one of the predicted miRNAs has been experimentally identified (Cui et al., 2006). The function of this HSV1 encoded miRNA is not known, but the possibility cannot be ruled out that cells might use this miRNA to target the virus. Therefore, we hypothesized that HSV1 induces the host’s RNAi response. The prediction of this hypothesis is that inhibition of the host RNAi pathway could enhance HSV1 replication. Indeed, knockdown of Ago2, the effector slicer in RISC responsible for mRNA cleavage, increased HSV1 yield of progeny, indicating that HSV1 is the target of the host’s RNAi response. These results demonstrate that in addition to the innate interferon response, mammalian cells also mount a RNAi response to inhibit HSV1 infection. These results combined with those of others (Lecellier et al., 2005; Pfeffer et al., 2004; Omoto et al., 2004; Bennasser et al.,

2005) further suggest that RNA silencing is a general mechanism utilized as a mammalian cell antiviral defense.

Establishment of optimum conditions to achieve maximum silencing

We predicted that HSV1 encodes one or more silencing suppressor(s) to antagonize the host RNAi response. To test the hypothesis, a transient silencing system was developed. In our initial studies, we observed that a perfect RNA hairpin decreased the steadystate target mRNA level compared with control hairpin (Fig. 3.1D) but the effect was not great and was highly variable from experiment to experiment (data not shown). However, imperfect RNA hairpin resulted in a remarkable reduction in the steadystate target mRNA level compared with the imperfect control hairpin (Fig. 3.1C).

These results indicate that imperfect RNA hairpin is more effective than perfect RNA

118 hairpin in silencing the target. Less efficacy of perfect RNA hairpin than imperfect RNA hairpin in silencing the target might be due to the function of adenosine deaminases

(ADAR). ADARs catalyze hydrolytic deamination of adenosines to inosines within perfectly doublestranded RNAs (dsRNAs), which results in degradation of dsRNAs within the nucleus (Källman et., 2003; Klaue et al., 2003). Therefore, perfect dsRNAs could be less stable than imperfect RNA hairpins.

Titration experiments indicated that silencing effects decreased when the molar ratio of the imperfect inducer hairpin to the target plasmid was lower than 1:1. However, no additional enhancement of silencing was observed when the molar ratio of the imperfect inducer hairpin to the target plasmid was greater than 1:1 (Fig. 3.2). These results suggest that 1:1 molar ratio of the silencing inducer to the target is sufficient to maximally silence the target.

HSV1 encodes one or more silencing suppressors

Although our results suggested that HSV1 infection induces the host’s RNAi

response, it was not known whether or not HSV encodes silencing suppressor(s) to

counter this antiviral effect. We observed that HSV1 (KOS) enhanced the steadystate

level of target mRNA level in silenced cells (Fig. 4.1). However, because the transcript

expressed from plasmid pEGFPC2, does not contain an intron, it was possible that this

enhancement of the target mRNA level might have been due to the more effective export

of intronless target out of the nucleus in infected cells (Chen et al., 2005; Chen et al.,

2002). However, this possibility was ruled out since HSV1 infection also increased the

steadystate level of the target mRNA with an intron in the transcript in silenced cells

(Fig. 4.3).

119 The increase in target mRNA amount in silenced cells by HSV1 could have been due to increased expression of the target gene, increased mRNA stability, or both. To address these possibilities, the effect of HSV1 infection on the halflife of target and no target mRNAs was determined. It was observed that HSV1 infection reduced the half life of a nontarget βactin mRNA in silenced and control cells, and decreased the half life of the target mRNA in control cells compared with mock infection (Fig. 4.4C and D).

These results are consistent with the inherent ability of wildtype HSV1 to generally destabilize mRNAs (Sorenson, et al., 1991). Nevertheless, HSV1 infection increased the halflife of the target mRNA in silenced cells and this halflife was comparable to that observed in nonsilenced (control) cells infected with HSV1 (Fig. 4.4E and F). Taken together, these results indicate that HSV1 is capable of completely reversing the silencing induced by an imperfect RNA hairpin directed to the target. The results noted above established that the halflife of the target mRNA in both silenced and control cells infected with HSV1 was shorter than that in mockinfected control cells (Fig. 4.4E and

F). Interestingly, it was found that viruses with lossoffunction mutations in host shutoff genes increased the steadystate target mRNA levels in silenced cells to an even greater extent than the isogenic wildtype (strain KOS) virus (Fig. 4.5B). Overall, the halflives of the target mRNA in silenced and control cells were comparable to that observed in mockinfected control cells (Fig. 4.6C and D), supporting my hypothesis that HSV1 can completely suppress imperfect hairpininduced RNA silencing.

HSV1 suppresses miRNAinduced silencing effectively

Small interfering RNA (siRNA) and miRNA pathways share several key components. HSV1 suppressed siRNAinduced silencing. Thus, we predicted that HSV

120 1 could suppress miRNAinduced silencing. It was found that HSV1 infection of silenced cells greatly increased the activity of the target reporter gene firefly luciferase, which contains miR30 target sequences in the UTR compared to controls (Fig. 4.7).

These results indicate that HSV1 infection can also suppress miRNAinduced silencing in mammalian cells. It is possible that silencing suppressor(s) encoded by HSV1 could target a common component shared by siRNA and miRNA pathways. However, whether silencing suppression is global and the mechanism for silencing suppression by HSV1 protein(s) is not clear and needs to be elucidated.

Silencing suppressor activity of HSV1 could be an immediate early gene product or a virion component

To identify the silencing suppressor(s) encoded by HSV1, we compared the timing of silencing suppression with the accumulation profiles of HSV1 immediate early, early and late genes, and determined the effect of HSV1 DNA synthesis inhibitor,

PAA, on the ability of HSV1 (KOS) to suppress silencing in the transient silencing system. Results indicated that enhancement of the target mRNA level in silenced cells occurred as early as 4 hr postinfection (Fig. 5.1B) and with a pattern similar to that of immediate early mRNAs (Fig. 5.2). Morevover, silencing suppression occurred with similar kinetics in the presence of the viral DNA polymerase inhibitor, PAA, and therefore, silencing suppression was not dependent upon viral DNA synthesis (Fig. 5.4).

These results suggest that silencing suppressor activity encoded by HSV1 could be an immediateearly gene or a virion component.

HSV1 encodes a silencing suppressor. However, silencing suppression was less effective at the late stages of infection (Fig. 5.1B). It has been shown that HSV1 encoded

121 premiRNAs are generated as early as 4 hr postinfection and a virusencoded mature miRNA accumulates beginning at 8 hr postinfection (Cui et al., 2006). These results suggest that HSV1 block the host’s RNAi response in favor of viral replication at early stages of infection. However, the decreased ability of HSV1 to suppress silencing at the late stages of HSV1 infection allows for cellular and viral encoded miRNAs to be produced. The virus might use the virusencoded miRNA to attack a cellular gene, thus modulating the cellular environment for optimum viral replication. Alternatively, the host might utilize the virusencoded miRNA or cellular miRNAs to target the virus, therefore, limiting viral replicarion (Fig. 4.8).

HSV1 US11 can function as a silencing suppressor in plants

To identify silencing suppressor(s) encoded by HSV1, we determined the effects of HSV1 immediate early candidate genes, ICP0, ICP4 and ICP27, and a virion component candidate gene, US11, on silencing in well defined silencing suppression assay systems in plants. It was found that US11 can suppress silencing in plants in the absence of other HSV1 genes (Fig. 5.3B, C and D). This finding is interesting because all identified silencing suppressor(s) encoded by mammalian viruses to date, are PKR antagonists. These results together with those of others suggest that PKR antagonists are good candidates for silencing suppressors encoded by mammalian viruses (Lecellier et al., 2005; Pfeffer et al., 2004; Omoto et al., 2004; Bennasser et al., 2005).

US11 might be involved in silencing suppression by HSV1 in mammalian cells

US11 can suppress silencing in plants in the absence of any other HSV1 gene.

This result prompted us to hypothesize that US11 plays a critical role in silencing suppression by HSV1 in mammalian cells. US11 is a γ2 gene, expressed very late during

122 HSV1 lytic infection (Roller and Roizman, 1992). However, US11 can function early during HSV1 infection since it is virion component (Roller and Roizman, 1992).

Moreover, US11 possesses all the functions of NS1, a known silencing suppressor encoded by influenza A virus (Chien et al., 1997; Chien et al., 2004; Wang et al., 1999;

Liu et al., 1997; Qian et al., 1995; Poppers et al., 2000; Roller et al., 1996). Both NS1 and

US11 have dsRNAbinding activity and interact with PACT to inhibit activation of PKR.

Therefore, we determined the effects of infection by a US11 null mutant, its rescued virus, and the wildtype virus on accumulation of the target EGFP mRNA and the half life of target mRNA in silenced cells. It was found that the US11 mutant (in 17syn+ background) almost lost the ability of HSV1 to increase the target mRNA level in silenced cells compared with mock infection. Also, it was found that infection of silenced cells by the US11 mutant resulted in a significantly shorter halflife of the target mRNA compared with infection by the wild type (17syn+). However, no solid conclusion can be made since the phenotypes of the US11 mutant with a compromised ability to suppress silencing were only partially rescued for the US11 rescuant. The deficiency in rescuing the phenotypes of the US11 mutant by the rescuant could be due to an unknown secondary mutation (personnel communication from David Leib at the University of

Washington). To understand the possible role of US11 in silencing suppression by HSV

1, a US11 mutant and its rescuant in KOS background need to be constructed, and the effects of the mutant and the rescuant on steadystate target mRNA level and the halflife of the target mRNA in silenced cells needs to be determined. Alternatively, experiments should be conducted to determine the effect of knockdown of the RNAi pathway on the mutant and the rescuant yields of progeny.

123 Surprisingly, no significant effect of expression of US11 on silencing in a transient silencing system has been observed to date (data not shown here). However,

US11 can replace HIV Tat protein as a silencing suppressor in a HIV mutant clone system (personal communications from Ben Berkhout at the Academic Medical Center of the University of Amsterdam, Netherlands). Results taken together suggest that US11 plays an important role in silencing suppression, but this function needs to be in the context of viral replication. One possible explanation is that US11 needs to work with another protein encoded by the virus to suppress silencing. Alternatively, phosphorylation of US11 by PKR, which occurs during HSV1 lytic infection, may be required for US11 to suppress silencing. Also, there is another possibility, i.e, US11 can prevent establishment of silencing but not reverse it since our assays for the function of US11 in silencing suppression were conducted in a transient expression system. To address the possibilities, a US11expression cell line should be generated and the effect of stable expression of US11 on silencing needs to be determined.

124

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