Cardiac Cell Type-Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN

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ABSTRACT

ZURNEY, JENNIFER M. Cardiac Cell Type‐Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN. (Under the direction of Dr. Barbara Sherry).

Viral myocarditis is an important human disease associated with a wide variety of viruses. The cardiac damage and inflammation associated with viral myocarditis can be immune mediated and/or due to direct cytopathic effect. Reovirus‐induced myocarditis reflects direct virus‐mediated apoptosis of cardiac cells, providing an excellent model to

study direct cytopathic effect in the heart. Previous work has found interferon‐beta (IFN‐β) to be an important determinant for protection against viral myocarditis. Importantly, IFN signals through the Jak‐STAT pathway to induce the expression of interferon‐stimulated genes (ISGs) and establish an antiviral state. First, we investigated the underlying

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described for any virus. The M1 gene has also previously been identified as a determinant of virus strain‐specific differences in the IFN response, and the M1 gene and the IFN response have been identified as determinants of virus strain‐specific differences in

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Cardiac Cell Type‐Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN

by

Jennifer Michelle Zurney

A dissertation submitted to the Graduate Faculty of North Carolina State University

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Microbiology

Raleigh, North Carolina July 1, 2008

APPROVED BY:

_______________________________ ______________________________ Dr. Barbara Sherry Dr. Michael L. Sikes

Committee Chair

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ii

DEDICATION

To my husband, Brian Shopmyer, and my parents, whose patience, understanding, and

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BIOGRAPHY

Jennifer Michelle Zurney was born in Michigan and spent her childhood moving around the

world. Finally settling in North Carolina, she began undergraduate studies at North Carolina

State University in the department of microbiology. After receiving her Bachelor of Science

degree in microbiology with a minor in genetics in 2003, Jennifer went to Washington DC to

intern with the federal affairs department for the University of North Carolina. After two

months in Washington DC, she returned to North Carolina State University to pursue

graduate studies in the Microbiology department under the direction of Dr. Barbara Sherry.

After completing her dissertation defense, Jennifer will continue her scientific training as a

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iv

ACKNOWLEDGMENTS

Special thanks to the following people, without whom this work could not have been

possible:

Dr. Barbara Sherry, for her guidance and support throughout this project, and most

importantly, for her open‐door policy. Thanks to all the members of the Sherry lab for their

support and contributions. I would like to extend my appreciation to Drs. Tim Petty, Rob

Smart, and Mike Sikes, for serving as committee members and providing recommendations

and suggestions that have been invaluable to this project. In addition, I would like to thank

all members of the Microbiology department for their guidance and direction. A special

thanks to my agent, TJ Schneeweis and to my undergraduate mentor, Dr. Michael Hyman.

Finally, thanks to my husband, parents, and friends, who endured this long process

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TABLE

OF

LIST

OF

TABLES

Page

Table 3.1. Repression of IFN‐induced IRF7 mRNA segregates with the T1L M1 and S2 gene segments and is associated with induction of myocarditis ... 125

Table 1. Summary of antibody cross‐reactivity with μ1/μ1c... 160

Table 2.1. Summary of VIB Formation and IFN‐β Production of

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viii

LIST

OF

FIGURES

Page

CHAPTER 1: LITERATURE REVIEW

Figure 1.1. TLR3‐dependent pathway for IFN‐β induction by dsRNA... 36

Figure 1.2. RIG‐I‐ and MDA5‐dependent signaling for IFN‐β induction by dsRNA ... 37

Figure 1.3. Positive feedback regulation of type I interferon genes ... 38

Figure 1.4. JAK‐STAT signal transduction pathway... 39

Figure 1.5. Schematic representation of the primary structure of Janus kinases (JAKs) and (STATs) signal‐transducer‐ and‐activator‐of‐ transcription, and interferon regulatory factor 9 (IRF9)... 40

Figure 1.6. Regulatory regions of ISG561 and IRF7 ... 41

CHAPTER 2: Basal Expression of IFNAR and Jak‐STAT Components are Determinants of Cell Type‐Specific Differences in the Cardiac Antiviral Response Figure 2.1. Cardiac myocytes express higher basal IFN‐β mRNA, nuclear activated ISGF3 components, and ISG561 mRNA expression than cardiac fibroblasts. ... 78

Figure 2.2. Basal expression of phosphorylated STAT‐1 in cardiac sections ... 79

Figure 2.3. Basal expression of IFN provides greater protection for cardiac myocytes than for cardiac fibroblasts... 80

Figure 2.4. Cardiac fibroblasts are more responsive than cardiac myocytes to IFN for induction of ISG561 mRNA ... 81

Figure 2.5. Multiple basal cytoplasmic Jak‐STAT components are expressed at higher levels in cardiac fibroblasts than in cardiac myocytes... 83

Figure 2.6. Basal IFNAR expression in cardiac fibroblasts and in cardiac myocytes ... 84

Figure 2.7. Cardiac fibroblasts are more responsive than cardiac myocytes to IFN for induction of STAT phosphorylation ... 85

Figure 2.8. Reovirus activation of multiple nuclear ISGF3 components is greater in cardiac fibroblasts than in cardiac myocytes... 86

Figure 2.9. IFN‐β dependent inhibition of T3D replication is greater in cardiac fibroblasts than in cardiac myocytes ... 87

Figure 2.10. Association of myocytes and fibroblasts in normal mouse myocardium... 88

Figure 2.11. Cardiac myocytes do not inhibit viral replication in adjacent cardiac fibroblasts... 89

Figure 2.12. Model for cell type‐specific IFN responses in the heart... 90

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CHAPTER 3: The Reovirus μ2 Protein Inhibits IFN Signaling Through a Novel Mechanism Involving Nuclear Accumulation of Interferon Regulatory Factor‐9

Figure 3.1. T1L represses IFN‐induction of IRF7 and STAT1, but not ISG56. ... 121

Figure 3.2. Analysis of reovirus reassortants for repression of IFN‐induced IRF7 mRNA ... 124

Figure 3.3. Analysis of reovirus recombinants for repression of IFN‐induced IRF7 mRNA ... 127

Figure 3.4. Reovirus T1L M1 inhibits IFN induction of an ISRE promoter ... 128

Figure 3.5. T1L does not degrade or inhibit translocation of STATs... 129

Figure 3.6. The T1L‐M1 gene mediates accumulation of IRF9 in the nucleus... 132

APPENDIX 1: Antibody Cross‐Reactivity with Reovirus μ1/μ1c Protein App. 1.1. Antibodies to STAT1α cross‐react specifically with reovirus μ1/μ1c... 149

App. 1.2. Antibodies to STAT2 cross‐react specifically with reovirus μ1/μ1c ... 150

App. 1.3. Antibodies to IRF9 cross‐react specifically with reovirus μ1/μ1c ... 151

App. 1.4. Antibodies to IRF3 cross‐react specifically with reovirus μ1/μ1c ... 152

App. 1.5. Antibodies to HSP90 cross‐react specifically with reovirus μ1/μ1c... 153

App. 1.6. Antibodies to actin, cdk4, cyclin D2, and GAPDH do not cross‐react with reovirus μ1/μ1c. ... 154

App. 1.7. Antibodies to GST, Rad50, tyrosine phosphorylated STAT1, and tyrosine phosphorylated STAT2 do not cross‐react with reovirus μ1/μ1c ... 155

App. 1.8. Antibody cross‐reactivity to reovirus μ1/μ1c is not due to the secondary antibody... 156

App. 1.9. Mass spectrometry results that identified μ1 as the viral protein cross‐reacting with STAT1α antibodies ... 157

App. 1.10. Visual representation of regions that antibodies were created against... 158

App. 1.11. Model for antibody cross‐reactivity with reovirus μ1/μ1c ... 159

APPENDIX 2: Viral Inclusion Body Morphology and IFN‐β Induction by Reassortant Viruses App. 2.1. Viral inclusion body morphology in reovirus infected L929 cells... 167

APPENDIX 3: Inhibition of HDAC Activity App. 3.1. Inhibition of HDACs by TSA treatment inhibits IFN induction of ISG561 mRNA... 175

App. 3.2. Inhibition of HDACs by TSA treatment inhibits IFN induction of IRF7 mRNA ... 176

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x

App. 3.3. Inhibition of HDACs by TSA treatment inhibits T3D induction of

IFN‐β mRNA... 177 App. 3.4. Inhibition of HDACs by TSA treatment inhibits T3D induction of ISG561 mRNA... 178 App. 3.5. Inhibition of HDACs by TSA treatment inhibits T3D induction of IRF7 mRNA ... 179

Summary

Figure 4.1. IFN‐β is anti‐proliferative for cardiac fibroblasts... 184

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CHAPTER

1 LITERATURE

REVIEW

Reovirus

Reoviruses (Respiratory Enteric Orphan viruses) are non‐enveloped, cytoplasmically

replicating viruses comprised of two concentric protein capsids surrounding a genome

consisting of ten discrete segments of double‐stranded RNA (dsRNA). These dsRNA

segments are divided into large (L), medium (M), and small (S) size classes, based on their

migration on a polyacrylamide gel (141, 162). Each dsRNA segment encodes a single

protein, except for the S1 gene segment, which is bicistronic, resulting in two translation

products, the nonstructural protein (σ1s) and the structural protein (σ1). Importantly, σ1

has been identified as the reovirus cell‐attachment protein (80) and the viral hemagglutinin

(165). The other three small gene segments (S2, S3, and S4) encode two structural proteins

(σ2 and σ3) and one nonstructural protein (σNS), respectively. The medium gene segments

(M1, M2, and M3) encode two structural proteins (μ2 and μ1) and one nonstructural

protein (μNS), respectively. The large gene segments (L1, L2, and L3) encode three

structural proteins, including the RNA‐dependent RNA polymerase (λ3 encoded by L1) (150)

and the guanylyltransferase (λ2 encoded by L2) (23). In addition to the dsRNA genome,

reovirus virions contain small, single‐stranded oligonucleotides comprising up to 25% of the

RNA in purified virions (11, 140). These oligonucleotides are phosphorylated (mono‐, di‐, or

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2

within the virion remain unknown (53), however, they appear to be dispensable for

infectivity (18, 39).

Reovirus virions enter a cell through receptor‐mediated endocytosis delivering the

virions into vacuoles that resemble endosomes or lysosomes (14, 15, 37). In these vacuoles,

reovirus outer capsid proteins (σ3 and μ1) undergo proteolysis mediated by a variety of

cellular proteases (36, 46, 47). This proteolytic degradation converts the reovirus virion into

an intermediate subvirion particle (ISVP) (5, 148, 152). During an infection, proteolytic

activation of virions outside of a cell can occur generating extracellular ISVPs (13). These

extracellular ISVPs may gain entry into a cell by deploying membrane‐interacting peptides

(myristoylated μ1N and φ; cleavage products of the outer capsid protein μ1) leading to pore

formation and particle recruitment to pores (66). Inside the cell, through an unclear

mechanism, ISVPs undergo further processing and are converted into transcriptionally

active cores that are released into the cytoplasm. Within the cytoplasm, reovirus

replication and assembly are thought to occur in distinct structures called viral inclusions

(10).

Under natural circumstances, mammalian reoviruses primarily infect the respiratory

and enteric tract. In the laboratory in a mouse model, reoviruses can induce pathology in a

variety of organs including the central nervous system, heart, and liver. The prototypical

laboratory strains of each serotype were isolated from children’s respiratory and

gastrointestinal tracts and are designated Type 1 Lang (T1L), Type 2 Jones (T2J), Type 3

Abney (T3A), and Type 3 Dearing (T3D) (129‐131). In mice, the serotypes show marked

(15)

and spreads well in intestinal tissue after oral inoculation, where as Type 3 Dearing (T3D)

does not (12, 73). Analysis of T1L X T3D reassortant viruses demonstrate the difference in

capacity of T1L and T3D to survive in the intestinal tissue is determined by both the S1 and

L2 genes (12). Until recently, identification of reovirus genes associated with a particular

phenotype has been accomplished by the use of reassortants (progeny virus derived from a

mixed infection with two different parent viruses). At present, a plasmid‐based reverse

genetic system has been developed to study reovirus replication and pathogenesis (74).

Viral

Myocarditis

Viral myocarditis is inflammation and damage to the myocardium, the muscular

portion of the heart. Viral myocarditis affects an estimated 5‐20% of the human population

(167). In infants, viral myocarditis can be fatal. As for the disease in adults, it usually

resolves, however, it can progress to chronic myocarditis, dilated cardiomyopathy, and even

cardiac failure (95, 167). Several viruses have been implicated in this disease, including

human immunodeficiency virus (HIV), enteroviruses, poxviruses, togaviruses, rhabdovirus,

herpesviruses, adenoviruses, paramyxoviruses, picornaviruses, arenavirus, and

orthomyxoviruses (40, 167).

The majority of human cases of viral myocarditis have been associated with

adenoviruses and enteroviruses (particularly group B coxsackieviruses) (16, 76, 94).

Adenovirus‐induced myocarditis is most likely not immune‐mediated (94). Enterovirus‐

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4

cytopathogenic effect on the heart (22, 57). In contrast, reovirus‐induced myocarditis is not

immune‐mediated, but instead reflects direct virus‐mediated apoptosis of cardiac cells (33).

Reovirus infection of nude (T‐cell deficient) (145) or SCID (T and B cell deficient) mice (144)

resulted in cardiac lesions, indicating that neither T nor B‐cells are required for reovirus‐

induced myocarditis. Instead, reovirus‐induced myocarditis was demonstrated to reflect

virus‐induced apoptosis of cardiac cells. Use of a protease inhibitor to block the cysteine

protease calpain, a known inducer of apoptosis in the heart, dramatically attenuated

reovirus‐induced myocarditis in neonatal mice. Moreover, treated neonatal mice had

reduced levels of serum creatine phosphokinase (CPK), a quantitative marker of cardiac

damage. In addition, these treated mice experience better weight gain compared to non‐

treated mice, suggesting the use of calpain inhibitors as a potential therapy for myocarditis

(32). Similar results were seen using agents that broadly inhibit caspases, also involved in

apoptosis, suggesting an additional novel therapeutic approach (33). Since reovirus induced

myocarditis is a result of virus‐induced apoptosis, it provides a unique disease model to

study the cardiac response to the direct cytopathic effect of viruses.

In examining the cardiac response to reovirus, the genes encoding viral core proteins

involved in viral RNA synthesis were found to be determinants of reovirus‐induced

myocarditis (143). In addition, in cardiac myocyte cultures the rate of viral RNA synthesis,

not the generation of progeny virions, correlated with viral myocarditic potential (142).

Furthermore, myocarditic reoviruses were able to spread more effectively through primary

cardiac myocytes cultures and induced a greater cumulative cytopathic effect than

(17)

induced antiviral cytokine interferon (IFN) in determining protection against reovirus

infection of spread between cardiac cells. To address the role of IFN in viral spread,

antibodies to IFN‐α/β were added to primary cardiac myocyte cultures. Addition of anti‐

IFN‐α/β antibodies to infected myocyte cultures benefited the spread of nonmyocarditic

reoviruses to a greater extent than that of myocarditic reoviruses, and was associated with

the M1 and L2 genes (146). This response to IFN‐α/β is cell type‐specific, as there was no

benefit to viral spread in differentiated C2C12 (skeletal muscle) cell cultures. In addition,

pretreatment of mice with anti‐IFN‐α/β antibodies enabled a nonmyocarditic reovirus to

induce cardiac lesions, demonstrating directly that IFN‐α/β is a determinant of protection

against viral myocarditis. Furthermore, in primary cardiac myocyte cultures,

nonmyocarditic reoviruses induce more IFN‐β and/or are more sensitive to the antiviral

effects of IFN‐β than myocarditic reoviruses. This difference in induction of, and/or

sensitivity, to IFN‐β was associated with the reovirus M1, S2, and L2 genes (146). Therefore,

these data indicate that reovirus induction of, and sensitivity to, IFN‐β is an important

determinant for protection against reovirus induced myocarditis.

For treatment of human cases of viral myocarditis, interferon alpha (IFN‐α) (28, 54,

100, 101) and interferon beta (IFN‐β) (75) have been shown to reduce the severity of

myocarditis, by inhibiting viral replication and improving cardiac function. However,

complete restoration of cardiac function has not been achieved with current therapies.

Therefore, further studies into the mechanisms of viral pathogenesis and viral response to

IFN are important for the development of better therapeutics.

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6

Induction

of

Type

I

Interferon

(IFN)

Interferons (IFN) were first described in the late 1950s, when it was discovered that

cells exposed to inactivated viruses secrete a chemical that interferes with the replication

and spread of subsequent viral infections (64, 65, 104). In addition to antiviral properties,

interferons have antiproliferative, proinflammatory, and immune modulatory functions

(117, 120). Interferons are classified into three classes, type I (IFN‐α/β, IFN‐ω,‐ε,‐τ, ‐δ, ‐κ),

type II (IFN‐γ), or type III (IFN‐λ), according to their amino acid sequence. Focusing on IFN‐

α/β, IFN‐alpha (IFN‐α) is represented by multiple structurally related subtypes (13 genes),

where as a single gene encodes for IFN‐beta (IFN‐β) (125). IFN‐α/β genes are subdivided

into two groups: 1) immediate‐early genes such as IFN‐β and murine IFN‐α4 and 2) delayed

type genes, which include the other IFN‐α subtypes (92).

Viral induction of IFN‐α/β starts with the detection of the invading virus by immune

system receptors, termed pattern recognition receptors (PRR). These receptors specifically

recognize molecules such as viral RNA or DNA (1). Viral RNA is recognized in the endosome

by membrane‐bound Toll‐like receptors (TLR), or in the cytoplasm by RNA helicases, such as

retinoic acid inducible gene‐I (RIG‐I, also known as DDX58) and melanoma differentiation

associated antigen 5 (MDA5, also known as IFIH1 or Helicard) (69, 71, 137, 172). Of the

TLRs, TLR3 recognizes viral double‐stranded (ds) RNA(2), TLR7/8 recognizes viral single‐

stranded (ss) RNA (34)and TLR9 detects unmethylated CpG‐DNA (56). The specific ligand for

RIG‐I is 5’‐triphosphate on either ssRNA or dsRNA that arises during viral RNA synthesis

(19)

activation can be stimulated by the dsRNA analog polyinosine‐polycytidylic acid (poly I:C) or

viral RNA, however, the precise ligand remains unclear (44). Following ligand engagement

(Figure 1.1 and 1.2), these intracellular receptors initiate signaling cascades leading to

activation of NF‐κB (nuclear factor kappa B), transcription factor‐2/c‐jun, and IRF3

(interferon regulatory factor 3) which bind to the IFN‐β promoter and stimulate

transcription(91).

Reovirus induction of IFN‐β in primary cardiac myocytes (108) and epithelial cell

lines (58) requires activation of IRF3. Reovirus activation of IRF3 is dependent on virion

disassembly in endosomes and requires the presences of viral genomic dsRNA, since empty

reovirus particles devoid of genomic dsRNA failed to activate IRF3 (58). As for the pattern

recognition receptors utilized by reovirus to induce IFN‐β, in 293T and HeLa cell lines

reovirus activation of IRF3 requires recognition by RIG‐I, not MDA5 (58). Transfection of

siRNA specific for RIG‐I strongly inhibits IRF‐3 activation by reovirus infection. In contrast,

MDA5 specific siRNA has no effect on reovirus‐induced IRF‐3 activity (58). In contrast to

IRF‐3 activation, reovirus activates NF‐κB via a mechanism that is independent of RIG‐I (58).

Reovirus activation of IRF3‐dependent gene expression in a different study, however, was

shown to occur via signaling through RIG‐I or MDA5 (86). Therefore, recognition of reovirus

infection by these various pattern recognition receptors may be cell type‐specific.

Indeed, reovirus induction of IFN‐β has been shown to be cell type‐specific. Cardiac

myocytes induce significantly more IFN‐β than cardiac fibroblasts in response to reovirus

(20)

8

infection of these cardiac cell cultures are unknown. In cardiac myocytes, reovirus

induction of, and sensitivity to, IFN‐β correlates with the capacity of certain reovirus strains

to cause myocarditis (146). Genetic analysis of the reassortant reoviruses identified the M1,

S2, and L2 genes as determinants of strain‐specific differences in the induction of IFN‐β in

cardiac myocytes (146). The viral core proteins encoded by these genes are μ2, σ2, and λ2,

respectively, and the mechanism(s) by which these proteins modulate induction of IFN is

unknown.

Positive

Feedback

Loop

for

IFN

Induction

Viral induction of IFN‐α/β occurs in three distinct phases (Figure 1.3). First, in the

immediate‐early phase, expression of the IFN‐β and murine IFN‐α4 genes is induced in

response to virus via a protein synthesis‐independent pathway through the activation of

IRF3. In the second phase, low level synthesis of IFN‐β and murine IFN‐α4 stimulate an

autocrine feedback loop by inducing IRF7 protein production through the IFN‐dependent

JAK‐STAT signaling pathway. Finally, in the third phase, continuing viral infection leads to

phosphorylation of the abundant IRF7, resulting in its activation and further induction of

IFN‐β and IFN‐α4 as well as induction of the other IFN‐α subtypes in a delayed manner

dependent on IRF7 production (92, 133). The induced IRF7 production appears to be

entirely responsible for augmenting the effects of autocrine IFN, as ectopic expression of

recombinant IRF7 alone confers viral induction of the delayed IFN‐α subtypes (171).

Furthermore, there is in vivo evidence that IRF7 plays a role in the positive feedback loop

(21)

addition, ablation of the IRF3 gene in transgenic mice demonstrated that IRF7 was still

capable of inducing IFN gene expression in the absence of IRF3 (134). Similarly, mice

deficient in either STAT‐1, IRF9, or the type I IFN receptor, were incapable of upregulating

IRF7 expression, resulting in an absence of the IFN amplification feedback loop (92, 133).

In addition to the feedback role of IRF7, low levels of IRF7 in the immediate‐early

phase may function as a primary transcription factor in IFN‐β induction in fibroblasts.

Mouse embryonic fibroblasts (MEFs) from either IRF3‐ or IRF7‐knockout mice show

impaired induction of IFN‐β (59, 134), indicating that, although neither transcription factor

is essential , both appear important in IFN‐β induction in this cell type. It remains uncertain

whether the IRF7 mediated effect is direct or indirect. However, it appears that activated

IRF7, like IRF3, exists as a dimer (81). Dimerization of active IRF7 has been detected by

several methods including velocity centrifugation (93), coimmunoprecipitation (164), and

interaction with recombinant bacterial fusion proteins (4). It remains controversial as to

whether IRF7 exists as a homodimer or as a heterodimer with IRF3 (81).

JAK

‐

STAT

Signaling

Pathway

The antiviral effects of IFN‐α/β are mediated through the induction of IFN‐stimulated

genes (ISGs), whose promoters are activated through a signal transduction pathway

following binding of IFN‐α/β to the IFN‐α/β receptor, composed of IFNAR1 and IFNAR2

subunits. Upon receptor engagement, tyrosine kinases, JAK1 and Tyk2, phosphorylate

signal transducers and activators of transcription (STATs), STAT1 (STAT1α and STAT1β) and

(22)

10

with p48/IRF9, forming the multimeric protein complex interferon stimulated gene factor 3

(ISGF3) (42, 160). ISGF3 complexes translocate to the nucleus and bind to specific

regulatory DNA sequences (ISRE; interferon stimulated response elements) to initiate the

transcription of several interferon stimulated genes (ISGs), including ISG561 and the gene

for the transcription factor IRF7, leading to the induction of an antiviral state (30) (Figure

1.4).

IFN

‐α

/

β

receptor

(IFNAR)

The IFN‐α/β receptor, IFNAR, is a heterodimer composed of IFNAR‐1 (α subunit) and

IFNAR‐2 (β subunit). In humans, IFNAR‐2 is expressed as three variants: a soluble receptor

(IFNAR‐2a), a short transmembrane form (IFNAR‐2b), and a long transmembrane form

(IFNAR‐2c) (35, 90, 110). In conjugation with IFNAR‐1, IFNAR2‐c is considered the

physiologically relevant receptor for IFN signaling. Expression of human IFNAR‐1 and IFNAR‐

2c, but not IFNAR‐2b, were able to reconstitute the antiviral IFN response (24). In addition,

in the mouse, the IFNAR‐2b chain is absent, instead two transcripts capable of encoding

soluble isoforms are generated by differential splicing (114). In vitro experiments have

demonstrated that soluble IFNAR‐2a can act either as an agonist or antagonist. In L929 cells

and mouse embryonic fibroblasts, soluble IFNAR‐2a can inhibit IFN signaling, where as in

primary thymocytes generated from IFNAR‐2 knockout mice, soluble IFNAR‐2a can bind

IFNα or IFNβ and generate an antiproliferative signal (52). Northern blot analysis of the

(23)

revealed that they were differentially regulated. Ratios of Ifnar2a:Ifnar2c range from 10:1

in some tissues, such as the liver, to approximately 1:1 in hemopoietic tissues (52).

Although, IFNAR‐2 has been shown to display intrinsic IFN binding activity, it cannot

transduce signals in the absence of IFNAR‐1 (114). Therefore, upon IFN binding initially to

IFNAR‐2, this complex then recruits IFNAR‐1 (43, 78). IFNAR‐1 associates with the Janus

kinase (JAK) family member, Tyk2, while IFNAR2 associates with JAK1 (158). Tyk2

phosphorylates a specific tyrosine residue at position 466 on the cytoplasmic receptor tail

of IFNAR‐1. This phosphotyrosine serves as a docking site for the rapid recruitment of

latent cytoplasmic STAT2 via its Src homology 2 (SH2) domain (169). Once anchored on the

receptor tail, STAT2 is then phosphorylated by Tyk2 on tyrosine 690, which serves to recruit

cytoplasmic STAT1. STAT1 is then subsequently phosphorylated on tyrosine 701, allowing

for heterodimerization of STAT1 and STAT2 (Figure 1.4) (30).

Janus

Kinases

(JAK)

Members of the Janus kinase (JAK) family, JAK1, JAK2, JAK3, and Tyk2, are

intracellular non‐receptor tyrosine kinases. Discovery of JAKs occurred at a time when PCR‐

based strategies and low‐stringency hybridization were being utilized to identify novel

protein tyrosine kinases. Initially, these novel protein tyrosine kinases were grouped into

the Just Another Kinase (JAK) family. Further investigation revealed that the JAKs were

markedly different from other protein tyrosine kinases due to the tandem architecture of

(24)

12

hallmark of JAK kinases. To denote this unique structural architecture, the JAKs were

renamed “Janus kinases” in reference to Janus, the two‐faced Roman God of gates and

doorways (126).

JAKs range in size from 120 to 140 kDa and feature seven conserved JAK homology

domains (JH1‐7). The two carboxy‐terminal JH regions represent the kinase (JH1) and

pseudokinase (JH2) domains (Figure 1.5). The pseudokinase domain (JH2) lacks catalytic

activity, however, is essential for normal kinase function. The four amino‐terminal JH

domains (JH7‐5 and half of JH4) constitute a FERM (four point one, ezrin, radixin, moesin)

domain that mediates association with receptors. Specifically, JAKs physically associate

with membrane‐proximal cytoplasmic domains (proline‐rich box 1/box2 domains) on

cytokine receptors following ligand binding. An SH2‐like domain (JH3 and half of JH4), of

unknown function, lies between the pseudokinase and FERM domains (126).

Signal

Tranducers

and

Activators

of

Transcription

(STATs)

Signal transducers and activators of transcription (STATs) are latent cytoplasmic

proteins that play key roles in cytokine‐induced signal transduction, requiring

phosphorylation to participate in transcriptional regulation. The spectrum of biological

systems that utilize the STATs extends beyond the interferon pathway, in which it was first

discovered, to include induction by several other cytokines [reviewed in (63)], involvement

in oncogenesis (17), proliferation, differentiation, and apoptosis (9, 103). In mammals,

(25)

Biochemical, genetic, and structural studies have identified seven conserved STAT domains,

including the amino‐terminal (NH2), coiled‐coil (C‐C), DNA‐binding (DBD), linker (LD), SH2,

tyrosine activation (Y), and transcriptional activation domain (TAD) (Figure 1.5) (21).

Due to alternative splicing, STAT1 exists as two isoforms, STAT1α (91 kDa) and the

splice variant, STAT1β (84 kDa). In contrast to STAT1α, STAT1β lacks a portion of the C‐

terminal transactivation domain (TAD) including the serine 727 phosphorylation site (Figure

1.5). Phosphorylation of serine 727 on STAT1α is required for maximal transcriptional

activity of STAT1α (166). The kinases that catalyze this phosphorylation in response to IFN‐

α/β, include protein kinase C‐delta (PKC‐δ) (157) and p38 MAPK (mitogen‐activated protein

kinase) (45). Both STAT1α and STAT1β are used interchangeably in the Type I IFN signaling

pathway for the generation of the ISGF3 complex (79). In addition to the formation of the

ISGF3 complex, STAT1 homodimers can be activated by IFN‐α/β signaling to generate the

IFN‐α activation factor (AAF) complex. AAF binds to the IFN‐gamma activated sequence

(GAS; TTTCCNGGAAA), whereas the ISGF3 complex recognizes another distinct sequence

called the IFN‐stimulated response element (ISRE; AGTTTNNNTTTCC) (125).

Formation

of

the

IFN

‐

Stimulated

Gene

Factor

3 (ISGF3)

ISGF3, is assembled from three proteins, STAT1, STAT2, and IRF9. The

transcriptional activation function of ISGF3 is provided by both STAT1 and STAT2. The

importance of the STAT2 TAD for ISGF3 activity was established in experiments using U6A

(26)

14

truncated C terminus (123). Further evidence that the TAD of STAT2 is require for the

function of ISGF3 was proven using GAL4‐based reporter systems and by the demonstration

of coactivator recruitment (115). Formation of ISGF3 requires the carboxy‐terminal half of

IRF9 (159). This region of IRF9 interacts with the amino‐terminal coiled‐coil domains of

STAT1 and STAT2 to form ISGF3 (79). Furthermore, UV cross‐linking experiments have

shown that STAT1 and IRF9 contact the ISRE at neighboring sites (AGTTTNNNTTTCC, IRF9

contacts the first two underlined T’s and the following T contacts STAT1) (124).

Nuclear

Translocation

of

the

ISGF3

Complex

Active transport of proteins into the nucleus requires the presence of a nuclear

localization signal (NLS) within the protein for transport through the nuclear pore complex

(NPC). The NLS of STAT1 and STAT2 becomes functional after phosphorylated STAT1 and

STAT2 heterodimerize via reciprocal SH2‐phosphotyrosine interactions (127). After

dimerization, IRF9 joins the complex forming ISGF3, which translocates into the nucleus as a

result of a gain of NLS function in the STATs that is recognized by importin‐α5 and importin

β (also known as karyopherin β) (96, 97). Nuclear translocation of ISGF3 induced by IFN‐α/β

treatment is observed as early as 30 minutes post‐treatment (7). In the nucleus,

dephosphorylation of STAT1 by TC45, a nuclear isoform of the T‐cell protein tyrosine

phosphatase (154) and dephosphorylation of STAT2 by an unknown protein tyrosine

(27)

(7). In the process, a nuclear export signal (NES) within the STATs is unmasked and

recognized by the exportin, CRM1 (chromosome region maintenance 1), resulting in export

of STAT1 and STAT2‐IRF9 back to the cytoplasm (127).

In addition, the nuclear export of STAT1 is regulated by JAK1. In JAK1‐deficient cells,

nuclear translocation of tyrosine phosphorylated STAT1 is impaired. However, this is

restored by the addition of leptomycin B (LMB), a specific inhibitor of CRM1‐mediated

nuclear export, indicating that JAK1 controls the nuclear export of STAT1. In addition, a

single point mutation within the nuclear export signal (NES) of STAT1 restored nuclear

retention of STAT1 in JAK1‐deficient cells. Furthermore, in the absence of JAK1, binding of

the transcriptional co‐activator CREB‐binding protein (CBP) to tyrosine phosphorylated

STAT1 is impaired (102). CBP is a transcriptional co‐activator with intrinsic histone

acetyltransferase (HAT) activity that has been shown to strengthen the activity of several

groups of transcription factors. All three components of the heterotrimeric transcription

factor ISGF3 complex (STAT1, STAT2, and IRF9) have been shown to be acetylated by CBP.

Acetylation of both IRF9 and STAT2 is critical for activation of the ISGF3 complex and its

associated antiviral gene regulation (153).

Regulation

of

the

JAK

‐

STAT

Pathway

Regulation of the cellular localization of STATs is one of the many mechanisms for

regulating the JAK‐STAT signaling pathway. Additional mechanisms for the regulation of the

(28)

16

component, inhibition by SOCS (suppressor of cytokine signaling) proteins, and nuclear

inactivation of STATs by PIAS (protein inhibitor of activated STAT) proteins (125). In

response to IFN, and after an initial activation of STAT1, PIAS1, but not other PIAS proteins,

specifically blocked the DNA binding activity of STAT1, thereby inhibiting STAT1‐mediated

gene activation (85).

Furthermore, regulation of histone deacetylase (HDAC) activity is another way to

modulate the JAK‐STAT pathway. HDAC activity is essential for transcriptional induction of

ISGs. Chemical inhibition of HDAC activity using trichostatin A (TSA) repressed both IFN‐

and virus‐induced global ISG expression (19, 132). Inhibition of HDAC activity prevented the

binding of RNA polymerase II to the promoter region of ISGs, thereby repressing ISG

expression (132). In addition to regulating ISG expression, HDAC activity modulates IFN‐β

expression. Specifically, RNA interference indicated that HDAC1 and HDAC8 repress IFN‐β

expression, while HDAC6 activates IFN‐β expression (111).

Interferon

Stimulated

Genes

(ISGs)

Interferon

Regulatory

Factor

7 (IRF

‐

7)

IRF7 was first identified for its role in silencing the Qp promoter region of the

Epstein‐Barr virus‐encoded gene, EBNA‐1, which contains an ISRE‐like element (109, 175).

Characterization of the IRF7 gene led to the discovery of two potential IFN responsive

elements, an IRF‐binding element (IRF‐E) in the promoter region and an ISRE (5’‐

(29)

inducibility to the IRF7 gene via the IFN‐dependent JAK‐STAT pathway by binding of the

ISGF3 complex to the ISRE (87). Further characterization revealed that the mouse IRF7 gene

consists of nine exons, which are translated to generate the full‐length protein, IRF7‐α,

along with two smaller variants, IRF7‐β and IRF7‐γ, both of which lack a portion of the

transactivation domain encoded by exons 4 and 5 (93). The IRF7 protein is composed of

four domains, including an amino‐terminal DNA binding domain (DBD) containing five

conserved tryptophan repeat sequences, an internal transactivation domain, an IRF

association domain (IAD), and a distal domain containing serine and threonine residues

required for both virus‐activated regulation and transactivation (TA/Regulatory) (81).

Basal expression of IRF7 has been detected in several types of tissue with

pronounced expression found in the spleen, thymus, and peripheral blood lymphocytes

(PBL) (175). In addition to basal expression, IRF7 can be induced to higher levels by IFN‐α/β,

lipopolysaccharide, tumor necrosis factor alpha (TNF‐α), tetradecanoyl phorbol acetate

(TPA), virus infection, and the Epstein‐Barr virus (EBV) oncoprotein LMP1 (3, 87, 88, 174).

The induced IRF7 serves to prime the cells for a second wave of a broader spectrum of IFN‐

α/β production, including IFN‐α2, IFN‐α5, IFN‐α6, and IFN‐α8 (92, 133). In addition to

regulating IFN‐α/β production, IRF7 has been found to control the production of type III

interferons (IFN‐λ1/ IL‐28A, IFN‐λ2/ IL‐28A and IFN‐λ3/ IL‐28B) (113). Thus, IRF7 is the

primary regulator of the IFN response in the local and systemic reaction of the host to viral

(30)

18

Therefore, viral regulation of IRF7 expression presents a significant means to

counteract the host IFN response. Kaposi’s sarcoma‐associated herpesvirus (KSHV) has

evolved several strategies to regulate IRF7, including inhibition of IRF7 phosphorylation and

nuclear accumulation via a tegument protein (ORF45) (176), inhibiting IRF7 DNA binding

activity via the encoded viral homolog of IRF3 (vIRF3) (68), and by targeting IRF7 for

proteosome‐mediated degradation (173). In addition, the degradation of IRF7, along with

degradation of IRF3 and IRF5 was observed in rotavirus infected cells (8). Furthermore, the

RING finger domain of ICP0 of herpes simplex virus type 1 was able to block IRF3‐ and IRF7‐

mediated activation of interferon stimulated genes (ISGs) (84).

Induction of ISGs is primarily due to IFN‐triggered activation of the JAK‐STAT

pathway leading to the ISGF3 complex binding to the ISRE. However, a subset of ISGs (such

as ISG15/54/56) is also directly induced upon virus infection, or by double‐stranded RNA,

independent of the IFN‐activated JAK‐STAT pathway by the binding of IRF3 to the ISRE (6,

25, 48, 50, 105, 118, 170). The induction of IRF7 was documented to only occur through

IFN, requiring the formation of the ISGF3 complex (133). However, recently, Sendai virus

infection was shown to directly activate IRF7 transcription by the binding of the virus‐

activated factor (VAF) containing IRF3, IRF7, CBP, and p300 to the IRF‐E and ISRE of IRF7

(107). Interestingly, in the absence of a functional IFN‐α/β receptor, reovirus serotype 3

Dearing (T3D) was shown not to directly induce IRF7 (151).

(31)

Interferon

Stimulated

Gene

561 (ISG561)

ISG561 (ISG56/IFIT‐1) was one of the first IFN‐inducible genes to be cloned (20, 77).

Further analysis of ISG561 found two ISRE consensus sites (ISRE I and ISRE II) in the

promoter (Figure 1.6) (163) that are necessary for induction by type I interferons, dsRNA, or

virus infection (50, 118, 155, 156) in either an ISGF3‐ or IRF3‐dependent manner (48, 118).

As for IRF3‐mediated activation of the ISG561 promoter, both ISRE sites contribute to

activation, since mutation of both sites was necessary for complete abrogation of the

induction of the promoter. When the ISRE sites were mutated independently, mutation of

the ISRE II site slightly decreased activation of the ISG561 promoter (26%), whereas

mutation of ISRE I resulted in a 76% reduction of activation of the promoter (48). In

addition, activation of ISG561 by IRF3 was shown to be independent of IFN. In HECB1 cells

that do not respond to type I IFN treatment, Sendai virus infection induced ISG561 protein

expression. Moreover, expression of a dominant negative form of IRF3 inhibited Sendai

virus from inducing ISG561 (83). Therefore, IRF3 is sufficient to induce ISG561 in response

to virus infection in an IFN‐independent pathway (48). In cardiac cell cultures derived from

IFN‐α/β receptor null mice, reovirus T3D directly induced ISG561, presumably through

activation of IRF3 (151). However, reovirus T3D did not directly induce ISG561 protein

expression (P56) in human glioblastoma cells (50). These differences may reflect cell type‐

specific induction of ISG561. Recently, an in vivo study in mice demonstrated that the

(32)

20

example, in the heart, dsRNA or IFN‐β could induce ISG561 protein expression (P56),

however, injected IFN‐α did not induce P56 (156).

The ISG561 encoded protein, P56, contains six 34‐amino acid tetratricopeptide (TPR)

tandem repeat motifs (147), which in other proteins have been shown to mediate protein‐

protein interactions (31). Recently, the N‐terminal domain of mouse P56, containing TPR1

and TPR2 was shown to interact with the eukaryotic initiation factor 3 (eIF3) “c” subunit,

eIF3c. This interaction interferes with the formation of the 43S preinitiation complex

leading to inhibition of translation (62). Interestingly, human P56 inhibits initiation of

translation by binding to a different subunit of eIF3, specifically the “e” subunit (eIF3e/

Int6/P48)(49, 61), selectively inhibiting the ability of eIF3e to stabilize the ternary complex

of eIF2⋅GTP⋅Met‐tRNAi (49, 61). Comparison of protein sequences revealed only 53%

sequence homology between mouse P56 and human P56. Despite differences in protein

sequence, the position of the TPR motifs on the protein were highly conserved between

(33)

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Cardiac Cell Type-Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN

ABSTRACT

DEDICATION

BIOGRAPHY

ACKNOWLEDGMENTS

TABLE

OF

CONTENTS

LIST

OF

TABLES

LIST

OF

FIGURES

CHAPTER

1

LITERATURE

REVIEW

Reovirus

Viral

Myocarditis

Induction

of

Type

I

Interferon

(IFN)

Positive

Feedback

Loop

for

IFN

Induction

JAK

‐

STAT

Signaling

Pathway

IFN

‐α

/

β

receptor

(IFNAR)

Janus

Kinases

(JAK)

Signal

Tranducers

and

Activators

of

Transcription

(STATs)

Formation

of

the

IFN

‐

Stimulated

Gene

Factor

3

(ISGF3)

Nuclear

Translocation

of

the

ISGF3

Complex

Regulation

of

the

JAK

‐

STAT

Pathway

Interferon

Stimulated

Genes