Journal of Leukocyte Biology Volume 57, May 1995 731

Interruption of cytokine networks by poxviruses: lessons from

myxoma virusGrant McFadden, Kathryn Graham, Kimberly Ellison, Michele Barry, Joanne Macen,

Martha Schreiber, Karen Mossman, Piers Nash, Alshad Lalani, and Helen Everett

Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

Abstract: Myxoma virus is an infectious poxvirus patho-gen that induces a virulent systemic disease calledmyxomatosis in European rabbits. The disease is rapidlyand uniformly fatal to susceptible rabbits and is charac-terized by generalized dysfunction of cellular immunityand multiple interruptions of the host cytokine network.A number ofvirus genes are classified as virulence factorsbecause virus constructs bearing targeted gene disrup-tions induce attenuated disease symptoms. Many of thesegenes encode proteins that interact directly with effectorelements of the host immune system. Included amongthese immunosubversive viral proteins are secreted mim-ics of host ligands or regulators (virokines) and homo-logues of cellular cytokine receptors (viroceptors). Fiveexamples of these immune modulator proteins encodedby myxoma virus are reviewed: ( 1) myxoma growth factor,a member of the epidermal growth factor ligand superfa-mily; (2) SERF-i, a secreted serine proteinase inhibitor;(3) Ml 1L, a receptor-like surface protein; (4) T2, a tumornecrosis factor receptor homologue; and (5) T7, an inter-feron-yreceptor homologue. The origin ofviral strategiesdesigned to subvert immune regulation by host cytokinesis considered in the context of the biology of myxomavirus within immunocompetent hosts.J. Leukoc. Biol. 57:731-738; 1995.

Key Words: TNF . serpins . IFN-y . virokines . viroceptors

INTRODUCTION

Many viruses that infect vertebrate hosts achieve sus-tamed host-to-host transmission by using specific strate-gies that evade or subvert the consolidated activities ofthe antiviral immune and inflammatory responses [1-4].Some of these viral strategies can be revealed by analysisof the interaction between viruses and the many classesof effector cells that directly mediate natural and acquiredimmunity, such as B and T lymphocytes, natural killem

cells, monocytes/macrophages, and antigen-presentingcells [5-8]. For example, many viruses down-regulate cellsurface major histocompatibility complex antigens of in-fected cells as part of a concerted strategy to ciicumvent

major histocompatibility complex-restricted recognitionof viral antigens [9, 10]. Related strategies have also beenuncovered by the identification of viral genes whose pro-tein products are not required for virus replication intissue culture, but instead allow for virus propagation in

host tissues that are normally visible to the imnmiiune amid

inflammatory systems of the host. The larger DNA virusesare of particular interest because they encode more pro-teins than are necessamy for the assembly of progenyvirions [1, 4]. Poxviruses provide an excellent example of

this, because they are among the largest eukaryotic DNAviruses and have the unusual capacity to mcplicate autono-rnously within the cytoplasm of infected cells [11-13].Many poxvirus proteins have been defined as virulencefactors, because they confer the virus with increased ca-

pacity to propagate within immunocompetent hosts andthereby contribute to viral pathogenesis. The deletion ordisruption of virulence genes frequently results in the

attenuation of the pathogenic profile in �‘iso, as deter-mined by reduced levels of poxvirus replication and host-host transmission [14-16]. In this review we consider howone poxvirus pathogen of rabbits, myxoma virus, inter-acts with the host immune system and specifically thecytokine network.

Myxoma virus and myxomatosis

Myxoma virus was first discovered as a novel infectiousrabbit pathogen in Uruguay at the end of the 19th cen-tury, when imported European rabbits (Oryclolagus cunicu-

Ins) were suddenly stricken with a previously undescribeddisease, later called myxomiiatosis [17, 18]. This diseasewas found to be transmitted by arthropod vectors, par-ticularly mosquitoes, and ss’as virtually 100% lethal to in-fected European rabbits. The myxomatosis diseasesyndrome was characterized by extensive fulminating le-sions, both internal and extermial, amid severe immunodys-function accompanied by supervening Gram-negativebacterial infections of the respiratory tract [17, 19, 20].The infectious viral agent, myxoma virus, was later shownto be a member of the poxvirus family and to have arisenfrom populations of the North ahd South American rab-bit (Sylvilagus sp.), a distinct genus from that of its Euro-pean counterpart [17, 18, 21, 22]. Interestingly, in theindigenous rabbits, i.e., Sylvilagus bachinani and Sylvilagus

brasiliensis, myxoma virus established a symbiotic yet non-pathogenic relationship, causimig only a persistent infec-tion �s’ith minimal cellular immune recognition and onlymiiinor symptoms [17, 21, 22]. Thus, it is only after infec-tion of the related but distinct European rabbit that thepathogenic syndrome that characterizes full-blown

Abbreviations: EGF, epidernuil growth factor; TN F, tumlior necrosis

factor; TUE. transforming growth factor; SF’s’, shope ht)roma virus; MRV,

malignamit ral)bit fibroma virus; NICE, iiwxomna gioss-th factor; SFGF, SF’V

growth factor; I FN, interferon.

Reprint requests: Grant McFadden, Department of Biochemistry, Uni-

versity of Alberta, Edmonton, Alberta, Canada TOG 2H7

Received November 1, 1994; accepted januai-y 12, 1995.

Cellularhomologue

Viral proteinlocalizationGene Copy No.a Function

Virokines NICF

SERP-l(SPI-4)�

I

2

EGF/TGF-ct

Serpinsuperfamily

Cellular/secreted

Secreted

Ligand of EGF receptor(stimulates mitogenesis)

Inactivates multiple host

serine proteinases

(inhibits inflammatory

response)

Yes

Yes

Viroceptors T2T7Unmappeif’NI IlL

22?1

TNF receptorIFN-y receptorInterleukin-l receptor?

SecretedSecretedSecreted

Cell sLirface

Binds and inhibits TNF-a/�3Binds and inhibits INF-’yBinds interleukin-I�l

Inhibits inflammation byunknown mechanism

YesNDND

Yes

Myxomatosisvirulence geneb

�virus genes that map within the viral terminal inverted repeat sequences are present as two copies.iv irulence genes are defined bs’ the attenuation of the disease syndrome of myxomatosis caused by infection with recombinant myxoma virus svith targeted inactiva-

tions of the gene in question (Nt�, not determined).

:jsPI_4 is an alternative nomenclature suggested for SERP-l [471.The presumptive equivalent gene from vaccinia (BI5R) encodes a soluble homologue of the cellular type II interlctikin-I receptor [86, 87J.

lb

,� HOMOLOG_‘s,� TO MGF

10000

85.00

41.46

38.89

TABLE 1. Virokines and Viroceptors Encoded by Nlyxonia Virus

732 Journal of Leukocyte Biology Volume 57, May 1995

myxomatosis manifests itself.The extreme virulence of myxoma virus was exploited

in the early 1950’s when the first attempt at irradicationof a vertebrate pest with an infectious agent was initiatedin Southern Australia. Myxoma virus was imported toAustralia from South America and was released into

populations of feral European rabbits that had overrunlarge areas of the Australian countryside [17]. Althoughmassive reductions in rabbit populations were initiallyregistered, resistant rabbits soon repopulated the infectedareas and the dominant field viruses became progres-sively attenuated [17, 18]. Today, myxoma-resistant rabbitpopulations in Australia approach the levels found beforethe original virus releases [23].

Recent studies have focused on the molecular mecha-nisms by which myxoma virus is able to exert such pro-found pathological effects on the immune system ofEuropean rabbits. Some of the virulence genes encodedby the myxoma virus have been identified and several ofthe expressed viral proteins have been shown to interactwith known components of the host’s immune system[ 24]. Here we review those secreted and cell surface pro-teins encoded by myxoma virus that modulate host cytok-me networks.

Virokines and viroceptors

Virus-encoded proteins that affect the activities of thehost immune system can function either intracellularly orextracellularly. The term virokine, initially coined in 1988to describe a novel virus-encoded epidermal growth fac-tor (EGF)-like growth factor and the 35-kDa secreted com-plement control protein of vaccinia [25], refers to viralproteins that mimic host ligands (e.g., cytokines orgrowth factors) or related soluble immune regulators(e.g., complement binding proteins). Later, the term viro-ceptor was proposed [26] to describe a class of viral pro-teins that are functional homologues of cellular receptorsand act by sequestering host ligands away from their tar-get cellular receptors. Although the first example of aviroceptor was the secreted tumor necrosis factor (TNF)receptor homologue encoded by several poxviruses[ 26-28], it is now apparent that some virus-encoded re-ceptor homologues are not secreted but function at thesurface of infected cells. For example, several human andprimate herpesviruses encode homologues of cellularchemokine receptors, also called serpentines, that are ex-pressed exclusively at the surface ofinfected cells [29-31].

Currently, the only known example of a poxvirus-encodedserpentine receptor homologue has been described in the

swinepox virus [32], but the ongoing sequencing studiesof other poxvirus genomes may yet reveal other membersof this class of membrane-bound viroceptor.

The use of virokines and viroceptors to modulate im-mune recognition has now been described for a variety olthe larger DNA viruses, particularly the poxviruses, in-doviruses, and herpesviruses [33, 34]. The specific exam-ples considered here that are encoded by myxoma virusare summarized in Table 1 and illustrate the diversitywith which one individual virus can utilize a spectrum olthese defense mechanisms to subvert immune recogni-tion and clearance of the infecting virus.

Myxoma growth factor

Currently, there is no example of a poxvirus-encodedcytokine homologue but there are several examples ol

ccc cc C

MGF N Is�AWAJ�1 37 45 51 5�bJ

Rec.ptor Binding Domain

POXVIRAI

mgi

1191

vgt

MAMMALIAN

h-btc (69) (104) 44.44

‘8” 44.44h-tgfa (47)

(174) 47.22

(210) 42.50

ti-Idgfl (75) (106) 56.25

Fig. 1. Comparison of MGF with other members of the EGF family o

growth factors. The NIGF putative signal sequence and the conserse�

cysteine-rich receptor binding domain is shown aligned to other poxviral

and mammalian EGF honiologues. The conserved cysteines are shown a�

black boxes and other conserved residues are indicated in white boxes�

The percentage of homology within the cysteine-nich comisers’ed domain

of a number of EGF family members are indicated: sfgf, Shope fibrom2

growth factor; vgf, vaccinia growth factor; vagf, variola growth factor

m-egf, munine epidermal growth factor; h-btc, human betacellulin; h-tgfahuman transforming growth factor a; m-sdgf, munine schwannoma-de.

rived growth factor; r-ndf, rat neu differentiation factor; h-tdgfl, htmma�

teratocarcinoma-derived growth factor. The accession numbers for thes

sequences are listed elsewhere [35].

I- c

()P2-P1iP1’-P2’-P3’-PA’-P5�)T AP�N

D l�l�L�J T� M V V R I KD S V �[ N K

� L�iL�lM �J L [i�J o S

%5t,is�iy, %.ciYTI’’

100 100528 287485 267530 280450 223

537 314953 7 31 0507 2714

Cons#{149}nsus A R S S P t E

McFadden et al. Interruption of cytokine networks 733

growth factor mimicry. The discovery and charac-tenization of poxvirus-encoded members of theEGF/transforming growth factor-a (TGF-a) superfamilyis reviewed elsewhere [14, 15, 35]. As illustrated in Figure1, all the poxvirus members of this family maintain the sixcysteine residues critical for correct folding of the recep-ton binding domain and all are secreted ligands for thecellular EGF receptor. In myxoma virus and other closelyrelated members of the leporipoxvirus genus, particularlyShope fibroma virus (SFV) and malignant rabbit fibromavirus (MRV), these viral growth factors are encoded bysingle-copy genes located close to the terminal invertedrepeat sequences in the genome [36-38]. The myxomagrowth factor (MGF) was shown to be a bona fide mimic

of EGF and TGF-cx in a variety of biological assays thatmeasured signaling events triggered by ligand binding tothe EGF receptor [39-42]. To assess the biological conse-quences of deleting the MGF gene from myxoma virus,and the related SFV growth factor (SFGF) gene in MRV,Opgenorth et al. [43, 44] created recombinant viruses inwhich the growth factor genes from myxoma and MRVwere disrupted by insertion of a selectable marker. Theresulting recombinant viruses were virtually normal forreplication in a variety of cultured cells but could onlyinduce an attenuated form of myxomatosis in susceptibleEuropean rabbits [43, 44]. In particular, rabbits infectedwith the MGF-disrupted myxoma virus exhibited de-creased levels of epithelial hyperplasia and metaplasia,which normally overlay viral lesions of the conjunctivaand respiratory tracts [44]. When similar MGF-disruptedmyxoma constructs were engineered to overexpress otherEGF-like growth factors, such as rat TGF-ct, the resultingviruses regained wild-type levels of pathogenicity in in-

fected rabbits [45]. These results clearly indicate thatMGF was indeed biologically equivalent to TGF-ct and is

therefore likely to function by stimulating host EGF re-ceptors.

A number of models have been suggested to explain� how poxvimus growth factors such as MGF might provide

a selective advantage for virus propagation in the tissuesof a vertebrate host [15, 35]. For example, mitogenicstimulation of quiescent cells in the vicinity of the virusinfection would dramatically up-regulate elements of thecellular macromolecular synthesis machinery and thus mi-prove subsequent virus replication and increase virus tit-ens. By mimicking a ligand for the ubiquitously expressedEGF receptor, vinal gene products like MGF would there-fore assist in virus spread within quiescent cells that nor-mally possess depressed pools of nucleotides and otherprecursors required for efficient viral replication andmorphogenesis.

Circumstantial evidence predicts the presence of other

growth factor homologues within the myxoma genome.One of the characteristic histological features of myxoma-tosis is the dysnegulated proliferation of endothelial cellsin the capillamy networks near myxoma viius lesions [20].Recently, a poxviral homologue of vascular endothelialgrowth factor has been described for poxvirus of sheep[46] and it is entirely conceivable that myxoma virus

might encode a related growth factor. Because the DNA-sequencing studies of myxoma virus are not complete,more examples of ligand mimicry may yet be uncovered.

SERP-1: a secreted serine proteinase inhibitor thatinhibits cellular inflammation

Currently, seven distinct serine pnoteinase inhibitor geneshave been discovered in poxvirus genomes all of which

encode proteins belonging to the superfamily of serineproteinase inhibitors (serpins) [14, 15, 47]. Members ofthe orthopoxviruses, such as vaccinia, cowpox, rabbitpox,and vaniola, each encode three known serpins that havebeen designated SPI-1, -2, and -3 [48-58]. Of particularnote is SPI-2, also designated crmA in cowpox, whichinhibits intenleukin-1�-converting enzyme [59], and thusregulates the infiltration of responsive leukocytes [15,55-58]. Although homologues of SPI-1, -2, or -3 have notbeen detected in myxoma virus, a related serpin, desig-nated SERP-1, has been described [60]. A schematic rep-resentation of the SERP-1 protein and its relationship toother viral and mammalian serpins is given in Figure 2.All active serpins function as pseudosubstrates for thetarget senine proteinases and form stable 1 : 1 complexesthat effectively remove the proteinase from the pool ofactive enzyme [47, 61]. Among the critical regions forserpin function is the PI-Pi’ site that forms the cleavablepeptide bond destined for hydrolysis by the reactive sen-me in the protease, and this peptide pair in part deter-mines the specificity of protease(s) that can be inhibited,

The Pi-PI’ residues ofSERP-1 are Arg-Asn, a unique pairnot found in any other serpin, and thus makes predictionof potentially inhibitable serine proteinases difficult [60].It has been shown that SERP-1 protein will form 1:1 in-hibitory complexes with a variety of human serine prote-inases, including plasmin, urokinase, tissue plasminogenactivator, and at least one of the components of the com-plement cascade [62]. However, it is still unclear ifany ofthese proteinases are actually targeted for inhibition by

SERP-1 in virus-infected tissues, and it is possible thatother potential target proteinases remain to be uncov-ered. SERP-1 is expressed from a late viral promoter and,unlike any other poxvinal serpin, is secreted from virus-in-fected cells as a 50- to 55-kDa glycoprotein [63]. Impor-tantly, virus constructs in which both copies of the

FIFACT Yr

NH2-�j�j

PCYYIRA� SrRPiss.

SERP-1 (MIX SPI-4)vv sP:-3

vs sP:-2 c�mAvv s�:-ispy SPH7

MA�MA1ASSr�rr’s:hPA � -

hGDN

rAIAF

Fig. 2. Comparison ofmnyxoma SERP-l witis other poxviral and mammal-

ian serpins. The upper illustration summarizes the consensus serpin

features, imicluding the distribution ofcommonly conserved a-helix struc-

tunes (light grey, putative helices labeled hA to hI). A portion of the

reactive center (black) is expanded to show the amnino acid sequence

surroumiding the scissile PI-PI’ bond (arrow). A comparison with a selec-

tion of other viral and mammalian serpimis indicates that SERP-l has aunique P1’ asparagine residue (italicized). The consemisus serpin reactive

center sequence is indicated and residues corresponding to the consensus

are boxed. Alignmnent is by consensus homology of the serpin reactive

center. Accession numbers used are: SERP-l, myxoma virus serpin

(P12393); SPI-3, vaccinia virus ORF K2L(P20532); VVSPI-2, vaccinia virus

cnmA/ORF B13R (M24218); VV SPI-l, vaccinia virus ORF B24R(M24217); SPV SPI-7, swine poxvirus ORF KIR(L2193l); hPAI-l, human

plasminogen activator inhibitor (P05121); hGDN, human glia-derived

nexin (P07093); rAIAF, rabbit al-antitmypsin (P23035).

21

© Cysleine

18 amino acid

membrane-spanning domain

166COOH

POXVIRAL MEMBERS OF THE TNF RECEPTOR SUPERFAMILY

VIROCEPTORS (TNF-R)

� ‘D- 1 mm m I d� mmI t I �i 1 mm m s�m� m I � I I I I flC

SFV” ‘1:1- rm�U- I I I It m i I 1 t IL I 11 m I 1 � 1 1 t t IIC

V�d.I�C2� � � m I [4 mti i I [I I 11 3 1L1� I I : m ii i i

C..p..C,,,.$ �-l1r:::::Il:I:TIIIIJI 1 1t I I I EI�I ti m i�m I �i : i i i m i i

V��d,�.AS3R .im #{149}mm I 1I_::f�1�IT m m [F1� fi II ii�i I1�

v.,Cw.l. C22t. .{I mm m i �i I m�Ti m rirrri i m

L�d� C,.&�.R1d� �

P.tyzoma 12 �-#{149}c::=m::::i::I:::i:::x__i�___1 IThre p75 �-c:i:::c:EJ=Iz�D- 4_-’��”TNFRp55

Ld.. Cy.wY�.RId� R.p..�. �

Fig. 4. Comparison of myxoma T2 protein with other poxviral members

of the TNF receptor superfamily. Characteristic features of the poxvirus

TNF receptor homologrmes including NH�-terminal leader sequences,

four cysteine-nich repeats, and COOH-terminal domains are comparedwith each other and the two humnan p55 and p7S TNF receptors [78].

Conserved cysteines are aligned ( I )‘ whereas (T) designate frameshifts

and ( � ) a stop codon in the discontimiuous open reading frames that

constitute the A5SR and C22L genes of vaccinia (strain Copenhagen).

734 Journal of Leukocyte Biology Volume 57, May 1995

Fig. 3. Predicted orientation of the myxoma NI I 1 L protein at the infected

cell surface. The COtl)H-terminal hydrophobic helix domain (amino acids

143-160), indicated by a shaded box, is presumed to span the plasma

meiiibnane once. The COOH-termimial six amino acids are believed to be

intracellular, ss’hereas the 142 amino acid NH,-tertninal is shown on the

exterior stmrface. The predicted extracellimlar domain has six cvsteine

residues (C). The amino acid residue numbers are imidicated on the

cliagiatn.

niyxoma SERP-1 gene have been disrupted grow normallyin cultumed cells but cause an attenuated myxomatosis byvirtue of a more effective infiltration of inflammatoryleukocytes to the site of the viral infection [63]. Thus,SERP-1 appears to function by inhibiting some aspect ofthe cellular inflammatory response. It is unknownwhether the direct target of SERP-1 function is the cytok-me network, but proimiflammatomy cytokines that requireextracellular proteolytic activation would be attractivecandidates.

More recently, the anti-inflammatory properties ofSERP-1 have been exploited for a novel experimentaltherapy in a rabbit miiodel for atherosclerosis. When puri-

fied SERP-1 protein was infused directly into the arterial

�ressel wall after balloon angioplasty-induced vascular in-

jury, the subsequent development of atheroscleroticplaque was inhibited [64]. This is the first evidence that

viral immune defense proteins such as SERP-1 can beexpressed, purified, and used as immunosuppressive re-

agents for therapy of disease syndromes associated withoverly exuberant inflammation or excessive immune ne-sponses.

Ml 1 L: a candidate cell surface viroceptor

Poxviruses are known to encode a variety of proteins thatare expressed omi the surface of infected cells [14-16],

however, the pathogenic roles of these cell surface pro-teins have only recently been investigated. One candidatefor a cell surface viroceptor encoded by myxoma virus is

�vI ilL, Sc) namiied because it is the 11th open readingframe from the left genomic terminus and is transcribed

to the left [44]. As shown in Figure 3, MilL is a relativelysmall viral protein (166 amino acid) expressed on thesurface of infected cells and possesses a single transmem-

brane region and a short (6 amino acid) intracellular

domain. M 1 1 L was originally discovered by accidentwhen the COOH-terminal end of M 1 1 L was truncatedduring the construction of an MGF deletion virus due toan overlap in coding sequences between these two genes[44]. Subsequent gene disruption analysis of the MilLgene indicated that Ml 1L was itself a significant virulencefactor for the induction of the myxomatosis disease syn-drome in susceptible rabbits [44]. When recombinantmyxoma virus with a disrupted M 1 1 L gene was used toinfect European rabbits, the standard disease symptomswere virtually eliminated and instead benign fibroma-like

E II t lesions characterized by massive influxes of infiltratingxtrace uar leukocytes were observed [44]. It is believed that cell sun-

face expression of M 1 1 L by the wild-type virus somehowPlasma prevents effective influx of inflammatory cells, panticu-Membrane larly heterophil lymphocytes and macrophages [44]. Sub-

sequent studies revealed that an M 1 1 L variant, which was

unable to traffick to the surface but instead was retainedIntracellular in the cytoplasm, was also ineffective at preventing cellu-

lan infiltration, suggesting a surface receptor-like functionfor Ml 1L [65]. Currently, no close homologue of Ml 1Lhas been described in the literature and therefore therelationship of Ml 1 L to the cytokine network must re-main speculative. One possibility is that Ml 1L is a nonsig-naling receptor that recognizes an unidentified ligandimportant for the inflammatory response. Another hy-pothesis is that expression of M 1 1 L prevents the elabona-tion of proinflammatory signals from infected cells,possibly by the regulation of the apoptotic response ofcertain immune cells to the virus infection.

T2: a secreted TNF receptor homologue

Many normal cellular cytokine receptors, particularly

those with single membrane-spanning domains, havebeen found to also exist in secreted soluble form pro-duced by either proteolytic cleavage from the cell surfaceor alternative splicing [66, 67]. These soluble receptorscan potentially function as ligand inhibitors by sequester-ing extracellular bioactive ligands away from the cell sur-face receptors. The use of a similar strategy by DNA�‘iruses to intercept host cytokines before receptor en-

gagement was first suggested when data base analysis of

i%IcFadden et al. Interruption of cytokine networks 735

the type II TNF receptor sequence revealed striking ho-mology to a gene from SFV [27, 28]. This poxvirus gene,designated T2 because it is the second open reading

frame from the viral genomic terminus, had been se-quenced in 1987 and shown to be transcribed as a typicalpoxvirus early gene [68]. It is now appreciated that thesecellular and viral proteins are all examples of a growingTNF receptor superfamily of proteins that share charac-tenistic cysteine-rich repeats in the ligand binding domain[ 69-71]. Myxoma virus has also been found to encode aclosely related T2 protein (Fig. 4), and the targeted dis-ruption of both copies of the myxoma T2 gene revealedthat the absence of T2 expression caused significant at-tenuation of myxomatosis in rabbits [26]. This was thefirst demonstration that a virus-encoded secreted recep-ton homologue was biologically important for virus propa-gation in a vertebrate host and lead to the proposal of the

generic term viroceptor [26]. Similar TNF receptor homo-logues have been detected in other poxviruses, notablycowpox and variola [72-75]. However, as indicated inFigure 4, in the Copenhagen and WR strains of vaccinia,the two T2-like genes are fragmented by virtue of internalstop codons and frameshift mutations [26, 76, 77]. Thegrowing poxvirus TNF receptor homologues family is re-viewed in greater detail elsewhere [78].

Studies using myxoma T2 secreted from a vaccinia vi-rus engineered to overexpress T2 indicate that the pro-tein binds and inhibits rabbit TNF-a but not mouse orhuman TNF-a [79]. Myxoma virus is one of the fewpoxviruses for which an evolutionary history within onevertebrate species is well established [17, 18], and the

� species specificity of the myxoma T2 protein is probablya direct reflection of its long symbiotic interrelationshipwith the South American rabbit [79]. It is thus reasonableto predict that other poxviruses with functional virocep-tons will also possess ligand species specificities that re-flect their evolutionary history.

T7: a secreted interferon (IFN)--y receptor homologue

�Given the well-documented importance of the IFNs (a, �3,and y) in combating viral infections, it is not surprising

� that many viruses have evolved distinctive anti-IFN strafe-gies [80, 81]. Currently, the only known examples of avirus strategy to inhibit IFN before ieceptor/ligand inter-action are the poxvirus-encoded soluble IFN-y receptorhomologues [82]. The first example discovered was theT7 gene of myxoma, thus called because it is the seventh

open reading frame from the tel-minus of the viralgenome [83]. The 37-kDa T7 protein is the most abun-dant protein species secreted from myxoma virus-infectedcells, and sequencing studies have revealed significantamino acid similarity to the human and mouse IFN-yreceptor (a-chain), particularly at the level of cysteimieresidues within the ligand binding domain (Fig. 5). Cross-linking experiments and direct inhibition studies revealedthat the T7 protein could bind and prevent the inductionof the antiviral state by rabbit IFN-y [83], indicating that

T7 was indeed another exaniple of a secreted viroceptor.Later studies indicated that, like the T2 protein, T7 was

highly specific for the rabbit ligand and could riot bind to

IFN--y from humans or mice [84]. Intelestingly, Scatchamd

analysis indicated that the K(f for soluble T7/rabbit IFN-ywas 1.2 nM, very similar to that reported for the soluble

versions of the cellular receptor a-chain for its cognatespecies IFN-y [82, 84]. It is therefore reasonable to predictthat the T7 protein functions by binding and sequestering

POXVMAL WNn�a

S wvwccw ci’ H CV cc c sc KIKDRSL i

�:Fm � E�S wvwccw c� H CY cc c sc KIKOR SI L

MYXOMA �I7 -ll-1II-EEIll-IHI-1ll---II-I---tI-Ellh1ll-llllfl��cELLuLARFNn�$ � wvwccw cP H CV cc c sc

=Fig. 5. Comparison of poxviral and niammaliami IFN-y receptors. Amino

acid residues conserved between the soluble poxviral IFN-y receptor

homologues amid the mammalian ligand binding domain are indicated

with boxes. Cysteitie residues critical for proper folding of the ligand

binding domain are highlighted with filled boxes. The length of the

tnature proteins are indicated on the right. Location of the transmem-

bramie domain (TNI) and cytoplasmic functional domain I are imidicated

for the cellular receptors. Further details and accesskn numbers for all

peptides are shown by Mossman et al. [82].

extracellular IFN-y produced by activated T lymphocytesand natural killer cells that infiltrate the viral lesions.

Currently, no direct biological data on the role of T7 inmyxoma-infected rabbits is available but experiments totest the effect of disrupting both copies of the myxomaT7 gene ame in progress. T7 also possesses a significantstretch of COOH-terminal sequences that have no ho-mologous counterpart in the data base, which raises thepossibility that the intact T7 protein has other functionsin addition to the bimiding and inhibition of IFN-y.

In addition to the T2 and T7 viroceptors, a third classof secreted viroceptom’ has been described for vacciniaand cowpox, viruses with homology to the intenleukin-1

(type II) receptor [85-87]. Pmcliminaly evidence suggeststhat cells infected with myxoma virus elaborate a similarinterleukin-i�i binding protein bin the myxoma virusgene remains to be miiapped and formally analyzed (K.

Mossnian, K. GIahani, A. Alcami, G. Smith, and G.N’IcFadden, unpublished obser1�ations). Given the rapidity

with which the first three secmeted viroceptors were dis-covered in the poxvirus systemli, it seems likely that miioreexamples of this stratetrv still memain to he umu-overt’d�

CONCLUSIONS

Poxviiuscs geneially replicate in tissues (skin, respiratomy

tract) that are accessible to niany of the effector elementsof the imniumie system. Thus, it is not surprising that theseviruses have evolved active countcrmiieasures to nullify at

least some of the effector mechanisms of immune recog-nition and cleaiance. What is perhaps surprising is theextent and breadth of the host ininiune repertoire thatcan actually be targeted fom subvemsion by specific viralgene products. The [angel’ DNA viruses have the luxury of

encoding moore proteins thami are minimally required for

�-ii-us replication, and it is believed that poxviruses,

through their evolutiomiamy histomy, have somehow ac-

quiied the codimig capacity fom a variety of genes that may

have been oiigimiall� deri�-ed froni the host [24, 33, 88,89]. The molecular nature of such acquisition evemits re-

main speculative. Because poxviruscs replicate exclusivelyin the cytoplasm of infected cells amid none of the viralgenes possess imitrons, omic hypothesis is that cellular

736 Journal of Leukocyte Biology Volume 57, May 1995

genes may have been acquired by necombination throughcytoplasmic cDNA intermediates. Even if this were a rareevent, if the captured gene conferred some protectionfrom the immune response, then the resulting viruswould acquire selective advantage within the infectedhost. In any event, the repertoire of immunosubvensiveviral proteins in many ways are elements of a collectivestrategic defense initiative that allows virus propagationeven in the face of mobilized inflammatory and immuneresponses. The total number of such poxviral anti-im-mune proteins is unknown but at least one poxvirus,vaccinia, has been shown to have at least 55 open reading

frames that are dispensable for propagation in tissue cul-tune [90]. In the case of myxoma virus, only five of theseveral dozen novel proteins secreted from infected cellshave been characterized, which further suggest that thestudy of myxoma anti-immune strategies is still in its earlystages.

The myxoma virus model for the study of poxviruspathogenesis is particularly attractive for a variety of rea-sons. The evolutionary host for myxoma is well estab-lished (American rabbit) and the myxomatosis diseasecaused when the virus infects a related but distinct host(European rabbit) has a well-defined pathology in an ani-mal well suited for biological experimentation [17-22].Furthermore, as a consequence of an important and fas-cinating set of field experiments in Australia and Europe,a collection of natural attenuated variants of the virus, aswell as resistant strains of host rabbits are now available.These attenuated viruses and resistant rabbit strains willbe invaluable for addressing important basic questionsconcerning the nature of virus virulence in outbred im-munocompetent hosts [17, 18].

With the myxoma model one can also begin to investi-gate the relationship between virus pathogenesis and thenature of the evolutionary pressures that selected for theacquisition of anti-immune genes. Myxoma virus infectionof native American rabbits is virtually asymptomatic, sug-gesting that the end product of virus-host co-evolution issymbiosis, rather than disease. In the course of this mu-tual accommodation between the virus and host,poxviruses such as myxoma have clearly adapted to theeffector mechanisms of the immune system. This accom-modation is directly reflected in the nature and scope ofthe viral genes that collectively render infected sites ap-parently invisible to effective immune clearance. How-ever, once the virus has crossed into a susceptible butgenetically distinct host, such as the European rabbit, thissymbiotic balance between virus and host is altered inways that are still poorly understood. Instead, the extremevirulence of the myxoma virus in the European rabbit isa powerful testament to the effectiveness of the virusstrategies to subvert immune recognition and clearance.Instead of mediating virus survival by promoting nonrec-ognition in American rabbits, the viral proteins becometransformed, in effect, into mediators of viral pathogene-sis in a disease of European rabbits. Hence, deletion stud-

ies within the myxoma genome reveal the existence ofgenes that are functionally characterized as virulence fac-tons by virtue of direct participation in the myxomatosissyndrome in susceptible rabbits, whereas evolutionarypressures undoubtedly selected only for the acquisition ofgenes that can be adapted to confer increased virus sur-vival. In either event, the resulting conspiracy of virusgenes so identified provide a rich repository with whichto evaluate the effector arms of the immune system itself.

In this review only those virus gene products that po-tentially interact with the host cytokine network havebeen considered. The collection of myxoma proteins cur-rently defined as viroceptors or virokines continues to

grow and it is conceivable that some of these will revealhost ligands or regulator proteins that have not beenuncovered by classical studies of the immune system. Acase in point is Ml IL, which has no counterpart in the

current data base, but which has all the characteristicsone might predict for a novel cytokine receptor homo-logue. Deletion analysis of M 1 1L in the myxoma genomereveals that this gene product is critical for inhibitingsome aspect of the inflammatory response, but themechanism of action remains to be deduced. Note that itis only because of the ability to perform direct biological

experiments with mutant constructs of virus that the cniti-cal importance of M 1 1L for immune inhibition was estab-

lished. Thus, data base amialysis of viral genes coupledwith in vivo experimentation in a biological system pro-

�‘ides powerful tools with which to address these issues.Finally, it is clear that some but not all of the myxoma

cytokine inhibitor proteins (e.g., T2 and T7) exhibit strictspecies specificity for the rabbit ligands. Given the possi-

bihity that new cytokine inhibitors are likely to be uncov-

ered in the poxvirus system, it is relevant to note that theonly two poxviruses known to be specific for humans aresmallpox (vaniola) and Molluscuin contagiosum. Neither ofthese two viruses are amenable for experimental analysisin animal models and hence acquisition of biological datafrom related poxviruses such as myxoma assumes greatenrelevance. By the same token, the human poxviruses alsoencode their own unique spectrum of immune defensemolecules, and the recent debate on whether to destroyexisting specimens of smallpox [91] should include atleast some consideration of the uniqueness of smallpox asa potential source of novel human-specific cytokine in-hibitons. Lessons from the myxoma virus system stronglysuggest that careful analysis of virus/host interactions,

both in vivo and in vitro, will provide an important re-

source for studying not only issues of virus replication

strategies, but the innermost workings of the immunesystem itself.

ACKNOWLEDGMENTS

G. McFadden is a Medical Scientist of the Alberta Hen-tage Foundation for Medical Research (AHFMR). K.Mossman and P. Nash are supported by studentshipsfrom the AHFMR and the MRC of Canada. This work wasfunded by an operating grant from the National CancenInstitute of Canada.

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