MYXOMA VIRUS INDUCED ACTIVATION OF CC-CHEMOKINE RECEPTOR 5 (CCRS)

Jennefer Masters

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Imrnunology University of Toronto

OCopyright by Jennefer Masiers ZOO0

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M-yxoma virus induced activation of CC-chemokine receptor 5 (CCRS)

Degree of Master of Science. 2000

iennefer Masters

Graduate Department of I m m u n o l o ~

University of Toronto

ABSTRACT

Vimses have evolved a number of suritegies to gain en- and replicate in host

target cells that. for HIV and the poxvirus, myxoma virus. involves appropriating

chemokine receptors. In îhis report we dernonstrate that activation of the humm

chemokine receptor. CCRS. in the context of receptor tyrosine phosphorylation. is a pre-

requisite for myxoma virus replication. Myxoma virus infection induces the rapid

phosphorylation of CCR5 on tyrosine residues. the association of Jalis and p56"' and

their phosphorylation-activation. Furthemore. inhibition of JrikZ through tyrophostin

BA2 resdts in decreased myxorna virus replication. whereas treatment of permissive ceils

with a p56'ck inhibitor results in an increase in viral replication. Additionally. we provide

e~ idence for myxoma virus inducible Stat and IRS activation. In contrast to CCRS

acti~ation effected by HIV Env protein. these myxomri virus-inducible phosphorylation

events rire not sensitive to pertusis toxin treatment. Moreover. in CCRS-espressing cells

that are non-permissive for myomi l virus infection. we provide evidence that myxoma

virus fails to rictivûte CCRS and invoke the prescribed tyrosine phosphorylation cascade.

Consistent with the observation that infection of CCRS-expressing cells is blocked by

herbimycin A. we infer that viral infectivity is dependent on subversion of non-G-protein

coupled CCRS-mediated signal transduction pathways triggered by the infecting myxorna

\.inis prtrticle. This provides a novel post-binding mechanism by which viruses can CO-

opt a cellular receptor to permit productive virus infection..

ACKNOWLEDGEMENTS

1 wouid like to thruik Dr. Eleanor Fish who has guided me throughout my career

as a graduate student and provided me with the opportun.@ to present my work at

international and nationd conferences. 1 also t h a d Dr. Tania Watts and Dr. Linda Penn

ueho have been supportive and understanding throughout my project. I would also like to

acknowledge Dr. Grant McFadden for providing me with a chance to continue workinz

nith such an interesting virus. To Dr. Alshad Lalani. who's crrizy science days finally

paid off. and also for his guidance and support and afternoon trips to the movies.

Thank you to the "Fish L a b for Listening to my pessimism and my crazy drearns.

Specifically. 1 would like to send my appreciation to Beata who is always available to

si\.-e her time. to Melissa for making those moming haircuts much more pleasant and for

alwriys volunteerin_a to passage my cells. To Jyothi for her support and sidance

throughout my strug~les and for her brownies and cookies that dways appeared when 1

was craving chocolate. Thank you Dr. Raj. for keeping me smiling and for k i n g a great

support in the Iab. and to Mark for his e n e r a and humor and for k i n g my chemokine

connection.

Thrink you to Dino for being so supportive and loving. and for runninz around to

=et my migraine medication when science became too much for my head to handle. Your

nizhtly chocolate bar runs have kept me sane.

Thrtnk you to my family for supporting me throughout my career ris ri student.

Especially, t h a d you to Mom and Dad for making the effort to understand my project

and belie~~ing in my talents.

1.7) Use of Chemokine Receptors by Myxoma Virus: ....................................................... Thesis Objectives 34-35

Chapter II

MATERIALS AND METHOD .................................................. 36-42

II . 1 ) Cells and Virus ......................................................... 36 II.?) Analysis Of Humm CCRS and CD4 Expression By

Flow Cytomeuy ...................................................... 36-37 ........ 11.3 ) Ce11 S taining for Flourescent and Electron Microscopy -37

11.4 ) Cs11 Lysis and immunoblotting .................................... ..3 8-39 11.5 ) Use of Pharmocotogical Inhibitors for Altering

Viral Lnfectivity .................................................. 3940

Chapter III

RESULTS AND DISCUSSION ............................................. 41-69

ILI . 1 ) Ectopic expression of human CCRS alone is not predictive of rn-yxoma virus infectitrity ........................................ 11 -5 1

IU.2) Myxoma virus induces CCRS tyrosine phosphorylation in NIH 3T3 . CCRS ceiis ...................... .... ............... -52-54

m.3) Initiation of myxoma virus infection is associated with tyrosine phosphorylsition of cellular proteins ................... - 3 - 6 0

III.4) Myxomsi virus induces Stat and IRS tyrosine Phosphorylation ...................................................... 6 1 -66

III.5) Tyrophostin B42 inhibits myxoma virus infectivity ........... -67-69

Chapter IV

................................................... CONCLUSIONS ........ ..... 70-73

........................................................... W . 1 ) Conclusions 70-73 ...................................... ........... iV.2) Future Directions .. 73

Chapter V

LIST OF TABLES

Introduction

Table 1. Human Chemokine Superfamily ......... .. ............ -17

Table 3. Human Chemokine Receptor Superfamily.. .......... 19

LIST OF FIGURES

Introduction

Figure i Schematic Representation of Poxvirus Morpholo-gy and Genome Organization.. ............................ 4

Fi-gure ii Poxvims Lifecycle.. ......................... .. - 8 .........

Figure iii Myxoma Virus immune Evasion Strategies. .......... 14

Fi+pre iv Schematic representation of signal uansduction pathways activated by chemokine-receptor interaction.. ...... - 2 3

Resul ts and Discussion

Fisure 1 Ectopic expression of human CCRS alone is not predictive of myxoma virus infectivity.. ......... -4243

Figure 2 RBLCCRS ceUs are not susceptible to rn-vxoma ............................................ virus infection . . M 5

Figure 3 RBL.CCR5 cells remain viable during infection and show no signs of viral mRNA production. ....... 48-49

Fisure 4 Viral progeny are evident only with .................................. NIH 3T3.CCR5 cells.. ..50-5 1

Ficoure 5 Mysoma virus induces CCRS tyrosine phosphorylation ............................... in NM 3T3. CCR5 cells.. -53-54

Fi+mre 6 initiation of rnyxoma virus infection is associated with tyrosine phosphorylation of cellular proteins.. -57-58

vii

Fisure 7 Inhibition of p56LC\esults in increased myxoma . . .......................................... virus replscauon 59-60

Figure 8 Myxoma virus induces Stat tyrosine .......................................... Phosphorylation .6 3.64

Figure 9 Myxoma virus induces IRS tyrosine .......................................... Phosphorylation ..6 5.66

Figre 10 Inhibition of J d 2 activity results in reduced ................................ myxoma virus replication 68-69

viii

LIST OF ABBREVIATIONS

Ab AIDS &-t/PKB AOP-RANTES APC AP- 1 BGMK CAMP CCR EEV ELR EPO ER ERK GAS 33120 HCMV HCV HHV-8IKSHV HW HOS IFN 1-id3 IL-6 IMV IRS- Il3 ISRE Jak JH JNK LTu$ MAPK MCP- 1 MEK MHC II Mn'- 1 a ' p M-T 1 M-T3 M-T4 M-T5 M-T7 M l IL

antibody acquired irnmunodeficiency syndrome protein kinase B aminooxypentane-RANTES. amino terminally modified RANTES antigen presenting ceii activatins protein- 1 baby green monkey kidney ce11 line cyclic ridenosine monophosphate CC chemokine receptors extracellular enveloped virus glutamic acid ( E ). leucine (LI. argïnine IR) erythropoietin endoplasmic reticulum extracellular-signal reglated kinase pmma activated sequences _olycoprotein 1 20. HIV envelope protein human cytomegalovims hepatitis C virus Kaposi's sarcoma associated herpes \ l ins human irnmunodeficiency virus human osteosarcomsi ce11 line interferon inhibitor of NF-KB interleufin 6 intrricellular mature virus insulin receptor subsuate- 1/2 interferon stimulated response elements janus kinase jak homology domains c-Jun N-terminal kinase lymphotoxin a /p mitogen activated protein kinase monocyte chernotactic protein- 1 MAPK kinase major histocompritibility complex class II macrophage inflammatory protein - 1 a$ myxoma chemokine binding protein T l myxoma TNFa receptor homolog myxoma anti-apoptotic protein myxoma anti-apoptotic protein myxoma EN-y receptor homolog myxoma anti-apoptotic protein

NF-& NIH 3T3 NSSA ORF PAGE PI-3' K PTK Pvk-2

RA RANTES RDEL RBL RL-5 SClD SH-2. SHP-2 S IE S N Stat TNFa ZAP-70

nuclear factor-& National institute of health. murine fibroblast nonstructural protein SA. HCV coat protein open reading frarne polyacrylamide gel electrophoresis phosphatidylinositol 3 kinase protein tyrosine kinase

?5FAK protein tyrosine kinase related to p l - lymphocyte kinase. src family of kinases 56 m a focal adhesion kinase. 125 D a rheurnatoid arthritis regulated upon activation. normal T ce11 expressed and secreted arginine (R ). aspartic acid ( D ). glutamic ncid ( E). leucine t L ) rat basophilie leukemia ce11 iine rabbit lymphocyte-5 severe combined irnmunodeficienc y Src h o m ~ l o ~ q - î domain phosphatase Stat inducible elements simian irnmunodeficiency virus sisna1 transducers and acti\xitors of transcription tumor necrosis factor a zeta associated protein. 70 D a

Chapter I

INTRODUCTION

In order to successfùlly invade a host. microorganisms must adopt a number of

strritegies, u-hich protect them against hostile host defenses. Some of the stealthiest of

microorganisms have adapted sevenl strategies. n-hich leave them virtually invisible to

the immune system. Viruses. having CO-evolved with the immune system. have become

estremely proficient at subverting host defenses. Interference with major

histocompatibility comples (MHC) presentation of antigen, blockade of apoptosis.

disruption of complement cascades and modulation of cytokine networks (Bany and

McFadden, 1997; Everett and McFadden, 1999; Lalani et al., 2000; Zuniga et al.. 1999)

are among the tactics utilized. A cytokine network frequently exploited by sevenl

microorganisms, including vinses, is the chemokuie system (Pease and Murphy. 1998 1,

( Lalani and McFadden, 1 999). Chemokines are involvsd in directing lymphocyte tnffic

to sites of infection and in activating immune cells to eliminate infectious pathogens.

Therefore. corruption of this system is an effective way of preventing the clearance of an

infectious agent.

Corruption of the chemokine system can take sevenl different forms. such as the

secretion of chemokine binding proteins (Lalani et al.. 1999a). subversion by vinlly

encoded chemokines and chemokine receptors and the use of chemokine receptors as a

portal for en- into cells (Lalani et al.. 1999b). Recently. it was demonstrated that

mysomri virus. a nbbi t specific poxvirus. utilizes chemohne receptors for iriral

infectivity ( Lalani et al.. t 999b). Therefore. the objective of this study was to determine

the dou-nstream sipal ing events, which result from the interaction between m p o m a

and CC chemokine receptor 5 (CCRS).

1 POXVIRUSES

Made infamous by the controversy surrounding the compiete eradication of the

remaining stocks of sxnallpos (varioln vil-zrs), the poxviruses have been the subject o f

intensi\.e researc h into the immune evasion phenomenon. Poxviruses are large double

stranded (ds) DNA. enveloped viruses that replicate within the cytoplasm of infected

cells ( Figure i ) ( Fenner. 2000). The genome ranges from 120 to 300 kb pairs and consists

of a single linear DNA molecute with closed hairpin ends (Figure i ) . This large genome

also enables the virus to encode its own replication and transcription machinery as well as

a battery of genes involved in abrogating host defenses (Fenner, 2000).

The pan-irus family is very large and diverse. causing disease in species ranging

from humans (\*accinia and molluscum contagiosum) and monkeys (monkeypos) to

camels (cameipos). ungulates (cowpos. sheepox. goatpos). swine (swinepox), squirrels

( squirrel fibroma ) and nbbits ( Shope fibroma. malignant fibroma. hare fibroma and

rn-noma) (Femer. 2000). Myxoma virus. the most notorious of the nbbit posviruses.

became the first viral agent released into the wild for the sole purpose of controlling the

rabbit population in Australia. From the beginning o f its release in 1950, the virus was

initially capable of killing 9994~ of infected rabbits ( Moss, 1996). However, with time the

\.inis soon became less virulent and therefore a less effective agent for controlling the

rab bit population. Furthennore. the selective pressure of the virus on the rabbit

population gave rise to a highly resistant species of rabbit. This prompted extensive

reserirch into m-orna virus pathogenicity. which will lead to a greater understanding of

\+ira1 tropism and infectivity.

Con

Figure i. Schematic representation of poxvirus morphology and genome organization. The morpholgy of poxviruses is brick shaped and large enough to be visualized by light microscopy (600 nm). Poxvirus genomes range between 120 and 300 kbp.. and encodr a DNA depcndent RNA polymerase and a DNA polymerase. Deletion o f these genes renden the virus unable to replicate. The virulence genes are found in duplicate copies and encode proteins involved in abrogating host defenses. (www- micro.msb.le.ac.uW33SIPoxvimses.html)

1.2.1 Poxvirus Receptors

Although there is v e y little knowledge about how poxviruses infect cells.

intensive research into vaccinia virus ïnfectiviiy has provided the greatest amount of

information. During an infection. wo stnictunlly different vims particles are expressed.

the intracellular mature vims ( M V ) and the extra cellular enveloped virus (EEV). The

IMV. constituting approsirnateiy 99Ob of the viral progeny are released fiom the cell

upon ce11 lysis. u-hereas the second forrn, EEV. is generated when the virus buds from the

cell. Despite the fact that this f o m of the virus makes up only 1°o of the total viral

progeny. it plays a crucial role in eliciting protective immunity (Smith et al.. 1997).

Furthemore. as a consequence of budding from the cell. EEV espress an additionai 10

proteins that are absent fiom IMV (Smith et al.. 1997 1. Therefore. it is likely that the hvo

different forms interact with different receptors to initiate infection. Unfortunately.

binding studies carried out to chri@ this point have been complicated due to difficulties

in purifving the two forrns of virus. To overcome this problem. confocal rnicroscopy \vas

utilized to differentiate between the EEV and IMV f o m s of vaccinia virus. As a result,

these studies demonstrated the nvo forrns do indeed bind differently to unrelated cells

( Vanderplasschen and Smith. 1997 ). Moreover, analyses involving deletion of several

\.irus coat proteins revealed that chondroitin sulfate and heparan sulfate mediate IMV ce11

binding (Hsiao et al.. 1999). Hotvever. it is not clear whether these binding events are

necessary for virus entry, since sulphate-mediated charge interactions are relatively non-

specific compared to viral tropism. which is highly variable.

Due to the complexity of poxviruses it is likely that more than one receptor is

utilized. In fact. accumulating data indicate 3 requirement for ce11 membrane chemokine

receptor expression for viral susceptibility ( Lalani et al.. 1999b). Since the cells utilized

by Lalani et ai., also expressed CM. it is also possible that chemokine receptor

engagement is enhanced by the expression of CD4. Like HIV. the poxviruses may

utilize chemokine receptors to increase their mobility and to activate the ce11 in order to

create a suitable environment for vin1 replication.

1.22 Powirus lifecvcle

Penetration of cells by enveloped viruses genenlly occurs by two distinct

processes. Once bound to the receptor. the viral envelope can fuse directly with the

plasma membrane. Altematively. the virus particle is internalized where a change in pH

allo\vs for fusion to an endosome. In either circumstance the viral particle releases its

core into the cytoplasm of the host ce11 (Vanderplasschen and Smith. 1997 ).

Data describing the penetration of IMV and EEV particles of vaccinia virus are

contro\.ersial. Ho\\-ever. it is understood that EEV particles enter cells much more

efficiently than do IMV particles. Treating cells with cytochalisn B. n-hich prevents

actin polymenzation. affects entry of IMV but not EEV. Furthemore. pharmaceuticals

sho\vn to inhibit PKC and protein tyrosine kinases ( P X ) were shown to significantly

inhibit IMV core release but had no effect on EEV core liberation (Locker et al.. 2000).

Recent evidence indicates that activation of a signaling cascade by IMV, leads to the

formation of ce11 surface protrusions, which allow the core to penetrate the plasma

membrane. Upon entry, viral cores remain surrounded by an imer membrane, which

must be disrupted in order for viral transcription and replication to occur.

Posvims genes are classified into 3 different categones: esirly, intermediate and

late and are espressed in a coordinated cascade. with each category ( Moss, 1996) being

dependent on the expression of genes in the previous category. Early genes are expressed

immediately afier the core is released fiom the plasma membrane. n-hereas intermediate

and late genes are only expressed after replication has commenced.

Early gene mRNAs are synthesized by DNA-dependent RNA polymense. ~vhich

dong lvith transcription factors. capping and polyadenylating enzymes. are packaged

uithin the core to allow for imrnediate transcription and replication ( Moss, 1996 1. Early

genes encode proteins needed for M e r uncoating of the viral core, and enzymes

responsible for nucleoside metabolism and DNA replication. Included in the 100 eariy

genes are virulence factors and transcription factors necessary for intermediate and late

zene espression (Figure ii)( Moss, 1996). *

The intemediate genes are the least numerous and encode pnmarily transcription

factors needed for late gene expression. Late expression of vin1 structural proteins and

enzymes to be packaged into virions for early transcription allows for the assembiy of

\viral progeny. As u-ell. additional virulence factors encoded by late genes permit the

virus's e~vrision from immune clearance. Once viral particles are assembled. early tubular

endsomes or trans-Golgi network membranes envelop them (Tooze et al., 1993. Schmelz.

1994) follom-ed by transport to the ce11 surface where the virus is released. Actin

polymerization is thought to be involved in transport of virus to the ce11 surface. however

direct evidence is not rivailable to support this claim.

Engulfrnent

Pbagocytic t'esicle

Productive

Cncoatin~ Gcnome replication

Es- - 4

intermediate

.4ssembiy

Re l ease 'ulatuntioni Golgi wnpping

Figure ii. Ponvirus lifecycle (adapted from Moss. 1996).

1.3 MYXOMAVIRUS

Myxoma virus belongs to the genus Ieporipo-ninw and causes a benign disease in

the North and South American brush rabbits (Sj-Iidagrrs). However. in European

( O~?rroIngzts) nbbits it causes a setvere systemic disease called myxomatosis ( McFadden.

1994 ). Mysomatosis is characterized by severe immune dysfiinction resulting in a

inortality rate of approsirnately 9996. The virus is tmnsmitted by mosquitoes and

disseminates throughout the rabbit in trafficking Iymphocytes, where it also replicates

( McFadden. 1 994). Some pathogenic features of my'romatosis include extensive

hemorrhagic lesions at the primary site of infection. which characteristically leads to

tissue degeneration and necrosis. Once dissemination occurs. seconda- lesions around

the ears. eyelids. lips. nares and genitalia become evident. Within nvo weeks. infected

rabbits succumb to supenrening gram-negative bacterial infections of the respintory tract

( Lalani et al.. 1999a ). Viral knock out studies have clearly demonstrated that the severe

immune dysfùnction seen in infected rabbits is due to the large repertoire of immune

evasion strategies encoded by the m y o m a virus genome (Lalani et al. 1999a; Hnatiuk et

al.. 1999: Mossman et al.. 1995). The timely expression of these proteins following

infection renders the virus virtually untouched by the host immune system and

consequently. impairs the rabbit's immunity to other pathogens. Considering the severity

of this disease it is evident that posviruses are amongst the stealthiest viruses hown.

1.3.1 Myxorna Virus Immune Evasion Strategies

i) Viroreceptors and Virokines

Through the CO-evolution with its nbbit host. mysoma virus has adapted several

very diverse stmtegies to evade. block or modulate key immune responses. With the

recent completion of the myxoma virus genome sequence (Cameron et al.. 1999). a great

num ber of immunornodulatory genes have been identified (Figure iii 1. Of these genes.

the best chancterized to date are products termed virokines and viroreceptors. Virokines

are usually secreted viral proteins that mimic cellular cytokines or gowth factors (Lalani

et al.. 2000). In contnst, viroreceptors c m be membrane bound or secreted. but are

general ly homologous to cellular receptors. One exception. however. is the myxoma

protein M-Tl. M-TI is a rnember of the T1/35 kDa fmily of posvirus chemokine

binding proteins (Graham et al., 1997). These proteins bind with high affinity to CC-

chemokines and inhibit their activities. Moreover, they have no h o w n homology to any

celliilar proteins including chemokine receptors (talani et al.. 1999a). M-Tl is secreted

continuously during virus infection. but when deleted fiom the virus genome. there is no

significant reduction in myxomatosis lethality ( Lalani et al.. 1999a )- The most notable

difference betn-een \\ild type and M-Tl knock out virus can be seen at the tissue level

u-here a greater number of infiltrating leukocytes. especially monocytes~macrophages

n-ere noted in rabbit tissues (Lalani et al.. 1999a). It is possible that the M-Tl protein

alone is not associated with the severe morbidity seen with myxomatosis. Rather. it is the

carefully orchestrated sequence of immune disruption. which results in my?coma's

lethality.

The most abundant. virally denved molecule expressed during infection is the M-

T7 protein. Although M-T7 \vas recently shokvn to be able to bind to chemokines at the

COOH-terminus ( Lalani et al.. 1997). it was first noted for its role in binding interferon y

( IFN-y). M-T7 is a tùnctional homolog of the cellular IFN-y receptor and it binds to

rabbit IFN-y n-ith high affrnity (Mossman et al.. 1995). Given the importance of IFN-y in

inducing an antiviral state. it is not surprising that viruses target this protein as a defense

mechanism. Moreok-er. in M-T7 nul1 mutants. m p o m a virus becomes significantly

attenuated in addition to exhibiting a decrease in viral dissemination and replication at

seconda- sites. Furthemore. significant cellular activation of germinal centers of the

spleen and lyrnph node c m be seen (Lalani et al.. 1997). Thus. M-T7 appears to function

in abrogating early activation of T lymphocytes by preventing communication between

antigen presenting cells (APC) and lymphocytes. possibly indicating a defect in APC

migration to the lymph nodes and spleen. As mentioned previously, M-T7 is also able to

bind to chernokines at the COOH-terminus. Although the fûnctional outcome for M-T7's

ability to bind to chemokines and prevent chernotaxis has not been clarified, it is possible

that its duaI role facilitates the severe pathogenicity seen in rabbits,

Tumor necrosis factor a (TNFu). lymphotosin a (LTa) and lymphotosin P (LTP)

are potent anti-viral cytokines implicated in the induction of apoptosis. inflammation.

necrosis and ce11 grouqh ( Aderka et al., 1985: Koff. 1986; Wong, 1986). Therefore.

inl-iibiting the function of these cytokines by viruses is an effective way to ensure virus

sunri\.al. Myxoma vims secretes a TNF receptor homologue terrned M-T2, which binds

u-ith high affinity to TNFu (Upton et al., 199 1. Schreiber. 1996 ). M-TZ acts by

sequestering TNFa and preventing signaling through the TNF receptor (Schreiber et al.,

1996 ). Biochemical chancterization of M-T2 revealed that the protein is secreted as both

a monomer and a disulfide-linked dimer (Schreiber et al., 1996). Furthemore. in

contrast to the cellular receptor. which binds as a trimer. Scatchard plot analysis has

show-n that bot11 forms bind effectively to TNFa but the dimer binds with higher affinity

(Schreiber et al., 1996). tnterestingly. data fiom deletion studies of the M-TZ protein also

demonstrated a roie for M-T2 in preventing apoptosis of virally infected CDJ+ T

lymphocytes (Macen et al.. 1996). M-T2 is therefore the second myxoma virus protein

if-itli hvo hnctional roles in mylcomatosis pathogenes is.

ü) Prevention of apoptosis

The ability of cells to undergo apoptosis or prograrnmed ce11 death is an intrinsic

property that can be triggered by a variety of inducers. Mysoma virus has evolved a

nuinber of stntegies to prevent apoptosis of target cells. Although the mechanics of how

the M-TZ protein rnay prevent apoptosis is still unclear. it is understood that this

inhi bit ion of apoptosis occurs independently of its activities related to TNF ( Schreiber et

al.. 1996 ). By means of mutational analysis. it was revealed that M-T2 variants could

prevent target ce11 apoptosis. yet they were not secreted and were unable to bind TNF

( Macen et al.. 1996). A second protein involved in the prevention of apoptosis is the

M 1 1 L protein (Everett et al., 2000). Recently, studies have shown this protein to be

associated Lvith mitochondria. where it may be involved in preventing loss of membrane

potential. cytochrome C release and caspase 3 activation (Everett et al.. 2000). M-T-4 and

M-15 rire furtlier examples of myxoma encoded anti-apoptotic proteins- However. their

mechmisms of action are yet to be resolved (Barry and McFadden. 1997; Mossman et al..

1996 1. Interestingly. the MOT4 protein contains an endoplasmic reticulum retaining ( ER)

motif. RDEL. and \vhen analyzed by confocd microscopy was sho\vn to be associated

LL-ith the ER (Hnatiuk et al.. 1999). Further analysis of M-T4-RDEL- virus n i I I indicate if

the RDEL motif is needed for the prevention of apoptosis.

Binds to and inhibits CC-chemokines

lnhibits IL-Ip action

t Reduces

Inhibits T\F action inflammatory

response

Failure

Reduces inflam matory

response (ligand unknown)

::; Myxoma Factories

(ceII surface viral protcins)

lntracellular blockade of cytokine and interferon signal trasduction

Figure iii. Myxoma virus immune evasion strategies (adapted from Nash et al. 1999)

1.4 CHEMOKINES AND CHEMOIUNE RECEPTORS

i.-L 1 Chemokines

The migration of Ieukocytes into areas of injury or infection is crucial for the

effective clearance of pathogens (Murphy et al.. 2000). Chemokines o r chernotatic

cytokines are potent attractors of various leukocytes such as neutrophils. monocytes and

lymphocyres (Baggiolini et al-. 1997: Murphy et al.. 2000: Rossi and Zlotnik. 2000).

More recently. chemokines have also been shown to be involved in the regulation of

hematopoiesis. angiogenesis. the clearance of pathogens. and in healing tissue injury.

These small pro-inflammatoxy proteins of 8- 10 kDa comprise a large family of over 40

chemokines. n-hich have been divided into 4 subfamilies based on the relative position of

the first hvo-consenred cysteine residues (Table 1 ) (Rossi and Zlotnik, 2000; Baggiolini

et al., 1997). These residues are important for forming disulfide bonds critical for

chemokine tertiary structure. The CXC. or a-chemokines have an intervening amino

acid between the first two cysteines, whereas the CC or B-chemokines have two

consecutive cysteines. Lyrnphotactin, a C chemokine, has only 2 cysteines in the mature

protein and is the only protein identified in this subfamily. Fnctalkine. the only member

of the CX;C family. has three intervening amino ricids between the first two cysteines

and is also the only known chemokine to be associated w-ith the ce11 membrane (Rossi

and Zlotnik. 2000).

In the past. chemokine subfamilies were biologically differentiated based on

\[.hich cells they are chernotactic for. The cx chemokines are fùrther subdivided based on

the presence or absence of an ELR motif. which precedes the first cysteine (Taub, 1996).

In general. ELR-CXC-chemokines induce chernotaxis and activation of neutrophils,

n-hereris a chernokines not expressing the ELR motif are chernotactic for

monocytes ( Taub, 1996 1. However. the expression of receptors, CXCR3 and CXCR4 on

lymphocytes indicates a role for CXC chemokines in the recruitment of lymphocytes.

The P chemokines were believeci to function primarily in attracting monocytes. however

emerging evidence indicates that CC c h e m o b e s are chemoattractant for T lymphcytes.

basophils. eosinophiïs, and mast cells ( Bacon and Schall. 1996; Schall and Bacon. 1994 ).

There is accurnulriting evidence suggesting that the status of receptor expression changes

throughout leukocyte differentiation and activation. As well. the expression of

chemokines changes throughout the course of an infection. Therefore. it is probable that

chemokine induced ce11 migration and inflammation is reguiated as much by the

chemokines being espressed. as by the ceils that express the appropriate receptors.

Tîb lc 1. Human Chemokine Supcrfamily Coiimion Nanie Full Namc Offlcial Name

\ICI'- I .-2.-3.4 XIIP- l a. ELC' XIIP-3P

\ l I P - 5 ~ . Lkn- 1

Eorasin Eotasin-i XIPIF-?

XIPIF-1 PARC DC-CK 1

\IDC STCP- I

Esodus- l X l I P - 3 ~ L X R C

E..;odus-2 6Ckinc SLC

HCC- 1

TECK

CS(' Cbemokine Ex.A--s

PF4 IP-10 1 1 lg IL-S SAP-' G RO-a.-fi.-.{ Cic'P-2 SDF-1 a. P PB P

PBP cIcav3ge products: CTAP-III

b- TG X1 IP-2

Regulated upon activation. normal T-ccll expresscd and sccrcted Monocyte chemotatic proteins 1 4 Xliicroplirige i n f i a m m a t o ~ prorcin- lu . EBI 1 ligand chcmokine hlacrophage inf lammato~. protein-3p

Macrophage infiammatory protcin-3u Leukotactin- l

Eosinophil chcmotactic factor Eosinophil chemotactic factor hlyeloid inhibitoc factor-? hlyeloid Inhibitoc factor- l Pulmonan and activation-rcgulated chemokine Dendritic ccll chemokine- Macrophage-dcrivcd chcmokine Stimulated f cc11 chcmotactic protein-l Thymus and activation-reyulated chemokinc Exodus- l hlacrophagc inilammator). protein-3cr Liver and activation-

reyulated chcmokine Exodus-2 6 cysteine chemokine Secondan Iymphoid-tissue chcmokine Hemofiltrate CC chcmokinc- l Thymus-expressed chcmokine

Epithelial-ceIl derived neutrophil- activating protein 7s Platelet factor 4 Interferon-y-inducible protein- IO XIonokinc induccd by interferon--{ Interlcukin-S Neutrophil aciivating protein-l Grov.-th relatcd oncoyenc-a.-p.-y G r a n u l o c ~ ~ e chcmotactic protein-, Stromal ccll derived factor- Iu.

Platclet basic protcin

Connccting-tissue-activatiny protcin III b-thromboglobulin Macrophage inflammato- prorein-2

CCL l CC15

CCLZ. CCLS. CCL'. CCL 13 CCL3. CCLJ CCL 1 9.

CCL 15

C C L I 3 CCCCL l S

CCL 1 -

CCLZO

CCL l J

CCLIS

C S C L S

C S C L J C S C L 1 O

C S C L 9 C S C L S P

C S C L 1. CSCL?. CSCL3 C S C L 6 C S C L 12 CSCL'

CSSSC cbcmoliints

Fracralkinc ncurotactin C S S S C L l

1.4.2 Chemokine Receptors

Chemokines interact with seven-transmembnne spanning. pertusis toxin

sensiti\.e. G-protein coupled receptors to induce their activities. To date. five human

CXC chemokine receptors (CXCR). nine CC chemokine receptors (CCRI and one each

of C and CX;C chemokine receptors (CR and CX;CR) have been identified (Table 2 )

( Rossi and Zfotnik. 2000: Baggiolini et al.. 1997). Chemokine receptors recognize

chemokines of the corresponding subfamily. One exception to this rule is the D u e

antigen receptor. which binds both CC and CXC chemokines (Rossi and Zlotnik. 2000:

Baggiolini et al., i 997)

Chemokine receptors are expressed on a wide range of ce11 types including

leukocytes. epithelial cells. endothelial cells. neurons. astrocytes and smooth muscle

cells. This diversity is indicative of the multiple roles chemokines play. For esample.

tfirough the activation of signaling cascades chemokines can activate T cells, recruit

lymphocytes. promote the degranulation of granulocytes. induce mitogenic effects and

regulate hematopoiesis ( Brosmeyer and Kim, 1999 ).

The role of various chemokines and chemokine receptors in the clearance of

pathogens is best-dissected using ligand and receptor knock out mice. For instance

MCP- 1 - - mice are unable to clear infections with Schisrosonrn nrarîsorti Howe\.er, these

mice appear to be indistinguishable fiom wild type mice in the clearance of systemic

.\~cobncrer.iunr rzrberctrlosis infection ( L u et al.. 1998). Schisrosomn niansoiii is a

parasite. n-liich sesually reproduces and releases eggs within the host's tissues. In order

Table 2. H u m Chmdcinc Rrreptor Superthniiy Rtu-cptor CeIl Expression L i m d s

CSC cbcmdiioe rmpiors

CSCR 1

CSCRl CSCR; C'SCRJ

C'CR6 CCR' CCRS c'CR9 CCR I O CCR 1 1

C cheniokioc rcccptw

SCR 1

c c - 1 . 1 5 3 - - CCLS . CCr'J

CCL2. '. S. 13 CCL 1 1 . 1;. 15. 2-1 ,- CCLS. -. S. 26 CCL 1 -. 22 c c = . J.5.S. - -CCL', II. 1;

to control the spread of parasite progeny. macrophages are recniited to surround the egg

and form a ,ganuloma. Therefore, MCP- 1 is n e c e s s q for the recruitment of

monocytesimacrophages. which is important for the formation of granulomas. resulting in

the eventual clearance of the Schistosome egg (Lu et al., 1998 ). The formation of

granulomas is also characteristic of A(~robacter-irrn tzrberctrlosis infection. It is

speculated that resistance to LM. trrber-crrlosis may be due to an intact Th- 1 response. As

well. CCRZ-- knock out mice are unable to clear infections with the intracellular

bacterium, Lister-irr nrorioq-rogenes ( Kunhara et al-. 1 997 ). Taken together, recruitment

of monocytes/macrophages for the clearance of Listeria nrono~~rogettes is important.

However. the hi@ rate of macrophage infection by these gram-positive bacteria may

indicate a need for the activation of chemokine induced intncellulrir killing. CCRS

deficient mice developed normally in pathogen free environrnents. However. when

challenged n-ith Listerin nlortoc\*togenes. they were unable to fight infection (Zhou.

199s ). Together ~vith the above data. this would indicate the need for the coordination of

CCR2 and CCRS mediated events in the clearance of Lisreria ard Shisrosonta. CCRS is

espressed on T cells of the Th- 1 subtype. which are involved in the activation o f

macrophages. Therefore the activation of macrophages is paramount in fonning

grandoinas and killing intra-cellular bacteria. Moreover. the inability of CCRZ and

MCP- 1 deficient mice to recmit monocytes to the site of infection prevents the activation

of monocytes and subsequent interactions n-ith Th- 1 lymphocytes. Consequently. these

mice are unable to mount an immune response towards the pathogens. Although there is

still a great deal of information to be revealed concerning chemokines and their receptors.

b o c k out studies will inevitably lead to a greater understanding of which immune

responses are more effective at clearing different infectious agents.

1.4.3 Simal Transduction Throueb Cbemokine Rece~tors

Studies of chemokine induced signal transduction have uncovered a number of

interesting findings. Firstly, it is becoming more apparent that the activation of G

proteins associated with chemokine receptors represents a fiaction of the level of

activities induced by the ligands. Also. accumulating findings on signal transduction

events induced by ligand-receptor interactions have shown that very different outcomes

may occur depending on the ce11 type-

The greatest amount of information regarding signaling through chemoliine

receptors is derived from G protein activation studies. In genenl. the G proteins

associated witli chemokne receptors are o f the Gai subtype. which are inhibited by

pertusis tosin (Rollins. 1997: Luster. 19%). Upon binding to the receptor. chemokines

induce the eschange of GTP for GDP leading to the dissociation of the a subunit form

the py subunit. This is followed by the activation of cAMP/protein kinase A pathway. the

MAPK pathway. the phosphatidylinositoL~calcium! protein kinase C pathway and the

JNK pathway ( Figure iv) (Iacovelli et al.. 1999: Hamm. 19%). Although most of the G

protein signaling induced by chemokines is pertusis tosin sensitive. implicating the Gai,

subunit. other pertusis tosin insensitive subunits cannot be over iooked- In fact, CXCRl

and CXCR2 activate Gu,, and Gaia subunits upon ligand binding. which leads to the

a&\-ation of MEK and ERK ( ShyamaIa. 1998). The Gp,subunit is also able to activate

the RasMAPK pathway and is therefore an alternative mechanism for regulating the

MAPK pathway (Gutkind. 1998). In addition to signal transduction resulting in

migration. chemokines have been shoivn to induce the activation of Aii- protein in kinase B

( PKB ). an important component of ce11 pro-sunrival machinery (Brunet et al.. 1999). The

activation of Ah by PI-3 kinase results in the downstream activation of IKB allowing for

the translocation of NF- KB to the nucleus and therefore up-regulating transcription of

sun-ivd genes (Ozes et al., 1999 1. Although this pro-sunfival characteristic of

chemokine receptor activation would appear to be beneficial for the maintenance of

inflammation. it poses problems for the induction of autoimmune diseases. such as

rheumatoid arthritis and for the survival of HIV infected cells. Specifically. HIV

activates G-proteins as gp 120 intencts with CCRS/CXCR;I resulting in the dissociation

of the py subunit. The py subunit can then activate PI-3'liinase resulting in the activation

of PKB. Thus it is possible that the secretion of high levels of soluble gp 120 results in

survival of cells infected with HIV. Moreover, the aberrant expression of RANTES and

MIP- 1 B were irnplicated in the persistent inflammation in rheumatoid arthritis (RA)

(Robinson et al.. 1995). Through the expression of high levels of chemokines in synovial

tissues, there is an increased number of cells infiltrating the affected joint. Furthermore,

sustained expression of these chemokines wouid resuit in an excessive inflammatory

response nith prolonged activation and s u ~ i v a l of the infiltrated leukocytes. This w u l d

ultimately lead to the destruction of the synoi-ial tissues.

In addition to the signals activated tfirough G proteins. there is emerging evidence

for the activation of various receptor-associated protein tyrosine kinases. Studies have

s hoivn that binding of chemokines to their-receptors stimulates receptor aggregation and

3icti\-ation of janus kinases (Jaks). followed by activation of signal tnnsducers and

activators of transcription ( Stats ).

The Jak-Stat sipaling pathvay was frrst identified using a genetic screen in

search of identifiing signaling molecules required for interferon induced genes ( Damell

et al.. 1994)- To date four mammalian Jaks; Jakl. J a E . J d d and TykZ (Wilks. 1989;

Fimbach-Kraft et al.. 1990; Rane and Reddy. 1994) and seven mamrnalian Stats; Stat 1.

Stat3. Stat3. Stat4. Stat 5a/b and Stat6 have been identified (DarneIl. 1997). Each

member of the Jak family is composed of seven Jak-homology (JH) domains. numbered

one to seven. starting at the carboxy terminus (Ziemiecki et al. 1994 ). The consewed

regions are found only in the Jak family members. where the JH 1 domain or kinase

domain. consists of nvo adjacent tyrosines. which allow for h-arts-autophosphorylation of

the associated receptor (Liu et al.. 1998 ). Subsequently. the phosphorylation of the

receptor allon-s for Stat association via SHZ-domain interactions (Liu et al.. 1998 1. The

phosphorylated Stat then dissociates from the receptor allowing for hetero- and homo-

dimer formation. Once dimerized, the Stats translocate to the nucleus where they activate

the transcription of genes (Darnell. 1997; Sekimoto and Yoneda, 1998 ). Stats bind to

consensus sequence eIements of target genes called. Stat inducible elements (SIE ).

interferon stimulated response elements ( ISRE ) and gamma activated sequences (GAS ).

The targeted deletion of several Jak and Stat genes has revealed a diverse

in\.olr-ement of these proteins in cytokine receptor signaling. Both Jak2 and Star3

deficient mice are embryonic lethal and Jakl deletion results in perinatal death of mice.

Various other knockouts have phenotypes mging fiom autosornal SCID (severe

combined iinmunodeficiency) for JaH-", indicating an involvement in leukocyte specific

signal transduction and loss of mammary gland development and lacto-genesis in StatSA

deficient mice (Liu et al.. 1998).

Aberrant expression of chemokines can be a factor in the induction of various

pathologies such as. rheumatoid arthntis (Robinson et al.. 1995 1. Therefore. therapies

using chemokine analogues. which act as receptor antagonists. could be very usefil in

preventing disease. AOP-RANTES. an NH;-tenninally modified analogue of RANTES.

has been crecited to act as a CCRS antagonist in hopes of blocking HIV infection

( Arenzana-Seisdedos et al.. 1996). This chemokuie analog was s h o w to bind sevenl

CC-chemokine receptors. including CCRS. How-ever. the status of receptor activation for

both RANTES and AOP-RANTES was not fùlly characterized until recently. Data

indicate thst acti\ation of CCRS by RANTES and AOP-RANTES is virtually identiccil

( Rodriguez-Fnde et al.. 1999). Both ligands induce CCRS dimerization. which leads to

the activation of tyrosine kinases and transcriptional factors ( Rodriguez-Frade et al..

1999 i. HEK-293 cells. when exposed to either RANTES or AOP-RANTES. induce the

association of Jak 1 with CCRS follo\ved alrnost simultaneously by Jakl activation.

Subsequentiy Jakl activation creates a docking site for SHZ-containing proteins such as

STAT and there is evidence that STATS associates with CCRS through its interaction

with JAK 1 ( Rodriguez-Frade et al.. 1999). Interestingly. evidence would suggest that the

acth~ation of late tyrosine phosphorylation events mediated by other tyrosine kinases is

absent with AOP-RANTES. The activation of p ' 2 S ~ ~ ~ and ZAP-70 has been described

afier RANTES stimulation (Bacon and Schall. 1996) and appear to be important in IL-?

receptor espression. Furthemore, it has been demonstnted that MIP- 1 a and RANTES

acti~-ate Stats in T lymphocytes (Mrong and Fish, 1998) and JakZiStat3 are activated by

MCP-1 interaction with CCR2 (Mellado et al.. 1998). CCRZ itself is tyrosine

phosphorylated at residue 139. which when mutated. prevents JakiStat activation and

calcium mobilization (Mellado et al.. 1998). It is becoming apparent that activation of

chemokine receptors does not exclusively result in G-protein mediated chemotasis. but

that other. as yet il1 defined biological outcomes are mediated by non G-protein coupled

signaling cascades.

1.4.4 Virallv Encoded Chemokine And Chemokine Receptors

Several viral open reading fiames of many of the poxvims and herpesvirus

genomes have revealed sequences that bare striking homolog-y to chemokines.

Mulluscum contagiosum virus is a poxvirus that infects humans but causes only benign

wart like lesions. Analysis of the genome sequence has revealed an open reading frame

( ORF ) MC 148R. ~vhich shares amino acid identity s-ith human macrophage

inflainrnatory protein 1 P (MIP- 1 P ) (Senkevich et al.. 1996 1. The predicted protein

encoded by MC 148R has retained correct cysteine positioning important for folding and

three-dimensional structure, however it lacks the N-terminal region. Deletion of the N-

terminus on several human chemokines has revealed a role in receptor activation (Clark-

Len-is et al.. 1995 ). Therefore. it is hypothesized that binding of MC 148R results in

antagonistic competition with the human ligand. As well. human herpes virus-8 (HHV-

S ). a 'J herpesvinis linked to Kaposi's sarcoma. posess three ORFs which share ZS40°'o

sequence identity to the human CC chemokine MIP- 1 a (Moore et al.. 1996 ). The newiy

identified genes. termed K4ivMIP-II. KGhMIP-1 and BCKivMIP-II have revealed a role

for \ inlIy encoded chemokines in disease pathogenesis ( Moore et al.. 1 996 1.

K4hrM1f -II has been shown by competition binding studies to effectively

displace human chemokine binding to CCRl, CC=. CCRS and CXCR-4 (Nichoix et al.,

1997. Moore et al.. 1996). vMIP-II has also k e n sho~t-n to interact mith the

cytomegalovirus chemokine receptor. US28 (Kledal et al.. 1997). Binding of vMIP-II to

chemokine receptors does not invoke activation of signal transduction and therefore

fünctions to sequester chemokine receptor away fkom the natural ligand. vMIP-II was

shown to reduce influx of inflammatory leukocytes and attenuate host inflammatory

responses in a rat mode1 of glomerulonephritis (Chen et al.. 1998 1.

One distinct characteristic of Kaposi's sarcoma is the formation of vascular

infrastructure associated ~vith tumors. Recent studies implicating chemokines in the

regulation of angiogenesis may implicate vMIP-1. II and III in the formation of vessels.

Moreover. vMIP-1 and vMIP-II induce angiogenesis in a chick chorioallantoic membrane

assay (Boshoff et al.. 1997). Thus. it is evident that virally encoded chemokines are

potent pathogenic factors.

The role of virus encoded chemokine receptors in disease pathogenesis has been

best described through charactenzation of the human cytomegalovirus (HCMVI encoded

chemokine receptors. HCMV genome sequence analysis revealed three ORFs, which

share sequence identity with chemokine receptors (Chee et al.. 1990). They are termed;

US27. US28 and UL33. of which, US28 has been the most charticterized (Gao and

Murphy. 1994). Binding studies have demonstrated that US28 interacts with sevenl CC

chemokines with high affinity, but exhibits no affinity for CXC chemokines (Gao and

Murphy, 1994: Neote et al.. 1993 ). Furthemore. cells transfected with US28 induce

intracellular calcium signaling in response to chemokine interaction (Gao and Murphy.

1994). Although it is possible that US28 fûnctions solely to prevent the formation of a

c hem0 kine gradient. thereby preventing infiltration of leukocytes, this does not explain

u-hy the receptor u.ould induce calcium s i p a l h g in response to chemokines. However,

it is possible that binding of chemokines to US28 resuits in events that are distinct from

events induced by activation of constitutive receptors.

A nurnber of y herpesviruses have also been shown to express fùnctional

chemokine receptors. HHV-8 encodes a chemokine receptor termed ORF7-4. which is

homologous to herpes saimiri's C X C E homolog. ECRF3 ( Arvanitakis et al.. 1997 ).

Although both receptors are able to bind CXC chemokines. ORF71 is also able to bind to

CC chemokines (Ahuja and Murphy, 1993 ). Furthemore. ECRF3 transfected cells

release c a 2 * when stimulated with chemokine. but in contrast to other viral and cellular

chemokines. ORF74 is constitutively active (Awanitakis et al.. 1997). It is unciear why a

receptor that is constitutively active would bind chemokines, but activation of these viral

chemokine receptors may play a role in augmenting viral replication.

1.5 EXPLOITATION OF SIGNAL TRANSDUCTION BY VLRUSES

The activation of signal transduction is paramount for cells reacting to an

infectious agent. The activation of the JaWStat pathway by IFN-IFN receptor

interactions. allows for the subsequent clearance of pathogens. Vinises have become

proficient at circum-enting the activation of signal transduction to enable their sunrival

within the host cell. Hepatitis C virus (HCV) interferes with TNF induced signal

transduction by preventing the activation of NF-KB (Shrivastava et al.. 1998 1. The HCV

core protein prevents 1-KB degradation, which is needed for NF-& activation and

nuclear translocation. Furthemore. HCV core protein activates AP- 1 associated with the

actiwtion of JNK and MAPKK. The mechanism by which HCV core protein achieves

this is still not understood (Shrivastava et al., 1998). However, it is evident that HCV has

become proficient at circumventing activities that should effectively result in v h s

clearance. Furthermore, HCV proteins are also able to inhibit JddStat signaling.

Reminiscent of the inhibition of NF-& seen with HCV core protein, the HCV

nonstructural protein 5A (NSSA) was shoum to inhibit activation of Stat induced

transcription (Heim. 1999). Although. NSSA does not exclusively inhibit JaUStat

signaling sictivated by IFN-ct. the inhibition did effectively ablate IFN-CL antiviral

activities ( Heim, 1999). The ablation of signal transduction and the redirection of

transcription factors to benefit viral survival is a strategy utilized by sevenl viruses.

including the inhibition of JabStat signaling that prevents espression of MHC II by

human cytomegalovirus (HCMV) (Miller et al., 1998).

By contrast. the activation of receptor mediated signal transduction by various

viruses has also been noted. For example, the herpes saimiri virus Tip-484 protein was

shonn to dramatically upregulate the activity of p56LCk, followed by the activation of

Stat 1 and StatS (Lund et al.. 1997). Further analysis of the target genes for Stat 1 and

StatS may lead to the identification of cellular genes required for virus replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) \vas shown to encode an IL-6

homolog. n-hich activates sipaling through the gp 130 receptor subunit ( Molden et al..

1997 ). The IL-6 receptor consists of two heterologous subunits. IL-6Ra and gp 130.

Antibodies to IL-6Ru effectively block human IL-6 intenction. However. the antibody

\\-as unable to block VIL-6 intenction and the activation of signal transduction. Indeed.

the activation of the Jak Stat pathway occurred npidly upon VIL-6-gp 130 intenction

(Molden et al.. 1997). Although it is not understood how gp lSO mediated signaiing

affects KSHV pathogenesis. it is believed to be essential for KSHV replication (Molden

et al.. 1997).

Friend virus. itVhich causes erythroieukimia in aduit mice. associates with the

erythropoietin ( EPO) receptor via gp55 envelope protein. The interaction of EPO with its

receptor results in the activation of JakZ and StatS (Yamamura et al., 1998). In contrast.

3355 induces the constitutive activation of Jak 1 and StatS. Interestingly, the activated

StatS is unable to bind to consensus DNA sequences and fails to translocate to the

nucleus (Yarnamura et al., 1998 ). Although. gpS5 activated cells become EPO

independent. the maintenance of Friend virus infection is not understood. Although

studies of HIV 23120 induced chemokine receptor signal transduction have received

much attention. it is evident that the exploitation of signaling intermediates by vimses is

not unique to HIV.

1.6 HIV INDUCED ACTIVATION OF CHEMOIUNE RECEPTORS

The exploitation of CD4 dong with chernokine receptors. specifically CCR5 and

CXCR-I. by HIV has been well established. Interestingly. individuals that possess the

C C R 5 S 2 mutation are not susceptible to HIV infection. Resistance to HIV in these

indi\-iduals is due to the requirement for monocyte!macrophage tropic HIV strains to

initiate infection (Hill and Littman. 1996). The use of chemokine receptors for viral entry

aIlou-s for selection of cells that \vil1 migrate throughout the organism. However, it is

apparent that the use of these receptors extends beyond the virus' need for dissemination.

In fact. e\-idence is mounting which elucidates a number of activities induced by HIV

interactions u-ith chemokine receptors.

Initial interaction benveen HIV- 1 coat protein (gp 120) and CD4 induces a

confonnational change that aliow-s for gp 120 interaction with one of the chemokine co-

receptors ( Berger et al.. 1999: Homk. 1999). Early studies have shown that pre-treating

cells n-ith chemokine could block HIV fusion and infection. Furthermore, pre-treating

cells u-ith pertusis tosin also prevents HIV infection. Subsequently. this led to the notion

that interaction ben\-een g p 120 and CCRSICXCRJ is necessary for the activation of

signaling intermediates. which \vould later be necessary for viral replication. Although

tliere are conflicting data testing this hypothesis. there is some evidence that ~ a ' ~

~nobilization is a consequence of gp 1 20-CCR5 interaction ( Kornfeld et al.. 1998 ). As

well. solubie g p 120 has been show-n to induce the chernotaxis of various ceils (Kornfeld

et al.. 1998). There is also mounting evidence that HIV envelope proteins are able to

activate protein tyrosine kinases. In fact, Pyk-2 activation was reported to be a

consequence of ,op 120 interaction with CCRS and CXCR-4 (Davis et al., 1997). PykZ is a

protein tyrosine kinase related to p 125-FAK and is expressed primarily in neuronal and

hematopoietic ce11 lineages (Lev et al.. 1995). Upon ligand binding to G-protein coupled

receptors. P y E becomes phosphorylated ( Lev et al.. 1995 ). Therefore the activation of

PykZ by MIV n.ould suggest that activation of chemokine receptors results from

interaction with gp 120. Furthemore. FAK association to CCRS is induced through

HIV-envelope interactions (Cicala et al.. 1999). suggesting that ligation of CD4 and

CCRS leads to the formation of an activation comples. This comples includes PyE.

ZAP7O. puillin. p56'Ck. F A K and CCRS. al1 of which are phosphorylated foliowing 5-

minutes esposure to HIV envelope (Cicala et al., 1999).

Evidence has been provided for the activation of p561ck through the CM-gp 120

interaction (Goldman et al.. 1994: Su et al., 1999: Wang et al.. 1998). Activation of

p56'c\timulates the association of p56'Ck with Raf- 1 and results in a Ras-independent

acti\ration of Raf-1 (Popik and Pitha, 1996). Interestingly. PKC has been shown in

clieinokine induced signal transduction to activate Raf in a Ras independent manner

(Kerine and Strieter. 2000). Therefore, it is possible that the activation of Raf- 1 may be a

consequence of G protein activation of PKC. Thus. the CO-association behveen p56'ck and

Raf- 1 maybe mediated through receptor complexing. In fact. G-protein activation

appears to be important, since pre-treatrnent of cells with pertusis toxin blocks HIV

infection ( Alfano et al.. 1999). However, this does not esclude the need for tyrosine

phosphorylation in HIV infectivity. Evidence does indicate the need for tyrosine

mediated signaling events. nevertheless it is not clear which stage of infection HIV

requires the activation of such intennediates. Data have s h o w that CXCR-i is

phosphorylated in CD-lT T lymphocytes and the downstream activation of p561Ck results in

receptor doivn regulation (Wang et al.. 1 998 ). Although. it is not understood h o ~ v

receptor d o i i regulation assists in vin1 entry o r replication. it is possible that the

activation of intermediates described above allows for intemalization of HIV and

enhanced replication.

There is considenble debate as to whether or not activation of chemokine

receptors is important for viral fision and entry. or whether the signals induced by HIV

envelope protein are necessary for donnstream replication. Studies using sevenl isolates

of CD4 and CCRS tropic simian immunodeficiency viruses (SIV) provide evidence for

actil-ation of serinejthreonine protein b a s e s . The activation of mitogen activated

protein (MAP) kinase kinase (MEK) and estracellular signal-regulated kinase 1 and 2

(ERK 1 2 ) \r-ere induced by CD4 tropic strains. whereas. p38 MAP kinase and c-Jun N-

terminal kinase (JNK) are activated by CO-ligation of CD4 and CCRS (Popik and Pitha.

1998 ). Moreover. the activation of ERK MAP kinase has been shown to induce the

actii-ation of AP- 1. promoting the sn-itch from latent to productive infection (Yang et al.,

1999). As tvell, the espression of constitutively active MEK or Raf kinases results in

HIV- 1 long terminal repeat (LTR) activation by NF-- (Yang et al.. 1999).

Unfortunately. the efSect of early activation of ERK-MAP kinase pathn-ays is not fully

understood. Hoivever, activation of NF-- by MEK or Raf following the treatment of

cells u-ith cytokines would implicate the activation of specific genes upon NF-KB

translocation to the nucleus (Yang et al.. 1999). Further evidence for a role of signal

transduction in mediating HIV replication derives from studies shon-ing the presence of

high levels of soluble gp 120 during the course o f productive HIV infection (Geiderblom

et al.. i 987 ): The implications are that circulating levels of gp 120 mediate CCR

acti\.ation. thereby promoting the switch from latent to productive infection.

1.7 USE OF CHEMOKINE RECEPTORS BY MYXOMA VIRUS:

TiiESIS OBJECTNES

When comparing the disease etiology o f AIDS with myxomatosis. it is apparent

that several similarïties exist. When it was deterrnined that HIV used chemokine

receptors as CO-receptor for infection, it became obvious that use of these receptors

contributed to systemic spread. The severe immune dysfiinction that follows HIV

infection. resulting in AIDS. can be explained by the deliberate induction of apoptosis in

CDJ* T cells. Myxomatosis also results in severe immune dysfunction. but in contrast to

HIV. the dysfunction is a direct result of myxomas' arsenal of anitimmune strategies.

Ne\-ertheless. the systemic nature of mysomatosis \vould indicate the use of leukocytes

for dissemination throughout the rabbit. Recently. it became evident that myxoma virus.

like HIV. also utilizes chemokine receptors to infect cells (Lalani et al.. 1999b). Rodent

fibroblasts (3T3 ) that cannot be infected with myxoma virus could be made h l l y

permissi\-e for myxoma virus infection by expression of any one of several human

chemokine receptors, inctuding CCRI, CCR5, and CXCR4. Conversely, infection of

3T3-CCRS cells can be inhibited by RANTES. anti-CCRS polyclonal antibody. or

herbimycin A but not by monoclonal antibodies that block HIV- l infection or by pertusis

tosin. These findings suggest that poxviruses. Iike W. are able to use chemokine

receptors to infect specific ce11 subtypes. notably migratory leukocytes. but their

mechanisms of receptor interaction are distinct.

The ability of herbimycin A to inhibit mysoma virus infectivity indicates a need

for the actimtion of tyrosine kinases. Our w-orking hypothesis is that intncelfular s ipa ls

transduced by myxoma \-inis-CCR intenctions have immunopathogenic consequences.

Therefore. a clearer understanding of the precise outcornes mediated by myxoma virus

interaction \vith CCRs n-il1 provide insights into potential therapeutic targets in human

disease situations where virus-receptor intenctions subvert and evade the host-immune

system. To elucidate the potential discrete sipaling pathways activated by myxoma

\-inis-CCR intenctions. we performed studies using both live and UV inactivated

rnysoma virus. Vin1 entry into cells is unaffected by LIV inactivation. Cells. both

permissi\-e and non-permissive to myxoma virus infection were employed. that express

the human chemokine receptor. CCRS. The objective of our studies was to determine

ix-hether viral activation of CCRS is a pre-requisite for vin1 entry andlor viral replication.

Cbapter II

MATERIALS AND METHOD

11.1 C e l s and Virus

NIH 3T3 cells (American Type Culture Collection) and NIH 3T3-T-l-CCR5 cells

obtained fiom D. Litman. New York University were rnaintained in Dulbeco's Modified

Eagle medium (DMEM) (Gibco-BRL ). supplemented with 10°,o fetal calf senim

( FCS )(Gibco-BRL). 100 unitslml penicillin (Gibco-BRL) and LOO m g ml streptomycin

(Gibco-BRL). The rabbit basophilie leukernia ce11 line, RBL-CCR5. were a gift fiom M.

Oppennan (Georg-August Universitat Gottingen, Germany ) and were maintained in

RPMI (Gibco-BRL ) supplemented with 10% FCS. 100 unitsirnl penicillin and lOOpg/ml

streptomycin and under the selective pressure of 600nglml of G4 18 (Gibco). All cells

were g r o m at 37 OC in an atmosphere of 5% CO 2. vMmyxlac. a myxoma virus (strain

Lausanne) derivative, bearing a P-galactosidase marker cassette in an intergenic location.

was a gift fiom G. McFadden (Robarts Research Institute, UWO). To determine the

activation of signaling pathways induced by myxoma virus. wnyxlac was W inactivated

nt Io9 PFUiml for 20 minutes using the Stratagene StrataLinker.

Ci.2 Analvsis of human CCRS and CD4 exwession bv flow cvtometrv

CeIl surface expression of CCRS and CD4 wris quantified by flow cytomety

using the monoclonal antibody ZD7(Pharmingen) and a monoclond antibody (SIM.1).

(National Institute of Health AIDS Research and Reference Reagent Program)

respectively. Cells were gated based on fonvard and side scatter. CD4 and CCR5

espression \vas detennined using an anti-mouse biotin conjugated secondary antibody

detected u-ith a Cy5 conjugated strepavadin fluorescent tertiary reagent. Staining \vas

conducted according to the manufacture's protocol. and flo\v cytometric data were

acquired using FACScan (Benton Dickinson Immunocytometry). CELLQuest sohvare

\{-as used to analyze al1 data.

11.3 Ceii Staining for Flourescent and Electroa Microscopv

To rnonitor viral RNA and DNA associated with cytoplasmic viral factories. as

ive11 as to determine ce11 viability. NIH 3T3-T4-CCRS and RBL-CCRS cells were

esposed to vmyslac at and m.0.i. of 1 and left to absorb for 1 hr at 37°C. Cells were then

\vashed once n-ith PBS and incubated for 16 hours in fiesh media. The cells were stained

n-ith 100 pglrnl acndine orange (Sigma) plus 100 pgml ethidiwn bromide (Sigma) and

\-isualized under fluorescence rnicroscopy at 40 X magnification..

For electron microscopy (EM) analyses. mycoma virus infected ceils were fixed

in 3 . 7 O O formaldeliyde + 1 .1°8 glutaraldehyde 16 hours post infection. The cells were

han-ested from plates and collected for staining with unnyl acetate and lead citrate. Thin

sections of the paraffin embedded cells were scanned by a transmission electron

microscope to identify viral particles ( Winter et al., 1995). (This work was done in

coilaborrition \vith Steve Doyle at the University of Toronto)

11.5 CeU Lvsis and Immunoblotting

Approxirnately 10' cells were exposed to the equivalent of 10' infectious units of

UV-inacti\-ated virus particles for the indicated times. Cells were washed tu-ice Lvith cold

PBS and iysed for 1 hour at. 4OC with rocking, using a lysis buffer cuntaining, 1O.o Triton-

S. 0 . 5 O 0 NP-IO. 150mM NaCl. 10 mM Tris pH 7.5. 1rnM each of EDTA and EGTA, 0.2

inM Na;V04. 0.2 rnM PMSF. I O p g m l aprotinin. 2 p g m l leupeptin. 2 p g m l pepstatin A

and 1 mM NaPO-. The whole ce11 lysates were pre-cleared with 4 pg of species specific

IgG (Jackson Labs) and 20 pl protein A-G plus agarose for 1 hour at 4°C. The

supematants were collected and immunoprecipitated over night at 4°C with 5 pg of one

of the follo\ving antibodies; a-CCRS (Santa Cruz. CKR-5(C-20) ), the a-phosphtyrosine

antibody. pY20 (Santa Cruz). a-Stat 1 (C- 136) and StatS (H- l9O)X (Santa Cruz), a-

JakI (HU-758) and u-Jak2 (Q-19) (Santa Cruz). u-Lck (3A.5. Santa Cruz). and a-IRS l

and IRSZ (Upstate Biotech). Following this. the antibody is collected using 20 pl protein

k G plus agarose for 2 houn at 4°C while rocking. The antibod-protein &G complex

\vas \\.ashed 3 times ~vith the above lysis buffer followed by the addition of 30 pl of ZX

sample reducing buffer. Finally. the immunoprecipitate \vas boiled and resolved by SDS-

PAGE and transferred to Hybond-C supported nitrocellulose (Arnersham Inc.).

To detennine the activation of phosphorylation of the immunoprecipitated

proteins. the anti-phosphotyrosine antibody -CG 10 (Upstate Biotech) \vas utilized.

Membranes Lvere blocked u-ith 1 O O,'o BSA in TBS (50 mM Tris pH 8.0 + 150 rnM NaCl)

for 1 hour and washed 3 time for 5 minutes with TBST (50 mM Tris pH 8.0. 150 mM

NaCl. O.? O Tween-20). A concentration of 1 pdml of 4G 10 in TBST (50 mM Tris pH

S.0. 150 mM NaC1. 0.2°h Tween-30) was used to probe the irnmunoblot for at least 1

hour. Membranes n-ere then \vashed 3 times for 5 minutes followed by the addition of

the HRP conjugated anti-mouse secondary antibody (Amersharn). at a 1 : 15000 fold

dilution in TBST. The secondan, antibody \vas incubated for 30 minutes followed by

tliree 5-minute ivashes. Detection was performed using ECL reagents ( Pierce ).

Imrnunoblotting for deterrnining the ioading of protein \vas perfonned as above

hou-e\-er. membranes were blocked in j O / O milk in TBST ( 10 mM Tris pH 7.5. 100 mM

NaCI + O. 1 O O Tween-20) and the primary antibody \vas also incubated in 90 milk TBST

for 1 hour.

Membranes \vere stripped for 10 minutes in a stripping solution containing: 7 M

guanidine-HCL, 50 mM glycine pH 10.8. 100 m M KCL, 5 pM EDTA and 138 mM P-

rnercaptoethanol. The membranes are then washed 2 times in ddHiO to remove escess

stripping solution and then incubated in TBST to equilibrate the membrane.

11.1 Use of Pharmocoloeical Inhibitors for Altering Viral Infectivitv

To examine the significance of virus induced activation of p56LCk (Src kinase) on

infecti~~ity. 4 s 1 o4 cells in 96-m-el1 plates were pre-treated for 15 minutes with varying

doses ( 1 nM. 5 nM. 10 nM, 50 nM in medium) of PP1(4-amino-5-(4-methylphenyl)-7-( t-

butyl )pyrazolo[3.4-dlpynmidine) (Biomol) diluted in DMSO. then incubated tvith PP 1

during virus adsorption. Cells were exposed to vmyxlac at an m.0.i of 10 for 1 hour. then

incubated in fresh medium containing PP 1, overnight. Approximately 16 hours post

infection. P-galatosidase activity was analyzed using a colorùnetric assay. Briefly. cells

m-ere lysed using a lysis buffer composed of 10 *,O NP40 and 50 mM Tris pH 7.5. After

one freeze thaw cycle. an aliquot of the ce11 lysates were tnnsferred to wells in another

plate containing. LOO mM NaH2PO~. 10 mM KCL. ImM MgSO4 and 50 mM B-

mercaptoethanol. The mixture kvas incubated for 5 minutes at 37 OC. To measure P-

galatosidase activity. o- nitrophenyl-9-D-gaiactopy~noside (ONPG) was added in at

concentration of 4 m2ml in t O 0 mM NaHzPO, pH 7.5. The breakdo\\n of ONPG by P-

datosidase results in a yellou- colour reaction. Afier l hour, the reaction was tenninated - by the addition of 1 M Na2CO;. hbsorbance was measured spectrophotometncally at 420

mn. using a THERMOma.. microplate reader.

Additionally. to examine the influence of virus induced activation of J a E on viral

infecti\*ity. the J a E inhibitor. tyrophostin B42 ( AG490 ) (Calbiochem ) was employed.

Cells tvere treated with varying doses ( 1 nM-1000 nM in medium) of AG40 diluted in

DMSO for 16 hours. and then infected as described above. The colonmetric assay for P-

galatosidase activity \vas performed as above.

Chapter III

RESULTS AND DISCUSSION

UI.1 Ectopic ex~ression of human CCRS alone is not wedicîive of mvxoma virus

infectivitv.

To determine whether infection by mysoma virus mediates CCW activation.

thereby rendering a ce11 permissive for viral replication. we undenook studies to examine

CCRS mediated signal transduction in two distinct ce11 lines that both express human

CD4 plus CCRS (Figure 1 ). yet are differentially sensitive to viral infection,

NIH 3T3-TJ.CCRS cells are murine fibroblasts transfected with a retrovims

espressing both human CD4 and CCRS. RBL.CCR5. a rat basophilic leukemia ce11 line

that expresses human C C W showed comparable levels of ce11 surface expressed CCRS to

the NIH 3T3.T-C.CCRS cells (Figure 1 ). Cell surface CD4 expression differs benireen the

tw.0 ce11 lines (Figure 1 ). To determine susceptibility to myoma infection. a

recombinant mysoma vinas that expresses the E. coli fi-galactosidase gene under the

control of a late viral promoter that drives a 1acZ reporter tnnsgene \vas utilized ( Lalani

et al. 1999b 1. Cells were infected with virus at an m,o.i. of 1 and stained with X-gal 16

hours post infection. The NIH 3T3-T4-CCRS cells were highly susceptible to infection

(Figure 2 ). However. the RBL-CCRS cells did not support viral replication. as revealed

in Figure 2

Figure 1 Ectopic expression of human CCRS alone is not predicîive of myxoma virus infectivity. Ce11 surface expression of human CCR5 and human CD4 were determined by by flo\r cytometric analysis Incubation with medium alone or 2' and 3 O reagents resulted in the negat i\-e cyto-m represented as theJ;lIed profle. Positive cytograms for CCRS (solid line) and CD4 (broken line) are represented as open profiles. Results are representritive of three experïments.

Figure 2. RBL.CCR5 ceils are not susceptible to myxoma virus infection. Susceptibility to rnysoma virus infection \vas detennined for both NIH 3T3-CCRS and RBL-CCRS. Cells were either mock infected or infected at a mul t ip i ic i~ of infection (m.0.i.) of I nvith Vm-lac for 1 h. Afier 16 h. infected monolayers were fised and stained for 1acZ espression. Results are representative of five experiments.

Mock lnfected Myxoma Virus Infected

Under the sarne conditions. when infected monolayers are ftved and stained with acridine

orange and ethidium bromide. the RBL-CCRS cells do not exhibit any significant changes

in nucleic acid localization in contrast to the NIH 3T3.CCR5 cells. which exhibit

increased cytoplasmic gene expression and altered morphology. characteristic of posvirus

replication ( Figure 3 ). Furthemore. we determined that exposure to myxorna virus does

not induce a cytopathic effect in the RBL-CCRS cells (Figure 3 1. Although increased

cytoplasmic nucleic acid is evident upon myoma virus infection of the RBL.CCR5 cells,

it is not representative of the esponential increase expected for virai replication and gene

transcription ( Figure 3 ). Furthemore. the activation of genes involved in viral clearance

may be responsible for the increased staining of nucleic acid in the RBL-CCR5 cells

(Figure 3 ). Notably. when NIH-313-T4-CCRO and RBL-CCRO cells are infected with

mysoma \-inis at an m.0.i. of 1. then esamined for cytoplasmic viral particles 16 hours

iater by electron microscopy. both ce11 srpes exhibit evidence of viral entry (Figure 4 ).

Esposure of NIH 3Tj.CCRS cells to mysorna virus leads to productive viral infection

and. as depicted in Figure 4. normal progeny at different stages of assembly are

\isualized. By contrast. the block of viral replication obsen-ed in the RBL-CCRS ceils is

not eserted at the level of viral entcy. Of note, when NIH 3T3.CCR5 and RBL.CCR5

cells rire adsorbed \.i.ith 1ix.e mysoma virus, and then the washed ce11 lysates are hanrested

for up to 4 hours folloiving esposure to virus. infective virus can be detected, indicating

tliat \-ims adsorption is not affected in non-permissive RBL.CCR5 cells (data not s h o w ) .

Additionally. intemalized virus particles are only obsenred sequestered within vesicular

stxwctures. ~vhich may be lysosomes in the RBL-CCRO cells (Figure 4.) We infer that

sequestration of mysoma virus particles in lysosomes of infected RBL-CCRS cells directs

their eventual de-mdation. Thus, the expression of CCRS and CD4 alone does not ensure

productive myxorna virus replication, as other cellular components are clearly required

Figure 3 RBL.CCR5 ceils remain viable during infection and show no signs of viral mRNA production. To \risualize RNA expression and DNA integriiy, cells were either mock infected or infected at an moi of 1 with vMyxlac. 16 h post infection cells were stained with acridine orange:ethidium bromide and visualized by fluorescence microscopy at 10X ~nagnification. Orange staining is indicative of fiagrnented DNA or RNA. The apparent morphology change and increased RNA localized in the cytoplasm of the NIH 3T3.CCR5 cells is indicative of viral infection. Data are representative of two different espenments.

M o c k Infected Myxoma Virus Infected

Figure 4 Vùal progeny are evident oniy with NIfi 3T3.CCR5 cells. Cells Lvere infected with myxoma virus at an moi of 1. then 16 h post infection were

u-ashed thoroughly with PBS and fixed. Cells n-ere visualized by electron microscopy. Amon-s in the NIH 3T3.CCR5 cells indicate viral progeny. Arrows in the RBL.CCR5 ceIIs indicate structures. which appear to be degraded vital particles.

NIH 3T3.CCR5

111.2 Mvxoma virus induces CCRS îvrosine phos~bowlation in NïH 3T3. CCRS cells. -

Since myxoma virus infection of NIH 3T3.CCR5 cells can be inhibited by

herbimycin A ( Lalani et al.. 1999b). at the outset we esamined whether adsorption of

r n p o m a virus induces CCRS phosphorylation. For these studies we employed UV-

inactil-ated mysoma virus in which the structural integrity of the viral particles are

retained. yet UV-induced damage to the viral genome irreversibly prevents viral

replication ( Ramsey-Ewing and Moss. 1998 ). When lysrites from myxoma virus esposed

NIH 3T3.CCR5 cells were immunoprecipitated with antibodies against phosphotyrosines

or CCRS and immunoblotted to detect CCRS or phosphotyrosines. we obsenred that even

1-5 minute esposure to UV-inactivated rnyxoma virus induces rapid phosphorylation of

CCRS on tyrosine residues (Figure 5 AB). Moreover. in NIH 313.CCR5 cells exposed

to myxoma \-irus particles. we routinely observed the CO-immunoprecipitation of a 56

kDa tyrosine phosphorylated protein with CCRS ( Figure 5C ). Immunoblot analysis

identified this protein as the Src kinase. p561ck (Figure SD). Our data would suggest that

p56'CL associated with CCR5 in a tyrosine phosphorylation dependent manner.

Figure S Mysoma vins induces CCRS ty-rosine phosphorylatioa in NIH 3T3. CCRS cells. NIH 3T3-CCR5 cells were untreated ( - ) or treated (+) with an equivalent of an m.0.i. of 10 UV inactiwted mysoma virus for the times indicated. Ce11 lysates were immunoprecipitated ( IP) with either an antiphosphotyrosine antibody (PY20) ( A ) or anti- CCRS antibody (B.C.D) and immunoprecipitated proteins were resolved by SDS-PAGE. then. sequentially immunoblotted (WB) as shown. for CCR5 (Al. phosphotyrosine (B.C) and p56'Ck (D). Results are representative of multiple experiments.

NIH 3T3.CCR5

vMyrlac

Time

K R . 5 35 kDa

IP: pY20 WB: CCRS

IP: CCRS WB: 4G10

IP: CCRS WB: 4G10

EP: CCRS WB: p56'Ck

111.3 Initiation of mvxoma MNS infection is associated with îwosuie

ln subsequent experiments we esamined the estent of mqiroma virus inducible

protein tyrosine phosphorylation in whole ce11 lysates fiom the permissive NIH

3T3 .CCRS and non-permissive RBL-CCRS cells. Immunoprecipitation of tyrosine

phosphorylated proteins followed by Western blot analysis reveal that a nurnber of

proteins consistently become tyrosine phosphorylated following 1-5 minute exposure of

NIH 3T3.CCR5 cultures to UV-inactivated myxoma virus. By contrast. the non-

susceptible RBL.CCR5 cells do not show any evidence of virus inducible tyrosine

phosphorylation of cellular proteins (Figure 6). The failure to invoke tyrosine

phosphorylation of cellular proteins in RBL-CCRS cells is consistent with published data

that the binding of the CC chemokine RANTES to CCRS on these cells leads to CCRS

phosphorylation that selectively occurs on serine but not tyrosine residues (Oppermam et

al. 1999).

Chemokine-mediated activation of chemokine receptors leads to the rapid

phosphorylation of receptor associated Jaks (Mellado et al.. 1998 1. PAGE mrilysis

re\.ealed inysorna virus-inducible phosphorylation of proteins that were candidate Jaks.

Stripping and reprobing these mtiphosphotyrosine immunoblots nith antibodies to Jaks.

confirmed the identity of tyrosine-phosphorylated Jak 1 and Jak2 (Figure 6 ). Although

the membrane \vas not tested for residual antibody. analysis of membranes treated

identically to the above resuhed in complete stripping of the membrane. Since p56 ICk

associated with CCRS in a phosphorylation-dependent manner (Figure 5C.D) it is

possible that p j 6 L c h a y be a s u b s a t e for the activated Jaks. Altematively, myxoma

virus-CCRS interactions may lead to cross talk between ce11 surface receptors that are

constitutively associated with CCRS, narnely CD4. which results in the subsequent

phosphorylation of CCR5 by other tyrosine kinases. like p56 ICk. Recently. evidence was

prolrided that shows the reciprocal desensitization of CCR5 and CD4 (Mashikian et al..

1999) by their respective ligands. As with HJV. mysoma virus rnay require both CD4

and CCR5 and the activation of p56 lCL may be a direct consequence of myxorna virus

interaction n-ith C M . Moreover, p56 lc"ctivation could lead to heterologous

desensitization of CCRS resulting in receptor down-regulation. This receptor

desensitization would effectively preclude additional viral particles fiom infecting an

dready infected target cell.

Indeed. when NIH 3T 3.CCR5 cells are treated with PP 1. û p56 ICk specific

inhibitor. Ive obsenred a dose dependent increase in myxoma virus replication (Figure 7).

( PP 1 inactileation of p56 ICk is specific at 10 nM ( Hanke 1996)). Although. the fold

increase in viral replication does not correspond to the fold increase in PPI concentration.

it is possible that, over the dose range examined. PP 1 may exert its effects on multiple

targets that n-ould include p56 lCk. As well. the rate of viral replication reaches a

masimun~ beyond \{.hich PP 1 can no longer esert an effect.

Figure 6 Initiation of myxoma virus infection is associated with tyrosioe phosphorylation of ceildût- proteins. NIH 3T3-CCR5 and RBL-CCRS cells were left untreated ( - 1 or treated (+) with an equivalent of an rn.0.i. of 10 UV inactivated my'toma virus. for the times indicated. Ceii lysates were imrnunoprecipitated ( IP) with anti-phosphotyrosine ( PY20 ) antibody and imrnunoprecipitated proteins were resolved by SDS-PAGE. then immunoblotted with 4G 10 anti-phosphotyrosine. ïhe NIH 313-CCR5 membrane was stripped and re-probed for Jakl and Jak2 with the respective antibodies. Results are representative of several experiments.

NIH 3T3.CCR5 Vmylac - + + Time (min) O I 5

r 1

IP:PY20 W B : Jakl

1P:PYZO W B : Jak2

Figure 7. Inhibition of p56Lck results in increased myxoma virus replication. NIH 3T3 .CCM ceils were either lefi untreated or treated with PP 1 ( 1 nM, SnM. 10 nM. and 50 nM ) for 15 minutes prior to myxoma virus adsorption. Cells were infected at an m.0.i. of I O and LacZ activity \vas measured 16 hours post infection. Results are representative of five different experiments.

III.4 Mvxoma virus induces Stat and IRS tvï-osine ~bosphowlation

In other cytokine receptor systems, the activation of Jaks results in the

engagement of multiple distinct proteins to tnnsduce signals and alter gene espression.

Accordingly. we esamined whether two of the best characterized downstream targets of

Jak acti\rrttion, the signal transducers and activators of transcription ( Stat ) ( Schindler et

al.. 1 999 ) and the insulin receptor substnte (IRS )-proteins (White 1998). \-t-ere

phosphorylated in NIH 3T3.CCRS cells exposed to myxoma virus. In an earlier

published report. evidence for RANTES-CCRS mediated phosphorylation-activation of

the Stat proteins. Stat 1 and Stat3 were provided (Wong and Fish 1998 1. The results in

Figure 8 N B indicate that exposure of NIH 3T3-T-I-CCRS cells to W-inactivated

m l o m a virus lerids to the npid phosphorylation of Stat 1 and Stat3 by 1 minute.

consistent nith the rapid kïnetics of phosphorylation-activation obsewed with the cognate

ligand. RANTES. Furthemore. in replicate experiments we observed that both Stat 1 and

Stat3 tyrosine phosphorylation is trrinsient. The transient nature of this Stat activation

may reflect the requirement for virus-inducible gene activation that is specific for viral

replication. yet precludes the sustained Stat-inducible gene activation required for viral

inhibition. The IRS famity of proteins includes IRS- 1 and IRS-2. which contain multiple

tyrosine phosphorylation sites in protein binding motifs for the SHZ domains of the p85

subunit of PI-3' kinase. the adapter protein. Grb-2. SHP-2 phosphatase and other

signaling elements (Ogawa et al.. 1998). These proteins play a central signaling role for

\various cytokine receptors by their ability to link these receptors to diverse downstream

signaling pathn-ays. We obsenled that myoma virus induces rapic! increases in the

tyrosine phosphorylation of IRS-1 and IRS-2 (Figure 9 N B ) . The high basal level of

phosphoylation apparent in the untreated control is indicative of a non-quiescent ce11

where multiple targets rnay activate IRS-1 and IRS-2.

Figure 8 Mynoms virus induces Stat tyrosine phosphorylation. NIH 3T3-CCR5 cells were untreated ( - ) or exposed (+) to an equivalent of an rn.0.i. of 10 UV inactivated myxoma virus. for the times indicated. Ce11 lysates were immunoprecipitated (IP) with anti-Stat 1 ( A ) and anti-Stat3 (B) antibodies. These immunocomplesed proteins were resolved by SDS-PAGE. then irnmunoblotted with -CG 10 anti-pTyr nntibody. The blots were stripped and re-probed with Stat 1 and Stat3 antibodies. respectively. Results are representative of three separate esperiments.

Vmyxiac Time (min.)

II': Statl WB: 4G10

IP: Statl Statl I, - WB: Statl

IP: S t . WB: 4G10

IP: stat3 StaO + WB: Stat3

Figure 9 Myxoma virus induces IRS tyrosine phospborylation. NIH 3T3-CCR5 cells were untreated (-) or exposed (+) to an equivalent of an rn.0.i. of 10 UV inactivated myxoma virus for the times indicated. Ce11 lysates were imrnunoprecipitated (IP) with anti-IRS 1 ( A ) and anti-IRS2 (B) antibody and resolved by SDS-PAGE. then irnrnunoblotted with 4G 1 O anti-phosphotyrosine antibody. The blots Lvere stripped and re-probed with IRS 1 and IRSlantibodies. respectively. Results are representative of two experiments.

IP: I l s 1 W R 4G10

III. 5 Tvrophostin BQ2 inhibits mvxoma v i r u s infectivity.

In subsequent expenments we examined the influence of Jak2 activation in

mediating viral infection. Specifically. when NIH 3T3.CCR5 cells were treated with

tyrop hostin B42, a dose dependent inhibition of myxoma virus replication was noted

( Figure 10A ). (Tyrophostin B42 specifically inhibits JakZ at 1 0 0 pM (Nielsen 1997,

Meydan 1996)). Therefore, the activation of JakZ by myxoma virus is required for

myxoma virus infectivity. However. it is unclear at which stage of infectivity Jak 2

activation is required. since in our system. the B-galactosidase gene is under the control

of a late promoter. which requires vin1 gene transcription and replication for expression.

in a recent study. IMV entry into HeLa celts was shoum to be sensitive ro protein tyrosine

kinase inhibitors ( Locker et al. 2000). Apparently, protein tyrosine phosphorylations

Lvere required for the formation of ce11 membrane protmsions that mediate \rirus

adsorption.

Figure 10. Inhibition of Jak2 activity results in reduced myxoma virus replication. NIH 3T3 .CCR5 cells were either lefi untreated or treated with AG490 ( 10 nM. LOO nM, 1 pbl. 1 O PM. 25 FM and 100 PM) for 15 minutes prior to myxoma virus adsorption. Cells n-ere infected at an rn.0.i. of 10 and Lac2 activity \vas measured 16 hours post infection. Results are representative o f four expenments-

Chapter IV

CONCLUSIONS

A major objective of this project was to determine whether, like HIV. myxoma

\-ims' utilization of chemokine receptors for infectivity, requires the activation of signal

transduction mediated by CCRS. Our results clearly show that initiation of a fully

productive myxoma virus infection in cells that express CCRS requires the tyrosine-

phosphorylated-activation of CCRS. As for HIV. interference with the signaling capacity

of CCRS appears to compromise the ability of mdv?toma virus to infect cells. Specifically.

the failure of myxoma virus to phosphorylate CCRS on tyrosines correiated with the

insensitivity of the RBL.CCR5 cells to myxoma virus infection. RBL-CCRS and NIH

3TS.CCR5 ceils are different species of cells both expressing the human CCRS receptor.

Therefore. it is possible that rat cellular intermediates are not able to intenct with the

human receptor. However. treatment of RBL-CCR5 cells with human RANTES does

induce c hemotaxis and phosphorylation of serineithreonine residues ( O p p e r m a ~ et al..

1999). Furthennore. the difference in susceptibility to my'roma virus infection cannot be

esplained based on ce11 type. since myxoma virus is able to replicate in BGMK cells. a

primate kidney ce11 line, RL-5 cells, a rabbit lymphocyte and HOS cells, a human

osteosarcorna ce11 line. Certainly, a pertinent expenment would be to over express a

CCR5 construct, which has mutated tyrosine residues into susceptible cells. and

determine subsequent sensitivity to myxoma virus infection.

Subsequently. it was important to demonstrate that the lack of viral replication in

the RBL.CCR5 cells was not due to viral-induced apoptosis. Acndine orange-ethidium

bromide staining revealed that RBL-CCRS cells exposed to my'roma virus did not

apoptose. When RBL-CCRS cells were exposed to myxoma virus. then lysed within 4

hours. live \-irus could be recovered. indicative of vin1 enhy into the cells. Under

electron microscopy we identified a number of degraded virus particles that appeared to

be in vesicular structures. which may be lysosomes (Figure 4). Thus. RBL-CCRS cells

do permit viral entry but are unable to sustain vin1 replication. In an earlier study using

the RBL-CCRS cells, RANTES-induced CCRS phosphorylation selectively occurred on

serine residues (Oppermann et al.. 1999). Whether the CO-expression of adequate levels

of hurnan CD4 are required for RANTES-inducible tyrosine phosphoiylation in these

cells. remains to be detennined. Certainly. there is no evidence that the RBL.CCR5 ceils

express a variant or mutant CCRS. Our data with the Jak 2 inhibitor. tyrophostin B42.

implicate Jak activation in mediating viral replication (Figure 10). Virus-inducible J&

activation may be requued for CCR5 phosphorylation. One possibility is that the

RBL.CCR5 cells express a mutant Jak2, or a constituitively active phosphatase. The use

of an alternative tyrophostin not specific for Jakt, will further address the role of j a k 2 in

myxoma virus replication.

Interestingly, treatment of NIH 3T3.CCRS cells with PP 1. a p56LCk specific

inhibitor. resulted in increased viral replication (Figure 7 1. Therefore, virus induced

activation of pj6LCk suppresses myxoma virus replication. Although the status of CCR5

phosphorylation wns not determined in our study. it is possible that the activation of

p56Lc\esults in receptor desensitization (Mashikian et al.. 1999). thereby preventing the

activation of signaling intermediates needed for continued myxoma virus replication. Of

note. down-regdation of CD4 and dissociation of p56LCk has been described for myxoma

virus infected CD4+ T lymphocytes (Barry et al.. 1995). Although. CD4 down-

replation by my?toma virus may be an effective mechanism for suppressing an immune

reaction. it is possible that dissociation of p56Lck from CD4 prevents CCRS

desensitization. which would allow for migration of infected cells. increasing

dissemination. as well as providing the necessary signaling intermediates required for

replication.

Esposure of NIH 3T3.CCR5 cells to W inactivated myxoma virus resulted in the

rapid activation. n-ithin 1 minute. of Jak 1 and Jak2. We also observed the coincident

phosphorylation of Statl and Stat3. It is unlikely that this Jak or Stat phosphorylation can

be attributed to virus activation of the IFN system. since the dsRNA viral intennediates

that mediate activation of the IFN system are not produced using UV inactivated

myxoma \rirus. Therefore phosphorylation of J a k l . JaE. Stat l and Stat3 is Iikely a

consequence of CCRS activation. The downstream targets for Stat 1 and Stat3 have yet

to be determined. It is Iikely that Stat activation results in transcriptional activation of

cellular genes that are needed for late vin1 gene expression. Additionally. Stats may be

required for the trans-activation of myxoma virus genes. However, ISRE (interferon

stimulated response element). SIE (Stat inducible element) and GAS (gamma activated

sequence) elements have not been identified in the myxoma virus genome.

As in other cytokine systems, cytokine-receptor interactions result in the

engagement of multiple signaling cascades. Myxoma virus induced phosphorylation of

the IRS proteins suggests that PI-3' kinase may be activated by virus. PI-3' kinase is a

potent mediator of ce11 survival and its activation by IRS 1 and IRS3 would provide an

immediate signal for the sumival of cells infected with myxoma virus. Furthemore, the

activation of other. as yet unidentified SH2 containhg proteins, such as GrbZ, may

implicate orher transcription factors in viral pathogenesis.

IV.2 Future Directions

The phosphorylation-activation of Jaks. Stats. and IRS proteins implicates

myxoma vinis in transcriptional activation of gene expression. To fûrther understand the

requirement for myxoma virus induced activation of CCRS it will become important to

identify the downstrearn targets of the different transcription factors. Using a strategy

that incorpontes suppressive subtnctive hybridization for differential gene expression in

uninfected verses infected cells. specific myxoma virus inducible genes will be identified.

Additionally. further scrutiny of the myxoma virus genome for Stat responsive gene

elements may provide evidence for Stat activation of myxoma virus genes.

An outstanding question is the role of CD4 in mycoma virus infection. Our

approach \vil1 be to dissect the different signaling events in NIH 3T3 ce11 variants.

Specifically. ive will examine susceptibility to virus infection in the parental NIH 3T3

cells. as 11-el1 as transfectants expressing human CD4 and CCR5 alone. Virus infection

if - i l1 be considered in the context of signaling events. In continuing studies we wili

establiçh ~vhether the tyrosine phosphorylation of mediators such as Jaks and p56Lck is

associated ii-ith activation of their kinase activity.

Cleariy. a greater understanding of virus-host interactions at the cellular receptor

and signal transduction levels \vil1 have wide spread implications for the development of

antiviral thenpeutics.

Chapter V

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