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Neuropharmacology 48 (2005) 1–13

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Mini-review

Virus-encoded chemokine receptors – putative novelantiviral drug targets

Mette M. Rosenkilde*

Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen,

Buildn. 18.6, Blegdamsvej 3, 2200-Copenhagen N, Denmark

Received 8 May 2004; received in revised form 19 July 2004; accepted 16 September 2004

Abstract

Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous

proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through controlof leukocyte trafficking, and have as such a paramount role in the antiviral immune responses. It is therefore not surprising thatviruses have found ways to exploit and subvert the chemokine system by means of molecular mimicry. By ancient acts of molecular

piracy and by induction and suppression of endogenous genes, viruses have utilized chemokines and their receptors to serve a varietyof roles in viral life-cycle. This review focuses on the pharmacology of virus-encoded chemokine receptors, yet also the family ofvirus-encoded chemokines and chemokine-binding proteins will be touched upon. Key properties of the virus-encoded receptors,

compared to their closest endogenous homologs, are interactions with a wider range of chemokines, which can act as agonists,antagonists and inverse agonists, and the exploitation of many signal transduction pathways. High constitutive activity is anotherkey property of some – but not all – of these receptors. The chemokine receptors belong to the superfamily of G-protein coupled7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many

chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets – ie. as novel antiviral strategies – willbe highlighted here together with the potentials of the virus-encoded chemokines and chemokine-binding proteins as novelanti-inflammatory biopharmaceutical strategies.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Virus-encoded chemokine; Chemokine receptors; Non-peptide antagonists for chemokine receptors

1. Introduction

Chemokines constitute a family of leukocyte chemo-attractants that exert their action through interactionwith 7-transmembrane (7TM) G-protein coupled recep-tors belonging to Family A (Rhodopsin-like) of thesuperfamily of 7TM receptors. Besides crucial roles inimmune system development, chemokines orchestrateleukocyte migration during homeostasis and inflamma-tion and control activation and differentiation of

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E-mail address: [email protected]

0028-3908/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2004.09.017

lymphoid cells, thus pivotal in the host immune responseagainst pathogens. In addition, chemokines regulateevents outside the immune system such as angiogenesisand organogenesis and different aspects of carcinogen-esis (Rossi and Zlotnik, 2000). The crucial importanceof the chemokine system in the defense against invadingpathogens is supported by the fact that several virusesinduce or encode for chemokine ligands, chemokinereceptors or chemokine-binding proteins that – indifferent ways – manipulate the immune system throughchemokine mimicry.

In addition to the subversion and exploitation ofthe immune system, the virus-encoded mimics within thechemokine system also contribute to the control of

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2 M.M. Rosenkilde / Neuropharmacology 48 (2005) 1–13

the local environments through influencing angiogenesisand cellular growth and differentiation. To date, morethan 50 different virus-encoded chemokines, chemokine-binding proteins or chemokine receptors have beenidentified in herpes-, pox and retrovirus presumablyobtained by the virus through an ancient act ofmolecular piracy (Wells and Schwartz, 1997) (Fig. 1,Table 1). The high conservation through severaldifferent virus strains is a strong evidence for thebiological importance of the chemokine mimics forvirus life-cycle. The sequence identity to their closestmammalian counterparts is quite low, giving testamentto an intense mutation-rate with ‘‘survival of the fittest’’as the dramatic theme. An understanding of themodifications in molecular properties of the chemokinesand their receptors – introduced by the virus – is veryimportant in the development of therapeutic strategiesagainst virus infection. However, going broader, thelessons learned from studies of virus-encoded proteinsprovide us with useful knowledge about structural andfunctional properties of proteins and about immunologyin general. From a drug-development point of view,viruses – and more broadly – microbial pathogens assuch, can be envisioned as pioneers. They have managedto identify the most important targets for own survivaland importantly, to design molecules that act on thesetargets. Current review is aimed at a detailed descriptionof the molecular pharmacology, ie. ligand-binding and

Fig. 1. Three basal mechanisms of viral chemokine exploitation in

terms of molecular mimicry. Left: the virus-encoded chemkines that

act either as agonists (vMIP-1 and -3, U83, vCXCL1, MCK1/2) or as

antagonists (MC148, vMIP-2) by binding to host-encoded chemokine

receptors. Middle: the virus-encoded chemokine receptors – some of

which are modulated by endogenous ligands acting either as agonists

or as antagonists. The majority of the virus-encoded chemokine

receptors are constitutively active – in addition to the ligand

modulation – and accordingly also inverse agonists are found among

the ligands for these (constitutively active) virus-encoded chemokine

receptors. Right: the virus-encoded chemokine-binding proteins

(scavengers) resulting in a broad-spectrum chemokine antagonism.

The poxvirus-encoded vCKBP2 family binds the N-terminal and

receptor-binding part of host chemokines whereas the Myxoma virus-

encoded vCKBP1 binds to heparin-like domains near the C-terminus

thus preventing the chemokine–GAG interactions. M3 interferes at

two distinct levels since it inhibits both the chemokine–receptor

interaction and the chemokine–GAG interaction through binding to

the N-terminus of the chemokines (see Table 1 for further details).

signaling properties of the virus-encoded chemokinereceptors. Initially, the endogenous chemokine systemwill be presented followed by a detailed description ofthe virus-encoded mimics: the chemokine receptors, thechemokines and the chemokine-binding proteins. Finally,the putative use of virus-encoded chemokine receptorsas novel innovative antiviral drug targets and the use ofthe virus-encoded chemokines and chemokine-bindingproteins as novel anti-inflammatory biopharmaceuticalswill be discussed.

2. Mammalian chemokine system

Chemokines comprise a large family of at least 50small polypeptides (8–12 kDa MW) that are divided intofour subfamilies – two large and two rather smallfamilies – dependent on the presence or absence ofresidue(s) between the first two of four conservedcysteines. The two large families consist of the CXC-and the CC-chemokines, whereas the CX3C- and theXC-chemokines represent the two small families. CXC-chemokines (CXCLs) (CXCL1 through 16) are furtherdivided based on the presence of an ELR-motif prior tothe CXC-motif with large functional implications. ELRCXCLs (Glu-Leu-Arg sequence prior to the firstcysteine) are induced under acute and chronic inflam-mation and mainly attract neutrophils, whereasnon-ELR CXCLs display a more constitutive expressionand exert their action on lymphocytes and cells outsidethe haematopoetic compartment. Angiogenesis is undercontrol by CXCLs since ELR CXCLs are angiogenic,while non-ELR CXCLs are angiostatic or angiomodu-latory (Strieter et al., 1995). The control of angiogenesisrepresents one way of influencing carcinogenesis; otherways involve control of leukocyte traffic in and outsidethe tumor and direct effects by chemokines as cancer cellgrowth factors. CC-chemokines (CCL1 through 28)influence most of the cells within the lymfoid andmyeloid compartments under inflammation as well asunder homeostasis. CX3CL1/Fraktalkine1 is the onlymember of the CX3C-family (three residues separate thefirst two cysteines), whereas XCL1/Lymphotactin rep-resents the second small family (this chemokine lacksone of the first two cysteines). A special refinement ispresented in CX3CL1/Fractalkine and in CXCL16 sincethe chemokine domain – in these two molecules – isanchored to a transmembrane a-helic in the C-terminusby a large mucin-stalk comprising 40–75% of themolecule. This enables the chemokine to remainattached to the membrane or shed from the cell by

1 In this review chemokine names will be given according to the

‘‘International Union of Pharmacology Nomenclature for Chemokines

and Chemokine Receptors’’ (Murphy et al., 2000) followed by their

‘‘old’’ names.

Table 1

Overview of virus-encoded chemokine ligands, chemokine scavengers and chemokine receptors

function References

emination Parcells, J. Virol. (2001) 75: 5159

lope Fraile-Ramos et al., 2002

usion

Kledal, FEBS Lett. (1998)

441: 209; Casarosa, J. Biol.

Chem. (2001) 276: 11331

Streblow et al., 1999

Margulies et al., 1996

Bankier, DNA Seq. (1991) 2: 1

Penfold, Proc.

Natl Acad. Sci. U.S.A.

(1999) 96: 9839

Penfold, Proc. Natl Acad.

Sci. U.S.A. (1999) 96: 9839

glands

Davis-Poynter et al., 1997

tion Oliveira and Shenk, 2001

ral Saederup, Proc.

Natl Acad. Sci. U.S.A. (1999) 96:

10881; Saederup, J. Virol.

(2001) 75: 9966

glands

Beisser et al., 1998; Gruijthuijsen,

J. Virol. (2002) 76: 1328

tion Beisser et al., 1999

Gompels, J. Virol. (1995) 209:

29; Isegawa et al., 1998

Milne et al., 2000

Zou, J. Virol. (1999) 73: 5926

is Yasukawa, J. Immunol. (1999)

162: 5417

y Forster, Cell. (1999) 99: 23

Nicholas, J. Virol. (1996) 70: 5975

Nicholas, J. Virol. (1996) 70: 5975

is Yasukawa, J. Immunol. (1999)

162: 5417

(continued on next page) 3M.M

.Rosen

kild

e/Neuropharm

acology48(2005)1–13

Family of virus Virus Gene name Molecule class Pharmacology Known or presumed

a-Herpesviruses Marek’s disease

virus

vIL-8 CXC-chemokine Unknown Virulence factor, diss

b-Herpesviruses Human CMV US27 Receptor (CCR-like) Unknown Located on viral enve

US28 CC and CX3C

receptor

Constitutively

active CC- and

CX3C-receptor

Chemokine scavenger

function, virus

attachment and cell f

Smooth muscle

cell migration

UL33 Receptor (CCR-like) Constitutively active,

no ligands identified

Expressed in viral

particle and on

virus-infected cells

UL78 Putative receptor Unknown Unknown

vCXCL1 CXC-chemokine Agonist for CXCR2 Unknown

vCXCL2 CXC-chemokine Unknown Unknown

Mouse CMV M33 Receptor (CCR-like) Constitutively active,


Virulence factor.

Important for viral

replication in salivary

M78 Putative receptor Unknown Virulence and replica

MCK1/2 CC-chemokine Agonist CaCC mobilization Pro-inflammatory, vi

dissemination

Rat CMV R33 Receptor (CCR-like) Constitutively active,


Virulence factor.

Important for viral

replication in salivary

R78 Putative receptor Unknown Virulence and replica

HHV6 U12 CCR Binds CC-chemokines, not

constitutively active

Unknown

U51 CCR Binds CC-chemokines, not


Downregulation of

CCL5/RANTES

expression

U83 CC-chemokine Agonist, CaCC mobilization Monocyte cell

line chemotaxis

CXCR4** CXC-receptor (host) Binds CXCL12/SDF Lymphoid homeostas

CCR7* CC-receptor (host) Binds CCL19 and -21 Homing to Secondar

Lymphoid Organs

HHV7 U12 Receptor (CCR-like) Unknown Unknown

U51 Receptor (CCR-like) Unknown Unknown

CXCR4** CXC-receptor (host) Binds CXCL12/SDF Lymphoid homeostas

Table 1 (continued)

esumed function References

econdary

rgans

Forster, Cell. (1999) 99: 23

Birkenbach, J. Virol. (1993)

67: 2209

ing,

omeostasis

Cook, Immunity. (2000) 12: 495;

Nakayama, J. Virol. (2002)

76: 3072

Homey, Nat. Med. (2002) 8: 157;

Nakayama, J. Virol. (2002)

76: 3072

ion,

lved in Kaposi’s

ation,

c,

d tumorigenic

Bais et al., 1998; Rosenkilde

et al., 1999; Yang

et al., 2000

Dairaghi, J. Biol. Chem.

(1999) 274: 21569

onocyte

elective

tractant

Kledal et al., 1997; Sozzani,

Blood. (1998) 92: 4039; Weber,

Eur. J. Immunol. (2001) 31: 2458;

Luttichau,

Eur. J. Immunol. (2001) 31: 1217

otaxis Stine, Blood. (2000) 95: 1151

Ahuja and Murphy, 1993;

Rosenkilde, J. Biol.

Chem. (in press)

Albrecht, 2000

ion,

rom

Rochford, J. Virol. (2001)

75: 4955;

Lee et al., 2003; Moorman

et al., 2003; Verzijl

et al., 2004

Interfere

okine–GAG

d the

eceptor

van Berkel

et al., 1999; Parry

et al., 2000; Webb

et al., 2004

Camarda et al., 1999

Telford et al., 1995

Telford et al., 1995

4M.M

.Rosen

kild

e/Neuropharm

acology48(2005)1–13

Family of virus Virus Gene name Molecule class Pharmacology Known or pr

g1-Herpesviruses Epstein–Barr

virus

CCR7* CC-receptor (host) Binds CCL19 and -21 Homing to S

Lymphoid O

EBI-2* Orphan receptor (host) Unknown Unknown

CCR6* CC-receptor (host) Binds CCL20 Mucosal hom

lymphocye h

CCR10* CC-receptor (host) Binds CCL26-28 Skin homing

g2-Herpesviruses HHV8 ORF74-HHV8 CXC-receptor Binding of and regulation

by CXC-chemokines,


Lytic express

possible invo

sarcoma form

anti-apoptoti

angiogenic an

vCCL1 (vMIP-1) CC-chemokine Agonist for CCR8 Unknown

vCCL2 (vMIP-2) CC-chemokine Broad-spectrum CC-, CXC-

and CX3C-antagonist. Agonist

for CCR3 and CCR4

Antagonize m

chemotaxis, S

Th2 chemoat

vCCL3 (vMIP-3) CC-chemokine Agonist for CCR4 Th2 cell chem

Herpesvirus

saimiri (HVS)

ECRF3 CXC-receptor Broad-spectrum CXC-chemokine

binding,


Unknown

Atheles

herpesvirus

(AtHV)

ORF74-AtHV Receptor

(CXC-like)

Unknown Unknown

Mouse gHV68

(MHV68)

ORF74-MHV68 CXC-receptor Broad-spectrum CXC-chemokine

binding, not constitutively active

Lytic express

reactivation f

latency

M3 (vCKBP3) Scavenger Broad-spectrum XC-, CC,

CXC and CX3C-chemokine

scavenger

Role in viral

pathogenesis.

with the chem

interaction an

chemokine–r

interaction

Equine HV2

(EHV2)

E1 CC-receptor Binds CCL11/Eotaxin, not


Unknown

E6 Receptor Unknown Unknown

ORF74-EHV2 Receptor (CXC-like) Unknown Unknown

5M.M. Rosenkilde / Neuropharmacology 48 (2005) 1–13
Poxviruses
Mollusc.

Contagiosum

MC148

CC-chem

okine

AntagonistforCCR8

Unknown

Luttichauet

al.,2000

Lepori-and

orthopoxviruses

vCKBP1

Scavenger

Broad-spectrum

CC-chem

okine

scavengers

Anti-inflammatory,viral

immuneevasion,binds

tothereceptor-interactionsite

Smith,J.

Virol.(2000)74:8460

Myxoma

vCKBP2

Scavenger

Broad-spectrum

C-,CC,

CXC-chem

okine

andIF

Ngscavenger

Anti-inflammatory,bind

totheGAG-interactionsite

onthechem

okines

Mossmanet

al.,1996;Lalani

etal.,1997

Swinepoxvirus

K2R

Receptor(C

CR-like)

Unknown

Unknown

Gao,Virology.(1995)209:207

Capripoxvirus

Q2/3L

Receptor(C

CR-like)

Unknown

Unknown

Gao,Virology.(1995)209:207

Abbreviations:*hostencoded

geneinduceduponvirusinfection,**hostencoded

genedownregulateduponvirusinfection.Thenew

lydescribed

7TM

receptors

encoded

byg1-herpesviruses(K

ledal,

2003)–are

notincluded

inthistable.

Thevirus-inducedor-downmodulatedchem

okinereceptors

are

alsoincluded

inthistable.System

aticdescription(from

left)ofvirusfamily,virus,nameofgene,moleculeclass

(ligand,scavenger

or

receptor),pharm

acologyofvirus-encoded

(orvirus-regulated)protein,knownorpresumed

functionandfinallyselected

references(see

textforfurther

references).

enzymatic cleavage (Bazan et al., 1997). The receptorsare divided into CXCR1-6, CCR1-11, CX3CR1 andXCR1. Chemokine binding varies from the highselectivity to profound promiscuity (Murphy et al.,2000). The main signaling of mammalian chemokinereceptors is via a pertussis toxin (PTx) sensitivechemotaxis and calcium release pointing at Gi proteinsas the main playmakers in this signaling. Furtheranalysis has revealed that the bg-subunits released fromactivated Gai mediate cell migration (Neptune andBourne, 1997; Arai et al., 1997; Neptune et al., 1999).Surprisingly, the cell surface of the migratory cellspresents a uniform distribution of chemokine receptors,and the directional migration depends on asymmetricdistribution on signaling molecules downstream to theG-proteins, like protein kinase B (PKB) (Meili et al.,1999). The downstream events include activation of themitogen-activated protein (MAP)-kinases being impor-tant for proliferation and adhesion molecule expressionand for chemotaxis in addition to Phosphatidyl-Inositol3-Kinase g (PI3Kg) (Bajetto et al., 2001). The signalingalso includes activation of tyrosine kinases e.g. RelatedAdhesion Focal Tyrosine Kinase (RAFTK) linkingreceptor signaling to activation of small GTPases(Ganju et al., 1998). Ultimately, transcription factorsare induced with an even more complex and poorlyunderstood regulation. Examples are Nuclear Factor-kB (NF-kB) and Janus kinase/signal transducer andactivator of transcription (Jak/STAT) – the latter ofpresumed importance for chemokine receptor-oligomer-ization (Rodriguez-Frade et al., 2001). For a recentreview on the signaling of mammalian chemokinereceptors, see Thelen (2001).

3. Virus-encoded mimics – the virus-encoded

chemokine receptors

The virus-encoded (7TM) chemokine receptors havebeen identified in the genomes of large DNA viruses, inparticular b-herpesviruses – CMV, HHV6 and -7 – andg2-herpesviruses – including Human Herpesvirus 8(HHV8), Murine g-herpvesvirus 68 (MHV68), EquineHerpesvirus-2 (EHV2), Herpesvirus Saimiri (HVS) andAteles Herpesvirus (AtHV) (Fig. 2). In addition, tworeceptors – still uncharacterized – have been identified inthe genomes of Poxviruses (Cao et al., 1995). Many ofthe virus-encoded receptors bind chemokines and signalupon chemokine binding – ie. are chemokine receptors –others remain to be ‘‘deorphanized’’. A selection ofcurrently known receptors is listed in Table 1. A generalpicture for the virus-encoded chemokine receptors isa broadening in ligand recognition as well as signalingpathway exploitation. Thus, the viruses have optimizedtheir receptors in the direction of promiscuity ratherthan selectivity with respect to ligand-interaction and


signaling initiation pattern. Importantly, many virus-encoded receptors are constitutively active throughmany pathways – a phenomenon that indeed distinguishthem from their endogenous homologous. The bindingand signaling characteristics reflect the differentbiological properties for the virus-encoded receptors asbeing important for virus life-cycle (cell-entry andsurvival, replication, dissemination, cell-survival and toa minor extent cell migration) (Rosenkilde et al., 2001;Holst et al., 2003).

4. Binding and signaling of g2-herpesvirus-encodedchemokine receptors

4.1. g2-Herpesvirus-encoded receptors

The open reading frame 74 (ORF74) of many ofg2-herpesviruses – HHV8, MHV68, HVS, AtHV andEHV2 – contains a CXC-chemokine receptor. Untilnow, the first three receptors have been characterized asbeing functionally related to mammalian CXCR2. Allthree ORF74 receptors bind a variety of CXC-chemo-kines and signal broad through several signal trans-duction pathways; however, the constitutive activityvaries dramatically among the different ORF74 recep-tors (Table 1).

ORF74 from HHV8 (or Kaposi’s Sarcoma associatedHerpes Virus (KSHV)) was discovered in 1994 from

U12 HHV7

U12U12

M33

R33

ORF74

EHV2

E1EHV2

US27US28

ORF74Mhv68

ORF74

HHV8

ORF74

HVS

ORF74AtHV

EHV2E6

UL78

UL33

R78

CMV

CMV

CMV

M78

U51 HHV7

U51U51 HHV6

a / b

HHV6a / b

Fig. 2. Dendrogram of human and animal herpesvirus-encoded

chemokine receptors. The receptors depicted are listed in Table 1.

The phylogenetic tree was constructed using Clustal W 1.5 alignments

(GCG, Genetics Computer Group, Wisconsin Package) of the full

amino acid sequence followed by an analysis by the Distance program

(GCG). The dendrogram was generously provided by Francois

Talabot, Serono Pharmaceuticals, Switzerland. Constitutively, active

receptors are depicted in red.

Kaposi’s Sarcoma lesions (Chang et al., 1994) and hasbeen associated with three human disorders, the primaryeffusion lymphoma, Kaposi’s sarcoma and the lympho-proliferative multicentric Castleman’s disease. In 1996,a chemokine receptor homolog was identified in ORF74in the HHV8 genome (Russo et al., 1996). This receptor,ORF74-HHV8, was quickly shown to possess trans-forming as well as oncogenic potential since highlyvascularized tumors developed when ORF74-HHV8transfected cells were transplanted into nude mice (Baiset al., 1998). The oncogenic potential was shown to bedependent on the secretion of an angiogenesis activator,vascular endothelial growth factor (VEGF) as down-stream event in the signaling cascade for ORF74-HHV8(Arvanitakis et al., 1997; Bais et al., 1998). Throughexpression in transgenic mice ORF74-HHV8 wasproven to be an aetiologic agent of Kaposi’s Sarcomasince these mice developed lesions with the samehistology and macroscopic appearance as Kaposi’ssarcomas (Yang et al., 2000; Montaner et al., 2003). Itis most likely that the early lytic-phase expressedORF74-HHV8 contributes in a paracrine manner inthe development of Kaposi’s sarcoma since it is onlyexpressed in 1–3% of cells expressing lytic genes(Kirshner et al., 1999; Sun et al., 1999). Studies of theconstitutive and regulated signaling of ORF74-HHV8have revealed an impressive plurality of pathwaysdownstream for a variety of different G-proteinsincluding Gq/16 (PLC-b activation (Arvanitakis et al.,1997; Rosenkilde et al., 1999)), Gi (inhibition ofadenylate cyclase (Couty et al., 2001)), and the G12/13(activation of the RhoA class of small GTPases(Shepard et al., 2001)). The downstream pathwaysinclude several members of the small GTPases, theMAP-kinase superfamily, RAFTK and a variety oftranscription factors, including NF-kB, Serum ResponseElement (SRE), AP-1, Nuclear Factor of ActivatedT-cells (NFAT), cAMP responsive element Bindingprotein (CREB) and Hypoxia Induced Factor (HIFa)(Sodhi et al., 2000; Montaner et al., 2001; Schwarz andMurphy, 2001; Smit et al., 2002; Couty et al., 2001;Shepard et al., 2001; Munshi et al., 1999; Cannon et al.,2003; Cannon and Cesarman, 2004). A diagram of thesedifferent constitutively active pathways is shown inFig. 3. The mechanisms upstream for gene-transcriptionare quite diverse, exemplified by the complex regulationof NF-kB activity through at least four pathways: (1)Gi, (2) G12/13/RhoGEF/RhoA pathway, (3) Gqand (4) bg-subunits activating e.g. PI3Kg (Shepardet al., 2001; Couty et al., 2001). ORF74-HHV8 inaddition possesses cell-survival properties throughactivation of Protein Kinase B (PKB), which in turninhibits downstream pro-apoptotic molecules (Monta-ner et al., 2001).

The constitutively active pathways are subjected toligand modulation by interaction with the majority of


human CXC-chemokines with nanomolar affinities aswell as by vMIP-2 encoded by HHV8 itself (Arvanitakiset al., 1997; Bais et al., 1998; Rosenkilde et al., 1999).The CXC-chemokines are divided into three groupsbased on their pharmacology: the ELR CXCLs – whichare pro-inflammatory and angiogenic in vivo – actingeither as agonists, CXCL1-3/GRO-a,b,g or as neutralligands, and CXCL5/ENA78, CXCL7/NAP-2 andCXCL8/IL8. In contrast, the non-ELR CXCLsCXCL10/IP10 and CXCL12/SDF-1a – characterizedby being angiostatic or angiomodulatory and tofunction during homeostasis – display inverse agonisticproperties (Fig. 4). Functionally, vMIP-2 represents aninverse agonist albeit with lower potency than CXCL10/IP10 and CXCL12/SDF-1a. Competition bindingexperiments have uncovered the existence of differentactive and inactive conformations that are not readilyinterchangeable. Thus, the neutral ligands do notcompete with either agonists or inverse agonists,whereas the agonists and inverse agonists competeinterchangeably (Rosenkilde and Schwartz, 2000).Mutants with reduced constitutive signaling displaya binding profile favoring inverse agonists over agonists,whereas mutants with increased constitutive signalingdisplay increased agonist binding and reduced inverseagonist binding (Rosenkilde et al., 2000). Finally,

G12/13Gq GqGi/o Gi/o

RhoA-GEFDAG

PKCPKA PKB/Akt

IP3

Ca++Ca++

RAFTKPLC-β PI3Kγ

Rho

JNK/SAPKErk1/2P38

VEGF Inactivation ofpro-apoptoticmolecules

AngiogenesisTransformation

Oncogenesis

Enhancedcell-survival

NFAT AP-1 NF- κB SREHIF-1 CREB

αα

AC

Calcineurin

α

Fig. 3. Signaling pathways activated constitutively by ORF74-HHV8.

Several levels of effector molecules have been implicated in the

signaling cascade elicited by ORF74-HHV8 from the initial G-protein

activation in the membrane (yellow) over a variety of intracellular

signaling molecules including protein kinases (PK‘‘X’’) (red), MAP-

kinases (purple) ending up with the activation of at least six different

transcription factors (orange) in the nucleus. These pathways are

regulated by a fine-tuned and delicate system of either stimulatory or

inhibitory mediators. The diagram is grossly simplified and several

suggested cross-regulations between depicted enzymes have been

excluded. The abbreviations are all mentioned in the text.

transgenic expression of different receptor phenotypesindicates that the constitutive as well as the regulatedactivities are important events for the tumor induction(Holst et al., 2001). Thus, strong evidence suggests thatORF74-HHV8 expression by virus, in a certain chemo-kine environment, provides the molecular basis forKaposi’s Sarcoma seen in humans.

The ORF74 receptor from HVS (ECRF3) was thefirst ORF74 receptor to be characterized (Ahuja andMurphy, 1993). HVS is a T-cell lymphotropic virusgiving asymptomatic infection in the natural host andfatal lymphoproliferative disease in New Worldprimates. ECRF3 also exhibits a broad-spectrumagonistic binding profile, however, in contrast toORF74-HHV8, ECRF3 only binds ELR containingCXCLs (all shared agonists by mammalian CXCR2)e.g. CXCL1-3/GROa,b,g, CXCL6/GCP-2, CXCL7/NAP-2 and CXCL8/IL8 (Ahuja and Murphy, 1993).Several pathways are activated in a ligand-dependentmanner, e.g. calcium release (Ahuja and Murphy, 1993),Gi, G12/13 and Gq as well as an array of transcriptionfactors (CREB, NFAT, NF-kB and SRE). Of thesemany pathways, only two (Gi and SRE, the latter beingdependent upon Gi as well as G12/13 activity) areactivated in a constitutive manner (Rosenkilde et al.,2004).

The ORF74 receptor from MHV68 binds a broadspectrum of ELR CXCLs as agonists (e.g. CXCL6/

ORF74HHV8

angiogenic

chemokinesGROα

GROγ

GROβ

IP10

SDF

IL-8

NAP-2

angiostatic /

modulatory

chemokines

acute

inflammatory

chemokines

-11 -10 -9 -8 -70

50

150

200

log conc.CXC-chemokine (M)

PI-tu

rnov

er, %

of b

asal

cons

titut

ive

activ

ity

100

Fig. 4. Ligand-regulation of ORF74-HHV8. The ORF74-HHV8 is

regulated by a broad spectrum of CXC-chemokines besides being

constitutively active through many different pathways (as depicted in

Fig. 3). The ligand-regulation shown here was measured as phospha-

tidyl-inositol (PI) turnover – indicating Gq activation – in transiently

transfected COS-7 cells. The agonistic ELR containing chemokines

CXCL1-3/GROa,b and -g are shown in green symbols. The neutral

ligands (also ELR containing) CXCL7 and -8/NAP-2 and IL8 are

shown in grey symbols and the inverse agonistic non-ELR CXC-

chemokines CXCL10/IP10 and CXCL12/SDF are shown in red

symbols. (Redrawn from Rosenkilde et al., 1999; Rosenkilde and

Schwartz, 2000). The same picture of ligand modulation with agonists

and inverse agonists has been shown for the Gi activity (Couty et al.,

2001) as well as for the transcription factor activation of ORF74-

HHV8 (Smit et al., 2003; McLean et al., 2004a, b).


GCP-22) and non-ELR CXCLs as antagonists. It alsoactivates a broad spectrum of signaling pathways,however, solely in a ligand-dependent manner (Verzijlet al., 2004). Thus, no constitutive activity has ever beenshown for this receptor, although the receptor has beenshown to induce transformation of NIH3T3 fibroblasts(Wakeling et al., 2001). ORF74 gene-deletion experi-ments in MHV68 have suggested that this receptorfunctions in the early reprogramming of a virus infectedcell and upon reactivation of the viruses from latency(Lee et al., 2003; Moorman et al., 2003).

The ORF74-EHV2 receptor displays high constitutivesignaling through Gi, but not Gq2 (similar to ECRF3),whereas the ORF74 encoded by AtHV remains to becharacterized (Albrecht, 2000; Telford et al., 1995).

The E1 CC-chemokine receptor encoded by EHV2signals through calcium release and chemotaxis uponselective binding of CCL11/Eotaxin (Camarda et al.,1999), whereas the E6 receptor from EHV2 remains tobe characterized. Structural studies of E6 reveal that this7TM receptor is closely related to a newly discoveredfamily of viral 7TM receptors encoded by theg1-herpesviruses (personal communication by T.N.Kledal and Kledal, 2003). No structural evidencesuggests that this family should be a chemokine bindingfamily (accordingly the g1-herpesvirus-encoded recep-tors are excluded from Table 1), yet this needs to beproven by deorphanization studies of these receptors.

5. Binding and signaling of b-herpesvirus-encodedchemokine receptors

5.1. CMV-encoded receptors

Human CMV encodes four 7TM receptors (US27,US28, UL33 and UL78) of which the first three sharestructural homology to chemokine receptors. Murineand rat CMV encode the UL33 and the UL78 receptor,whereas they lack the US27 and US28 gene.

US28 signals constitutive through many pathwaysincluding Gq and the transcription factors NF-kB,CREB and NFAT in a constitutive manner (Casarosaet al., 2001; McLean et al., 2004a, b). The regulatedactivity of US28 is somewhat more complex andincludes additional pathways. Initially, US28 wasdiscovered as a receptor that induced calcium releasein response to CC-chemokines (Neote et al., 1993; Gaoand Murphy, 1994). Competition binding studiesuncovered a broad-spectrum high affinity CC- andCX3C-chemokine binding. The CC-chemokines havebeen shown to regulate ERK1/2 MAP-kinase activityand to induce calcium release (Billstrom et al., 1999).

2 Rosenkilde MM, unpublished observation.

The constitutive signaling is reduced, although notabrogated by CX3CL1/Fraktalkine (Casarosa et al.,2001) and the partial inhibition (by CX3CL1) has beenshown to depend upon the predominantly endosomallocalization of US28 (Waldhoer et al., 2003). Thisintracellular expression of US28 is in contrast to thesurface expression of ORF74 from HHV8 (Schwarz andMurphy, 2001; McLean et al., 2004a, b). A chemokinescavenging function has been suggested for US28(Bodaghi et al., 1998) and the constitutive activity hasbeen suggested to influence early cellular programming(Pleskoff et al., 1998). Finally, US28 has been shown tomediate smooth-muscle cell chemokinesis and chemo-taxi towards CC-chemokine gradients. Considering theexpression of a variety of CCLs (including US28ligands) in atherosclerotic plaques, it is tempting toassign US28 a role in the development of CMVassociated vascular diseases (Streblow et al., 1999).

US27 is located immediately proximal from US28and – despite a close structural relationship to US28 –this putative receptor has never been reported to bindany chemokine or signal constitutively (Fraile-Ramoset al., 2002; Waldhoer et al., 2002).

5.2. UL33/M33/R33 family

As for US28 and some ORF74 receptors, constitutiveactivity has been described for this family – yet with nochemokine modulations. UL33, M33 and R33 signalsconstitutive through Gq and several transcriptionfactors, whereas the activation of UL33 in additionseems to involve Gs (Waldhoer et al., 2002; Casarosaet al., 2003a). Concerning the transcription factors, bothNF-kB and CREB are activated in a Gi dependent way(Waldhoer et al., 2002; Casarosa et al., 2003a). UL33 isincorporated in viral particles and expressed on virusinfected cells (Margulies et al., 1996). In vivo studieshave indicated importance for M33 and R33 in virusdissemination to – or replication in – salivary glands andfor the general virulence (Beisser et al., 1998; Davis-Poynter et al., 1997).

5.3. UL78/M78/R78 family

No ligand-binding or signaling events have ever beendescribed for this family. Yet, gene-deletion experimentsin recombinant viruses for M78 and R78 have provenfunctional importance in viral replication and virulencein general since animals infected with the deleted mutantviruses had decreased mortality (Oliveira and Shenk,2001; Beisser et al., 1999).

5.4. HHV6- and HHV7-encoded receptors

Two different receptors with structural homology toendogenous CC-chemokine receptors, the U12 and the


U51 receptors, are found in the genomes ofHHV6 and -7.U12 from HHV6 is a functional chemokine receptor,which induces calcium release in response to variousCCLs (Isegawa et al., 1998). No constitutive activity haseven been shown for this receptor. In contrast, U51 fromHHV6 has been shown to bind various CCLs; however,no ligand-dependent or ligand-independent signalinghas ever been described (Milne et al., 2000). HHV6 hasbeen suggested as possible pathogen in multiple sclerosis(MS) since HHV6 replication has been detected in MSlesions and in cerebrospinal fluid of patients with MS;however, HHV6 could also ‘‘just’’ be an innocentbystander since it normally establishes latent infectionsin CNS.


chemokines

The virus-encoded chemokines are found within theCC- and the CXC-chemokines, and show in generala large diversity in their pharmacological properties –from agonistic to antagonistic (and inverse agonistic)properties (Table 1). The complexity grows with thestrong dichotomy in the ligand–receptor interactionwith profound selectivity for some ligands, e.g. MC148encoded by the poxvirus Molluscum contagiosum beingselective antagonist on CCR8 (Luttichau et al., 2000),and large promiscuity for others, e.g. vMIP-2 encodedby HHV8 being antagonist on a broad spectrum ofendogenous and virus-encoded CC and CXC-chemo-kine receptors (Rosenkilde et al., 1999; Geras-Raakaet al., 1998a,b). Thus, vMIP-2 antagonizes CCR1, -2, -4,-5 and CCR10 – and on the CXC-branch, vMIP-2antagonizes CXCR3 and -4 among the endogenouschemokine receptors (Kledal et al., 1997; Luttichauet al., 2000). This chemokine is not the only one encodedby HHV8, since in fact, vMIP-2 represents the antag-onistic sibling of a total of three CCLs (vMIP-1-3) ofwhich vMIP-1 and vMIP-3 act as agonists for CCR3, -4and -8 (Luttichau et al., 2000; Kledal et al., 1997;Boshoff et al., 1997). Due to the fact that chemokinereceptors are differentially expressed on Th1 and Th2cells (Sallusto et al., 1998), the outcome of the agonisticand antagonistic actions of vMIP-1-3 on this carefullyselected group of chemokine receptors (described above)is an immune-polarization in favour of Th2 cells overTh1 cells. The balance of Th1 and Th2 cells is essentialfor adaptive immune responses, and the antiviralimmune response against HHV8 is dominated by Th1cells. Thus, these three chemokines (vMIP-1-3) togetherprevent the host from mounting an efficient antiviralTh1 dominated immune response and can be consideredas elegant examples of biopharmaceuticals created bynature’s own bioengineering specialist – the large DNAvirus HHV8 (Lindow et al., 2003). Due to the

redundancy in the endogenous chemokine system, itwould – in general – be an advantage to block severalreceptors with one drug for efficient anti-inflammatoryresponses. The broad-spectrum biopharmaceuticals likevMIP-2 from HHV8 are attractive for this purpose, andvMIP-2 has accordingly been shown to inhibit theinflammatory reaction in, for instance, a rat model ofglomerulonephritis (Chen et al., 1998) and in spinal cordcontusions (Ghirnikar et al., 2000). In summary, themany different properties of HHV8 encoded chemokinescomplete the picture of complex pharmacology andbiology for the virus-encoded chemokines as such, andpoint towards important roles of the virus-encodedchemokines in the corruption of the immune systemduring the virus–host interaction (Wells and Schwartz,1997; Rosenkilde et al., 2001).


chemokine-binding proteins

The virus-encoded chemokine-binding proteins(vCKPBs) are encoded by members of the poxvirusesand in a single case, by a herpesvirus (MHV68). Theyfunction as chemokine scavengers, ie. neutralize chemo-kines, and have no homologs within the mammalianchemokine system. Accordingly, they represent a uniqueclass of anti-inflammatory chemokine modulators withthe antagonistic chemokine function being ‘‘invented’’by the virus. The vCKBPs are divided into differentclasses based on structural differences. vCKBP1 – todate only one member, M-T7 – is encoded by Myxomavirus. It binds with low affinity to a broad range of XC-,CC- and CXC-chemokines at the heparin-binding site,and thereby inhibits the interaction of these chemokineswith glycosaminoglycans (GAGs) – an event necessaryfor proper chemokine action (Mossman et al., 1996;Lalani et al., 1997). vCKBP2 consists of severalmembers encoded by different poxviruses. These pro-teins interact with high affinity with most, but not all,CC-chemokines at the receptor-binding site, and therebyselectively inhibit the action of these CC-chemokines(Alcami et al., 1998; Smith et al., 2000). Gene-deletionexperiments with members of vCKBP1 and -2 lead toincreased leukocyte infiltration into the sites infectedwith the knock-out strain compared to wildtype virus(Mossman et al., 1996; Graham et al., 1997). From thesegene-deletion studies it was observed that the mostsevere decrease in virulence was observed for thevCKBP2 knock-out strain – suggesting that the mostefficient way to inhibit chemokines is by means ofa perturbation of the GAG–chemokine interaction.vCKBP3 – to date only one member, M3 – is encodedby MHV68. M3 is unique since it interferes at twodistinct levels with both the receptor binding and theGAG binding site within all four classes of chemokines


(XC-, CC-, CX3C-, and CXC-chemokines) – thus todate the most broad-spectrum chemokine inhibitionobserved within the different vCKBPs (van Berkel et al.,1999; Parry et al., 2000). Structural studies have shownthat M3 interacts with the N-terminus of the chemo-kines (Webb et al., 2004) and studies with MHV68carrying inactive M3 have uncovered importance of M3for the establishment and maintenance of latency insecondary lympoid tissues (Bridgeman et al., 2001). Fora recent review of vCKBPs, see Alcami (2004).

8. Conclusion

Over the last two decades we have learned thatchemokine mimicry is common and important forherpes- and poxviruses. The virus-encoded chemokinesand the chemokine receptors have been obtained fromthe host and through ‘‘combinatorial chemistry’’ struc-turally and functionally varied by random mutagenesis.The anti-inflammatory virus-encoded chemokines (e.g.vCCL2 from HHV8) and chemokine-binding proteins(scavengers) block the host immune system and promotevirus evasion from the host immune response. As such,these virus-encoded chemokines and chemokine-bindingproteins serve as elegant examples of biopharmaceut-icals – developed and engineered by the large DNAviruses (herpes- and poxvirus) – that in the future couldserve as important lead compounds for novel principlesof anti-inflammatory treatment in humans. Otherviruses encode pro-inflammatory chemokines (e.g.vCXCL1 from HCMV) that function opposite andpromote virus growth and dissemination.

From a molecular pharmacology point of view, thechemokine receptors (and ligands) constitute a uniqueopportunity to study basic principles of ligand recogni-tion, activation mechanism of 7TM receptors (many ofthem being constitutively active as described in currentreview), internalization and recycling pathways as beingexamples of targeted evolution. The studies of virus-encoded chemokine receptors have uncovered that thesemolecules are important although not mandatory forvirus survival in vivo. Thorough studies of the receptor–ligand interaction and the signaling properties of thevirus-encoded chemokine receptors have revealed regu-lation by chemokine agonists as well as inverse agonists,thus strengthening the fact that these receptors indeedare subjects for modulation. 7TM receptors are ingeneral excellent drug targets3, and several non-peptidebased antagonists are currently under development forendogenous chemokine receptors (e.g. CXCR4 andCCR5 antagonists (De Clercq and Schols, 2001)). Manychemokine (peptide) antagonists have been identified forthe US28 and the ORF74-HHV8 receptors (Rosenkilde

3 http://www.7TM-Pharma.com and http://www.axovan.com.

et al., 1999; Arvanitakis et al., 1997; Geras-Raaka et al.,1998a,b; Casarosa et al., 2001) and in fact, also non-peptide antagonists have been identified for these tworeceptors – although ‘‘only’’ with low (micromolar)potency (Rosenkilde et al., 1999; Casarosa et al., 2003b).The development of high-potency antagonists/inverseagonists for the virus-encoded 7TM receptors will beessential for the elucidation of the roles of thesereceptors in virus life-cycle. Taken with the putativeroles of these receptors in virus pathogenesis and virusassociated pathology (e.g. HCMV as playmaker inatherosclerosis, HHV6 as playmaker (or innocentbystander?) in multiple sclerosis and HHV8 as beinginvolved in Kaposi’s sarcoma) these non-peptide antag-onists/inverse agonists may serve as putative leadcompounds for a novel class of antiviral therapy.

Acknowledgements

The author wishes to thank Thomas N. Kledal forcritical reading and discussion of the manuscript. Thisreview was supported from the Danish Cancer Society,the Danish Medical Research Council and the Novo-Nordisk Foundation.

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