VIROLOGY

184, 370-382 (1991)

Myxoma Virus Expresses a Secreted Protein with Homology to the Tumor Necrosis Factor Receptor Gene Family That Contributes to Viral Virulence C . UPTON, 1 . L . MACEN, M . SCHREIBER, AND G . MCFADDEN' Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 21-17 Canada

Received April 26, 1991 ; accepted June 3, 1991 Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture . However, many of these these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection . Recently it has been shown that Shops fibroma virus encodes, within the terminal inverted repeats, a protein (1-2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNFa and TNF$ . We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein . To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker (Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid . The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52-56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at early times after infection . Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated . The majority of infected rabbits were able to mount an effective immune response to the Infection and completely recovered . These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF . We propose the term "viroceptor" to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection . e 1991 Academic Press, Inc . INTRODUCTION All poxviruses share a number

of

essential for growth in tissue culture and encode many of the proteins that are determinants for virus virulence, tissue specificity, and host range (Pickup at at, 1984 ; Kotwal and Moss, 1988a ; Boursnell etal., 1988 ; Perkus at at, 1991) . Without the selective pressure im-

common charac-

teristics which include replication within the cytoplasm of host cells and possession of a large doublestranded DNA genome with hairpin termini (Moss, 1990) . It

is becoming

posed by continued propagation in an animal host, those genes in the terminal regions of continuously passaged laboratory poxvirues, such as vaccinia, have

apparent that the central portion

of the genome of poxviruses from the different genera encode the genes required for the processes essential for virus growth, such as transcription, replication, and maturation (Broyles and Moss, 1986 ; Niles etal ., 1986 ;

diverged considerably in both information content and genome position from terminal regions of poxviruses from the different genera that cause defined disease syndromes in their native host (Upton at at, 1 987a ; Upton and McFadden, 1986a ; Howard et al., 1991) .

Earl et al., 1986) . As such, many of the genes in this region of the viral DNA have been relatively well conserved throughout the evolution of the different poxviral genera (Gershon et al., 1989 ; Tartaglia at al.,

Recently it was shown by computer homology scores that the T2 open reading frame (ORF) of Shope fibroma virus (SFV), which maps very close to the termini of the genome (Upton at al., 1987a), encodes a protein with significant homology to the ligand binding domain of the human type I tumor necrosis factor receptor (TNFR I) (Smith et al ., 1990a) . When recombinant SFV T2 protein was expressed in COS cells, this gene product was shown to be secreted as a heterodisperse 58-kDa species that specifically binds TNFa and TNFff with high affinity (Smith at al., 1991) . Vaccinia virus (strain

1990 ; Upton and McFadden, 1986b ; Upton et at, 1990b) . On the other hand, at least for the poxviruses for which sequence information is available, the genes closer to the termini of the genome appear to be non-

Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No . M37976 . ' To whom correspondence and reprint requests should be addressed . 0042-6822/91 $3 .00 Copyright 0 1991 by Academic Press, Inc . All rights of reproduction in any form reserved .

370

MYXOMA VIRUS ENCODES TNFR-HOMOLOG

WR) has also been reported to contain DNA sequence homologies to the TNFR (Howard at al., 1991), but the identified ORF (Sal F19R) was interrupted by a frameshift and a termination codon that suggests the elaboration of a single intact TNF-binding protein is unlikely . Tumor necrosis factor a (TNFa) and the related lymphotoxin (TNF$) play pivotal roles in inflammation and the orchestration of immune responses against such diverse stimuli as bacterial and viral pathogens, physical trauma, and neoplasia (Cerami and Beutler, 1988 ; Beutler and Cerami, 1988, 1989 ; Rosenblum and Donato, 1989 ; Balkwill, 1989 ; Fong and Lowry, 1990) . Among the other activities attributed to the TNFs are inhibition of polymorphonuclear leukocyte migration (Salyer et at, 1990), stimulation of the transcription of HLA class I genes alone and synergistically with interferon y (Johnson and Pober, 1990), and coordinated autocrine regulation with interleukin 1 in monocytes (Smith at al., 1990b) . Although many of the effects of TNF are well characterized, the mechanisms of signal transduction (Schutze et al., 1990) and killing by TNF are still unclear, although cytoxicity seems to be correlated with the induction of toxic superoxide radicals since manganese superoxide dismutase can protect against TNF killing (Wong and Goeddel, 1989 ; Asoh et at, 1989). In addition to the more general roles of the TNFs in immune regulation . TNFa has been shown to be active in the killing of virus-infected and tumor cells (Melton et at, 1986 ; Wong and Goeddel, 1986) and also to synergize with interferons to augment the antiviral state (Adreka et al., 1985 ; Hughes et al ., 1988 ; Campos et al., 1988) . Therefore it should not be surprising to find examples of viruses that have evolved strategies to counteract the anti-virus effects of the TNFs . For example, a protein from the E3 region of a group C human adenovirus has been shown to inhibit cytolysis by TNFa and cytotoxic T cells (Gooding et al., 1988 ; Wold and Gooding, 1989) . Recently two distinct, but related, cellular receptors which bind both TNFs have been cloned and sequenced from endothelial (TNFR 1/75-80 kd) (Smith et al ., 1 990a) and placental (TNFR 11/55-60 kd) (Loetscher et al., 1990 ; Schall et al., 1990) tissue . Although the two receptors share little homology on their cytoplasmic domains, their cysteine-rich TNF-binding regions share demonstrable similarities and form the basis for a new family of immunoglobin-like surface molecules, of which the SFV T2 protein is a member (for review, see Sprang, 1990) . This growing family contains a number of surface proteins with related symmetry of cysteine-rich repeats, including the receptor for nerve growth factor and CD40, but to date the only member other than TNFR I and 11 to specifically bind TNFa and 0

37!

is the SFV T2 protein (Smith at at, 1991) . Soluble TNF inhibitors have also been purified from the urine of febrile patients and N-terminal analysis of these proteins suggests that they are truncated forms of the normal TNFRs (Seckinger et al., 1990a,b ; Engelmann et aL, 1990) . It is likely that these secreted soluble forms of the TNFRs are part of the complex control mechanisms required forthe regulation of this potent cytokine during normal immune functions . Since the secretion of a virus-encoded receptor homolog for an important cytokine such as TNF could clearly play a major role in the evasion of the host immune response we investigated the evolutionary relationship of this TNFR-like family in several poxviruses. SFV induces benign fibrornas in adult rabbits, but the virus generally remains strictly at the site of innoculation and tumor regression invariably follows, unless the host is immunocompromised (reviewed by McFadden, 1988) . Several related poxviruses, however, are able to migrate from the initial infection site and set up a systemic infection which is poorly recognized by the host and causes fatal disease . Two such Leporipoxviruses, myxoma and malignant rabbit fibroma virus (MRV), induce, in addition to primary and secondary tumors, a severe immunosuppression that leads to overwhelming infections by opportunistic Gram negative bacteria (Strayer at at, 1 983a,b) and are normally lethal for adult immunocompetent European rabbits . Here we show that myxoma and MRV express and secrete a common T2 homolog to the TNF receptor superfamily and that inactivation of both T2 gene copies in myxoma results in dramatic attenuation of myxomatasis in infected rabbits . Thus, the secretion of the T2 gene product from cells infected with these viruses is one pathway used by the Leporipoxviruses to enhance virus virulence by abbrogating the anti-viral effects of TNF-dependent immune response pathways of the host . MATERIALS AND METHODS Cells and viruses The source of viruses (SFV, strain Kasza ; MRV ; and myxoma, strain Lausanne) has been described (Block at al., 1985) . Viruses were propagated in BGMK cells grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum . Cloning and sequencing of DNA Previous studies (Upton et at, 1988 ; Upton et at, 1990a) suggested that the TIRs of myxoma and MRV were quite similar to the equivalent regions in the SFV genome (Fig . 1), suggesting that any myxoma counterpart of SFV T2 would likely be present within or near

37 2

UPTON ET AL.

the penultimate myxoma virus BamHl S fragment (Russell and Robbins, 1989) . Thus, myxoma DNA was isolated as previously described (Wills et at, 1983), restricted with BamHl, and the S fragment was isolated and cloned into BamHl-digested pUC19, using standard procedures (Sambrook et at, 1989) . Preliminary DNA sequencing at the ends of the myxoma BamHl S fragment revealed homologous sequences to those closely flanking the SFV ORF T2 . Therefore, an internal Sall fragment containing the T2 homology was subcloned into Sail-digested ml 3mtl21 (Chambers et al., 1988) and both orientations were obtained . Nested deletions were made for each orientation (Henikoff, 1984) and sequenced using Sequenase (USB Corp ., Cleveland, OH). To obtain the sequence at the C-terminus of the T2 gene the appropriate Pstl/EcoRl fragment was blunt ended with T4 DNA polymerase and cloned into the Smal site of ml3mtl21 . Sequencing from the Pstl end of this fragment completed one strand and a deduced oligonucleotide primer was used to confirm the sequence of the opposite strand . The equivalent BamHl fragment was also cloned from MRV and was shown by DNA sequencing that the MRV T2 ORF is identical to that of myxoma . Computer facilities of the Genetic Computer Group Sequence Analysis Software Package (Devereux et at, 1984) on the Canadian National Research Council Molecular Database System were used to analyze these DNA and translated protein sequences . Construction of myxoma T2 deletion mutant The construction of plasmid pMT2G used for the inactivation of both TIR copies of the myxoma T2 gene is shown in Fig . 2 . Briefly, a deletion of 30 nucleotides within the cloned myxoma T2 gene was achieved by digestion with Smal, which cuts pMT2KH only twice, at adjacent sites near the 5'-end of T2 . A selectable marker was then inserted into the inactivated myxoma T2 gene afterthe addition of Bglll linkers . In preliminary experiments, the S-galactosidase gene under control of the vaccinia virus p11 promoter was used for identification of novel viruses ; however, individual isolates expressing O-galactosidase were not stable, presumably because "repair" of the altered locus using the other viral copy of the TIR caused loss of the ,B-galactosidase gene. Therefore, the Escherichia coli guanosine phosphoribosyltransferase (Eco-gpt) dominant selectable marker driven by the vaccinia virus 7 .5K promoter (Falkner and Moss, 1988) was used . Since Lepori- and Orthopoxviruses utilize the same transcriptional regulatory mechanisms, the 7 .5K promoter from vaccinia functions at both early and late times in Leporipoxviruses as well (Macaulay and McFadden, 1989) . The fu-

sion of the 7 .5K promoter, as a 289-bp EcoRl fragment from pW5 .1 (gift of D . Hruby), to the Eco-gpt coding sequence, as a 650-bp Bglll-Ddel fragment from pSV2gpt (gift of R . Godbout), was achieved by cloning these cassettes into the Smal and BamH1 sites of pUC19, respectively, to create p7 .5gptA. The p7 .5-gpt fusion fragment was released with EcoR1 plus Hindlll, bluntended, and ligated to Bglll linkers for insertion into pMT2KH . To facilitate identification of recombinant viruses, a parent virus (vMYX-lac) was chosen which had already been engineered to express ,e-galactosidase . The construction and biological characterization of vMYX-lac will be presented elsewhere (A . Opgenorth et al., in preparation) . In this virus the #-galactosidase gene is present between the growth factor and M-T9 ORFs of myxoma virus (Upton et at, 1987b) and appears to have no effect on virus growth in vitro or virulence in vivo. The control virus vMYX-GF- AM-11 contains a p-galactosidase cassette at the growth factor locus of myxoma and will be described separately (A . Opgenorth et at, in preparation) . To isolate the T2-minus virus BGMK cells infected with vMYX-lac were transfected with Hindi 11-linearized pMT2G using standard protocols . After 2 days viruses were harvested and passaged twice with selection medium (25,ug/ml mycophenolic acid, 20 Ag/ml xanthine, 15 µg/ml hypoxanthine) ; individual plaques were picked and amplified three times after identification under 1 % low melting point agarose with selection medium plus 500,ug/ml X-gal . The polymerase chain reaction was used to check for correct insertion of DNA into the myxoma T2 gene, using appropriate primers flanking the T2 gene . Stocks of the gpt-positive viruses were grown up and Southern blotting of the final stock virus, denoted vMYX-T2gpt, confirmed that both copies of the myxoma T2 gene were interrupted by the selectable marker (data not shown) . Infection of rabbits with vMYX-lac and vMYX-T2gpt All animal experiments were performed using C-level containment facilities underthe guidelinesof Canadian Council on Animal Care to avoid any possibility of spread of the virus to other rabbit stocks . Female New Zealand White rabbits (Oryctolagus cuniculus) (12-16 weeks old) were obtained from a local supplier . The rabbits were observed for 7 days to confirm they were healthy and then were injected intradermally on each side of the back with a 0 .1 ml dose of virus (5 x 10 2 PFU) in DMEM . The rabbits were monitored daily for symptoms of myxomatosis (see Fenner and Ratcliffe, 1965, for review of the disease syndrome) . If the rabbits experienced severe difficulty in breathing due to supervening infections of Gram negative bacteria



MYXOMA VIRUS ENCODES TNFR-HOMOLOG

which frequently preceeds death, they were sacrificed immediately . Detection of viral proteins secreted from myxomainfected BGMK cells BGMK cells were grown in T150 flasks until approximately 80% confluent, the medium was removed and cells were infected with virus (0 .5 ml in DMEM) at a multiplicity of 10 . After 1 hr adsorption at 37 0 , 20 ml DMEM plus serum was added (time = 0) . At time = 120 min, the medium was removed, cells were washed twice with phosphate-buffered saline (PBS), and 15 ml methionine-free DMEM (no serum) was added and supplemented with 120 1sCi Trans 35S-label (35S-McUCys mixture ; ICN Biomedicals) . At time = 180 min, the labeling medium was removed, cells were washed twice with PBS, and 15 ml DMEM (no serum) was added . At T= 300 min, the medium was collected (EARLY sample) and replaced by fresh 15 ml methionine-free DMEM (no serum) with 120 pCi Trans 35 S -label . In order to collect secreted proteins at late times of infection, at T = 360 medium was removed, cells were washed twice with PBS, and 15 ml unlabeled DMEM (no serum) was added . At 480 min, medium was collected (LATE sample). The samples were concentrated 30-fold using Centriprep-10 filters (Amicon) and aliquots equivalent to the protein secreted from 5 x 10" cells over the 1-hr chase period were loaded into lanes of a 7-15% gradient polyacrylamide gel (Laemmli, 1970) . After electrophoresis, the gel was stained with Coomassie blue, destained, dried onto Whatman 3MM chromatography paper, and autoradiography performed . RESULTS Cloning and sequencing of the myxoma and MRV T2 homologs We have previously mapped and sequenced the myxoma and MRV homologs of the Shope fibroma growth factor (SFGF) and T9-L genes and found them to reside at similar positions in the myxoma and MRV genomes (Upton and McFadden, 1986a ; Chang et al., 1987 ; Upton et al., 1987b)) . Since sequence analysis suggested that myxoma also contained homologs of SFV ORFs T5 and T8 at similar positions within the TIR, we therefore predicted that if myxoma and MRV contained a counterpart of the SFV T2 gene, it would probably be located within or near BamHl S, which maps as a 2 M fragment at a site beginning less than a kilobase from the hairpin terminus (Russell and Robbins, 1989) . Accordingly, this BamHl fragment was purified and cloned from both myxoma and MRV . T2 homologs were identified in both viral DNAs and sequenced as described under Materials and Methods . The map po-

373

sition of the myxoma T2 ORF, shown in Fig . 1, demonstrates that its context within the viral genome is the same as that of the SFV T2 gene . A Kpnl-Hindlll fragment containing most of the T2 ORF plus 5'-upstream sequences was subcloned into pMT2KH and adjacent Smal sites at the 5'-end of the gene used to construct a plasmid, pMT2G, suitable for inactivation of the viral T2 gene with a selectable E. colt gpt cassette (Fig . 2) . The DNA sequence of the myxoma T2 OFF and its flanking regions are presented in Fig . 3 together with the deduced amino acid sequence of the ORF . DNA sequencing of the MRV T2 ORF determined that it is identical to myxoma T2, as would be predicted from the recombination pathway that created MRV from myxoma and SFV (Upton et al., 1988) . Therefore, these T2 ORFs in MRV and myxoma will be simply referred to, hereafter, as myxoma T2 . The myxoma T2 ORF shares 76% identity with, and is one amino acid longer than, the SFV T2 counterpart (Fig . 4) . This degree of conservation between equivalent ORFs from different Leporipoxviruses is typical of previous findings with other genes at or near the viral TIRs (Upton at al., 1987a, b ; Upton et al., 1990a) . The SFV T2 mRNA has been shown previously to be regulated temporally as an early gene, and expected mRNA 5'-start sites immediately upstream of the T2 coding sequence are detected before DNA replication (Macaulay et al., 1987) . The region immediately 5' of the myxoma T2 OFF corresponding to the predicted promoter domain for this ORF is very similar to its SFV counterpart and has all the important features of an early gene . However, between 52 and 182 nucleotides upstream of the myxoma T2 ORF there is an unusual series of nine related tandem repeats (Fig . 2), each 12-20 nucleotides long (Fig . 5) . These repeats are of note because they are not present as such in the SFV genome and could potentially affect expression levels of T2 in myxoma compared to that in SFV . Although there is weak homology to two of the myxoma repeats in the same region of SFV, it is impossible to determine whether one region is the progenitor of the other . The function, if any, of these tandem repeats is at present unknown, but it will be of interest to determine if they affect promoter function of myxoma T2 or are the result of fortuitous amplification of intergenic DNA . No other comparable tandem repeats have been found elsewhere in the TIRs of myxoma or SFV . Since these two viruses are quite different biologically, it will be important to quantitate T2 expression in terms of steady state message levels and secreted protein (in progress) . Analysis of the myxoma T2 protein sequence The TNFRs I and II are part of a growing gene family that include the nerve growth factor receptor (Johnson



UPTON ET AL.

374

1

2

4

6

8

10

12

14 (kb)

Inverted Terminal B

Repeat

B

L IT 0

SFV

TI

T2

T3AT3CT4

I

T5

T6

Inverted Terminal B

T7

TB

K DD

cc

E

SFGF

BB

S

P S

T9-L

Repeat

BB

L

MYX

E

U3

B

SE

B

1 kb K

Sm Sm

H

Myx T2 FiG . 1 . Mapping and sequencing of the myxoma virus T2 gene. BamHl restriction fragments IT, 0, and Ears indicated for the left end of the SFV DNA (DeLange et al., 1984) and fragments AA, S, CC, K, DD, and U3 for myxoma DNA (Russell and Robbins, 1989) genomes . Open reading frames are shown as arrows. The SFV gene T2 is within the viral TIR, in the terminal BamHI IT fragment . (Upton et 1987a) . The striped box depicts the Sall-Pstl DNA fragment sequenced and presented in Fig . 3 . Restriction enzymes : B, BemHl ; E, EcoRl ; Kpnl ; P, Pstl ; 8, Sall ; Sm, Smal . al., K,

et al., 1986) and a number of other cell surface molecules predicted to be ligand-binding receptors (Stamenkovicetal., 1989 ; Mallettetal., 1990) . The aligned protein sequences of these cellular molecules indicate a number of elements in common, including a signallike sequence, a characteristic cysteine-rich extracellular domain, and a membrane-spanning region, although their cytoplasmic domains are totally unrelated . In contrast to these cell surface proteins, the SFV and myxoma T2 proteins lack transmembrane domains while retaining the signal-like sequence and the putative ligand binding cysteine-rich domains (Fig . 6) . The method of von Heijne (1986) predicts that both SFV and myxoma T2 proteins contain signal sequences at their N-termini and could be cleaved between amino acid residues 16 and 17 during secretion to yield proteins of 33 kDa (if unmodified) . Recombinant SFV T2 protein in the absence of replicating virus is found to be secreted from COS cells and the protein isolated from medium migrates as a diffuse band of approximately 58 kDa (Smith et al., 1991) . This difference between predicted and observed sizes is believed to be due in large part to glycosylation of the SFV protein. The SFV and myxoma T2 both have four potential sites for N-linked glycosylation, three of which are at similar positions in the proteins . After the initial discovery that the SFV T2 contained substantial homology to the ligand-binding domain of

TNFR I (Smith et al., 1990a), it was reported that vaccinia virus (strain WR) contained a discontinuous string of three mini-ORFs that, when linked together, produced an amino acid sequence (SaIF 1 9R) with a significant degree of homology to this superfamily (Howard et al., 1991) . Since the complete sequence of another vaccinia strain (Copenhagen) has now been reported (Goebel et al., 1990), we wished to compare SaIF1 9R between the two strains and in Fig . 6 show the Copenhagen version (designated A53R) . The DNA sequences of A53R from vaccinia Copenhagen and SaIF19R from vaccinia WR were identical . Unexpectedly, the database analysis revealed a second homology to the TNFR family in the TIR of vaccinia strain Copenhagen at precisely the same genomic location as the Leporipoxvirus T2 genes (Fig . 6) . This second ORF, C22L, possesses a similar array of conserved cysteines and is clearly a closer homolog to SFV/myxoma T2 than is A53R but, like A53R, is a fragmented ORF and unlikely to be expressed as an intact polypeptide . It is tempting to speculate that, because these vaccinia strains have been passaged extensively in tissue culture, some of its genetic repertoire designed for propagation in living host has evolved away from active expression in the absence of the selective pressure exerted by host immune response . The Leporipoxviruses SFV and myxoma, on the other hand, are still maintained in rabbits as well as tissue culture and the



MYXOMA VIRUS ENCODES TNFR-HOMOLOG Myx T2 B

P SS

_

S E

B --

K SmSm

H

Kpnt+Hindifragment

pUC19Kpn1+HindHI

KE

SE

H p7o s-Eco gpt

3'-T2

5*-r2

a KE

~

SE

H

p7 .5-Eco/ gpt

FIG. 2 . Construction of the T2 inactivation plasmid pMT2G . The striped box shows the Sall-Pstl DNA fragment sequenced and presented in Fig . 3 . The myxoma T2 gene was inactivated by deletion of 30 nucleotides (between two Smal sites) and insertion of the p7 .5/ Eco-gpt selection cassette as described under Materials and Methods .

important gene products required for growth in target tissues exposed to immune surveillance remain intact . An alignment of the cysteine-rich domains from the SFV and myxoma T2 proteins with the two TNFRs (Fig . 6) reveals several important features . The highest homology within this group is present in the first three of the four cysteine-rich repeats . Over this region, the SFV and myxoma T2 proteins share 40 .6 and 43 .196 identity with the TNFR I, respectively . Of all members of this family, myxoma T2 and human TNFR I are the most closely related pair. It will be interesting to ascertain if the rabbit TNFR I is more closely related to T2 than the human version . Although there is no direct proof that poxviruses can capture cellular genetic information, for example via cytoplasmic cDNAs, the close sequence similarities of many poxviral proteins with cellular counterparts suggest that a cellular origin of some poxvirus genes is possible . Construction of T2-minus myxoma virus Preliminary experiments to inactivate the T2 gene of myxoma with ,B-galactosidase were unsuccessful due

375

to a still poorly understood copying mechanism which spontaneously duplicates terminal sequence alterations from one TIR copy to the other . In order to exert dominant selection pressure, the E, coil guanine phosphoribosyltransferase marker found useful for vaccinia virus recombinants (Falkner and Moss, 1988 ; Boyle and Coupar, 1988) was used to create the insertion vector pMT2G (Fig . 2) in which a p7 .5-gpt cassette has interrupted the myxoma T2 open reading frame . Since the ,B-galactosidase marker is very useful in identifying sites of viral replication in cell culture and in rabbit tissues, the recipient virus used was vMYX-lac, in which a j3-galactosidase marker was placed in an intergenic location between the myxoma growth factor and the downstream M9 ORF . This virus is indistinguishable from wild type parent virus (strain Lausanne) both in vitro and in infected rabbits (A . Opgenorth et al., in preparation) . As described under Materials and Methods, in the presence of mycophenolic acid recombinant virus clones were grown up, proper insertion of the p7 .5-gpt marker into both copies of the T2 locus was checked by PCR and Southern blotting, and one virus isolate, designated vMYX-T2gpt, was chosen for further analysis . In vitro, no difference was observed between vMYXlac and vMYX-T2gpt in terms of viral growth in any cell line tested (not shown) . Plaque morphologies, growth kinetics, and virus yields were indistinguishable, suggesting that no major impairments to the replication potential of the virus outside an animal host were created by the T2 inactivation . The T2-minus myxoma virus fails to secrete a 55-kDa protein When the SFV T2 was expressed as a recombinant protein in transfected COS cells, the majority of the T2 protein produced was secreted into the medium as a heterogeneous species that migrated on SDS-polyacrylamide gels at an estimated size of 58 kDa (Smith et at, 1991) . Since the myxoma T2 has a very similar N-terminal signal sequence, and also has several potential N-linked glycosylation sites, we anticipated that any expressed T2 protein would . also be secreted into the medium as a highly modified species . Previously it has been shown that several strains of myxoma virus do cause secretion of several polypeptide species from infected cells (Pennington et at, 1982) and so it was of interest to carefully examine the protein secretion profile for the parent virus in this study (Lausanne) and its T2-minus derivative . To label secreted proteins, infected cells were exposed to S35 -labeled methionine plus cysteine for 1 hr, and the labeled medium was replaced with cold medium for 1 hr . Any labeled polypeptide species that



376

UPTON ET AL . 1 GTCGACAGTTCCGAATCTCTCATTCGAGCTATGTTGCGTCACGACAGTTACATCACCGAG 61

GCCTGCGCCATCATTGGCTTATTGGCGCGGGTCGCCGAGTACTGGGGGTACGACGATCCT

121

ATCCCCACCTCGGTGAGAGTGATCGAGTTGTTCATGAGATTGATCACCGACTACGAGGTC

181

CCGCATCTCAGGTCCGTGATTAATATTCGTTTACAACGCCTTTGCTCTCAGGAACGTCGT

241

GACGACTAATTGGCTCCTCTCTAATTCGGCTCCTAATTCGGCTCCTCTCTAATTCGGCTC I I II 2---- II 3 II 4 ____ I

301

CTAATTCGGCTCCTAATTGGCTCCTCTCTAATTCGGTTTTTATCTCTCTAATTGGCATCT I 5____II 6 II 7 II 8 1

361

CTAATTGGCACCTTCTACGTCTTTTTCTCTTCGTGTAAAATATAAAAATAACTACGGTAT 1 9 1

421

ATGTATCATGTTTCGTTTAACGCTACTACTCGCGTACGTCGCGTGCGTATACGGGGGCGG M F R L T L L L A Y V A C V Y G G G

481

TGCCCCGTATGGCGCGGATCGAGGAAAATGTAGAGGGAACGACTACGAAAAGGACGGACT A P Y G A D R G K C R G N D Y E K D G L

541

GTGTTGTACCTCCTGTCCTCCCGGGTCGTACGCCTCTAGGTTATGCGGACCCGGGTCCGA C C T S C P P G S Y A S R L C G P G S D

601

CACGGTATGTTCTCCGTGCAAGAACGAAACCTTTACGGCGAGTACGAACCACGCTCCCGC T V C S P C K N E T F T A S T N H A P A

661

GTGCGTAAGTTGTCGAGGGCGGTGCACAGGCCACCTATCCGAGTCTCAATCGTGTGATAA C V S C R G R C T G H L S E S Q S C D K

721

AACCCGCGATAGAGTCTGCGACTGTTCTGCGGGGAACTATTGTCTGTTGAAAGGACAGGA T R D R V C D C S A G N Y C L L K G Q E

781

GGGGTGTAGGATATGCGCTCCCAAAACGAAGTGTCCCGCGGGGTATGGCGTCTCCGGACA • C R I C A P K T K C P A G Y G V S G H

841

TACGCGTACGGGCGACGTGCTCTGCACAAAATGTCCTCGGTACACGTATTCCGACGCCGT T R T G D V L C T K C P R Y T Y S D A V

901

ATCCTCCACGGAGACGTGTACCTCGTCGTTTAACTACATCAGCGTGGAATTCAACCTATA • 5 T E T C T S S F N Y I S V E F N L Y

961

TCCCGTAAACGACACGTCTTGTACGACGACCGCCGGACCCAACGAAGTGGTTAAAACGTC • V N D T S C T T T A G P N E V V K T S

1021

GGAGTTCTCGGTTACGCTAAATCACACGGATTGTGATCCCGTCTTCCACACGGAATACTA • F S V T L N H T D C D P V F H T E Y Y

1081

CGGAACGAGCGGCAGCGAGGGCGCGGGAGGATTCTTCACCGGGATGGATAGGTACCAGAA • T S G S E G A G G F F T G M D R Y Q N

1141

TACGACCAAAATGTGTACGCTTAATATAGAGATACGGTGCGTCGAGGGAGACGCCGTGCG T T K M C T L N I E I R C V E G D A V R

1201

TACTATACCGAGGACGAGCGACGGGGTCGGCGTCCTATCTCATTCGGAAACGATTACCGT T I P R T S D G V G V L S H S E T I T V

1261

GATAGGAGGGTGCCTGTCCGACGTGAACGTAGATATCGAGTACAGCGACAGTAATCATCC I G G C L S D V N V D I E Y S D S N H P

1321

CGAGGAGGTCGACGACTTCGTGGAATACCATTGGGGTACACGCCTCCGTCTCTTTCCCTC E E V D D F V E Y H W G T R L R L F P S

1381

ACCCAAACGATGTAGACTCGTTTCATAGATTACGGATTTTCTTCTAGTTAAATTCTTAAA • K R C R L V S

1441

AAAAAGTCGAATTATAATAAAACGTGGGCGTATAGAAGAACTCTATCATGAAACGCCTGT

1501

GTGTATTATTCGCGTGCCTGGCCGCGACCCTCGCGACGAAGGGCATCTGCAG 1552

FiG . 3 . The DNA sequence (1552 nucleotides) of the Ss/l-Pstl region indicated in Fig . 1 . Translation of the myxoma T2 ORF is shown below the DNA sequence . A series of nine tandem-related DNA repeats that have no counterparts in SFV are underlined upstream of the myxoma T2 ORF . The MRV T2 sequence was identical to that of myxoma .



MYXOMA VIRUS ENCODES TNFR-HOMOLOG

37 7

1 MFRLTLLLAYVACVYGGGAPYGADRGKCRGNDYEKDGLCCTSCPPGSYAS

1 :II

II

:I

:III : :

ll :

:

III

I :IIIIIIIII

II

II

50

MYX

50

SFV

III

1 MLRLIALLVCVVYVYGDDVPYSSNQGKCGGHDYEKDGLCCASCHPGFYAS

51 RLCGPGSDTVCSPCKNETFTASTNHAPACVSCRGRCTGHLSESQSCDKTR 100

1111111 :111111

: :IIIIIIIIIIIIIIIII

I!Ilillll

MYX

II :I :

51 RLCGPGSNTVCSPCEDGTFTASTNHAPACVSCRGPCTGHLSESQPCDRTH 100

SFV

101 DRVCDCSAGNYCLLKGQEGCRICAPKTKCPAGYGVSGHTRTGDVLCTKCP 150

MYX

1111 , 11

111IM1111111

11

11

Ill

101 DRVCNCSTGNYCLLKGQNGCRICAPQTKCPAGYGVSGHTRAGDTLCEKCP 150

SFV

151 RYTYSDAVSSTETCTSSFNYISVEFNLYPVNDTSCTTTAGPNEVVKTSEF 200 MYX

III

:I

II

lillll :lllllll :llllllll

I

III :II

II

151 PHTYSDSLSPTERCGTSFNYISVGFNLYPVNETSCTTTAGHNEVIKTKEF 200

SFV

201 SVTLNHTDCDPVFHTEYYGTSGSEGAGGFFTGND)RYQNTTKMCTLNIEIR 250

MYX

IIII

IIIIIIIIIIII :III

IIIIIIIII

111111 :1111 :11

201 TVTLNYTDCDPVFHTEYYATSGKEGAGGFFTGTDIYQNTTKVCTLNVEIQ 250

SFV

251 CVEGDAVRTIPRTSDGVGVLSHSETITVIGGCLSDVNVDIEYSDSNHPEE 300

MIX

I

III

: :I :

:I

:I

:

:

1111111 :I :IIIIIIIII

III

lil :l

251 CSEGDDIHTLQKTNGG-STMPHSETITVVGSCLSDVNVDIMYSDTNHPGE 299 301 VDDFVEYHWGTRLRLFPSPKRCRLVS 326

IIIIIIIIIIIII :II

IIII

SFV

MYX

II

300 VDDFVEYHWGTRLRFFPLPKRCTPVS 325

SFV

Fie . 4. Alignment of the myxoma T2 and SFV T2 ORFs . Vertical lines indicate identity and semicolons show conservative amino acid changes . The two protein sequences are 7696 identical .

were secreted during the 1-hr chase were analyzed by PAGE . As shown by the autoradiogram in Fig . 7, the profile of secreted proteins from wild type virus-infected cells contains multiple species at early (lane 1) and late (lane 2) times that are dramatically different from uninfected cells (lane 7) . Experiments with the parental myxoma virus tagged with the ,e-galactosidase cassette at an innocuous site (vMYX-lac) showed an identical secreted protein profile (data not shown) . Since the T2 gene is expressed from an early promoter, it was anticipated that, if T2 were secreted from virus infected cells in a fashion similar to that observed in

transfected COS cells, there should be a difference in the early proteins secreted from vMYX-T2gpt-infected cells (lane 3) but not necessarily at late times (lane 4) . As can be seen by comparing the early profiles in lanes 1 and 3, it is clear that a diffuse band of the expected size (52-56 kDa) is, in fact, missing from cells infected with the vMYX-T2gpt mutant and no such alterations are observed with an unrelated deletion mutant (vMYX-GF7 SM-11) at a site just outside the viral TIR (lanes 5, 6) . We conclude that the T2 protein in myxoma is expressed and secreted from virus-infected cells at early times of infection and that the

5' CTAATT .000TCCTCT CTAATTCGGCTC CTAATTCGGCTCCTCT CTAATTCGGCTC CTAATTCGGCTC CTAATT .000TCCTCT CTAATTCGGtTttTaTCTCT CTAATT .000atCT CTAATT .GGCaCCT .TCT ACGTCTTTTTCTCTTCGTGTAAAATATAAAAATAACTACGSRATATGTATC

. . .

FIG. 5 . DNA sequence upstream of myxoma T2 gene . The nine repeat elements (see Fig . 3) have been aligned: gaps in the alignment are shown as () and nucleotides that do not fit the consensus are shown in lowercase . The first methionine codon of the myxoma T2 reading frame is underscored .



3 78

UPTON ET AL .

MYX T2 SFV T2 TNFR I TNFR II W C22L W A53R MYX

T2

c

K R G K C • G T C R L V C • Q K

C

S

N D H D R E G K

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P C P C S C

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A S E N • L - R H C S R N N • L - P A C T+N+P Y S - N G C

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TNFR II

G



C22L

E

C C



A53R

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SFV T2 TNFR I TNFR II W C22L W A53R

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G G G

C

P G S D T P G S N T K T S D GPGQD T

P

R G R-- C-- T G H L S E S Q • P-- C-- T G H L S E S Q S R- C-- S S D Q V E T Q • --- C R K E M G 0 V E I S • R R ++ + + + + + + + + D • ---L41--P T G S F D K V

C C C C .Q

T R D R V

C C C C C -Q-

C A P K T K C C A P Q T K C C A P L R K C C L - - - C C • S Q T K C C • P K R RC -



- P A G

Y G V

-

Y F T Y Y

P A G R P G L N G G I G P C C

G G G F

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G N D N

C • K P C • R A C T R S C T V S C • T K £ T G S

A V S S T-

E T C T S S

F N Y

T E D T T

H Q R H Q

G H T R T G

G H T R A G V ARP G T E T S V • L S C Q E K Q 3L • ------ G G-- G I D E Q G

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Y S D S L S P T- E R C G T S F N Y I S V

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D N D N N

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N L Y P V N D T S T T T A G F N L Y P V N E T S E T T T A G E F L R N E C V S C S N--- CA -- K K S L- - - - E A T K-Y S H T V S S A - D K,.EPVPBNTF N Y I DA E I N L Y P V N D T s[A A T R TTTTG Y C D Y L R N Y R L D P F P P C K L S KM N

R Y T Y S

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G • Y A S R L C G G F Y ASR L C • G • H A K V F C T K G T Y L Y N D C • G T Y A S R L C S E F K V RC •

• S K T N

C R I C R I A L-- S K Q E C R L R H Y W S E N L F Q C F N • L-- K G S S G C • A A T D S S Q T E D • N

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FiG. 6 . Alignment of homologous regions of myxoma T2, SFV T2 (Upton et at.., 1987a), TNFR I (Smith et al., 1990a), TNFR II (Loetscher et al., 1990 ; Schall etal., 1990), and two related ORFs from vaccinia virus strain Copenhagen (ORF C22L (Goebel et al., 1990) and OAF A53R (Goebel et al., 1990 ; Howard et al., 1991)) . For comparative purposes, both of the vaccinia virus ORFs shown in this diagram have been juxtaposed to link downstream mini-ORFs which have been segregated by frame shifts between the + symbols and a STOP codon (`) . The presented sequences start at amino acid number 27 (myxoma T2) ; 27 (SFV T2) ; 39 (TNFR I) ; 43 (TNFR II) ; 36 (W ORF A53R) ; 31 (VV ORE C22L) . Boxed amino acids indicate high conservation of residues between the four repeats . Gaps introduced into the sequences are shown as '-' .

secreted form is highly modified, probably by glycosylation, into a heterogeneous species of around 5256 kDa . The T2-minus myxoma virus is significantly attenuated in rabbits In order to ascertain the affect of the T2 deletion on the disease course caused by the mutant virus, immunocompetent adult New Zealand White rabbits were injected intradermally with infectious virus and the rabbits were monitored for the well-established clinical symptoms associated with myxomatosis . Rabbits infected with vMYX-lac parental virus routinely succumb within 10-11 days to the progressive viral infection at multiple secondary sites, both internal and external, as has been described for the classic virulent strains of myxoma virus (Fenner and Ratcliffe, 1965) . However, the eight rabbits infected with vMYX-T2gpt demonstrated considerably moderated symptoms in that primary and secondary tumor sizes were consistently less and the extent of supervening Gram negative bacterial infections was reduced (Table 1) . Although three of the rabbits showed increasing severity of symptoms by Day 10 and were sacrificed, five animals were only moderately afflicted and able to completely clear the infection . These survivors were totally recovered within 4 weeks and became immune to

challenge by wild type myxoma virus . Although detailed immunopathology remains to be conducted, it is tempting to speculate that because the rabbits are not genetically inbred and may very well show considerable polymorphism at MHC loci which determine TNFa and )4 expression, the heterogeneity observed in the response to the mutant virus infection may reflect the inherent variability of TNF responses in the different individuals . It will be of interest to determine if the ability to elaborate TNF by a given individual host can be correlated with the capacity to recover from infection by T2-minus myxoma . In any event, it is clear that the T2 gene in myxoma is a potent virulence factor for the virus in the rabbit host, and its deletion results in attenuation of the disease syndrome by compromising the ability of the virus to deflect the TNF-dependent aspects of the immune response .

DISCUSSION Poxviruses of vertebrate hosts can induce a wide spectrum of disease symptoms ranging from minor lesions that are restricted to the site of inoculation right up to lethal systemic infections that are rapidly invasive and overwhelm the host immune response (reviewed by Turner and Moyer, 1990 ; Buller and Palumbo, 1991) . Although the precise number of viral genes that are devoted to the problem of circumventing the differ-



MYXOMA VIRUS ENCODES TNFR-HOMOLOG 1

2

3

4

5

6

7

Secreted proteins from myxoma-infected cells . Cultures labeled with [S'slcysteine plus methionine as described under Materials and Methods and chased for 1 hr at early or late times of infection . Labeled protein secreted during the chase was concentrated and analyzed on a 7-15% polyacrylamide gel . Lanes 1 and 2, myxoma, early/late ; lanes 3 and 4, vMYX-T2gpt, early/late ; lanes 5 and 6, vMYX-GF - AM-11, early/late ; lane 7, mock-infected, The arrow indicates, in lanes 1 and 5, the predicted myxoma T2 protein that is absent from the vMYX-T2gpt mutant, lane 3 . Positions of marker proteins detected by Coomassie staining are indicated by bars ; 97 .4, 66 .2, 45 .0, 31 .0, 21 .5, and 14 .1 kDa . FIG. 7 .

were

ent elements of cellular and humoral immunity is still unknown, studies on near-terminal deletion mutants suggest that in many poxviruses at least 30 kb of sequence information at or near the viral TIR is not needed for replication in tissue culture and that many of the genes that map in this region are presumably devoted to some aspect of propagation in animal hosts . For example, in the case of vaccinia virus, up to 55 open reading frames near the viral termini can be eliminated without loss of infectivity in tissue culture (Perkus, et at, 1991), suggesting multiple poxviral gene products have evolved whose function will be to either dampen or directly intercept host-directed antiviral responses . The possibility that TNF might be one such target for poxviral-specific countermeasures was first suggested by the observation that the deduced primary sequence for one of the recently cloned cellular receptors for TN F bore striking similarity with the T2 OFF of SFV (Smith et at, 1990a) . Even more strikingly, the SFV T2 protein,

379

when expressed in transfected COS cells, could be shown to be specifically secreted into the medium and could bind extracellular TNFa and $ with high affinity (Smith et at, 1991) . TNF is known to be a principal mediator of inflammation, an important regulator of cellular immunity and, along with the interferons, is a critical element in mediating the cellular antiviral response (Wong and Goeddel, 1986 ; Rosenblum and Donato, 1989 ; Larrick and Wright, 1990) . The importance of TNF-dependent pathways in combatting viral infection is illustrated by the fact that adenoviruses utilize an E3 protein (14,7 kDa) to specifically combat TNF cytolysis (Wold and Gooding, 1989 ; Gooding et at, 1988) . In view of the potential importance of TNF responses to poxviral infections, we sought to identify T2-related gene products in two poxviruses, myxoma and MRV, that have been successfully adapted to evading host antiviral responses and cause systemic lethal infections . In this communication we show that these two viruses encode an identical T2 gene, with over 80% homology to the SFV T2 version, that possesses all of the structural motifs predicted for secretion and TNFbinding . When both copies of the T2 gene of myxoma are inactivated with a selectable Eco-gpt marker, the resultant virus is deficient at secretion of a heterodisperse 52-56 kDa species, but is otherwise normal in terms of growth and replication in tissue culture cells . When the disease profile of the mutant virus was studied in rabbits, the primary and secondary lesions were less extensive due to an apparently more vigorous immune response and the majority of infected rabbits were able to completely clear the viral infection . A minority of rabbits still succumbed to the disease, however, suggesting that not all of the rabbits in this particular strain (NZW) are equally capable of producing TNF in response to myxoma infection, and hence cannot combat even a crippled T2-minus variant . It is clear from these data that T2 protein is an important virulence factor in myxoma virus and underscores the importance of studying pathogenic poxviruses with a defined host disease syndrome . Vaccine virus is a prototype Orthopoxvirus that encodes a variety of gene products that are secreted and can be shown to ameliorate virulence in experimentally infected animals (Buller et al., 1988 ; Kotwal et al., 1989), but this virus has been selected for efficient propagation in tissue culture and undoubtedly has lost pathogenic markers due to the absence of selective pressures incurred by propagation in a living host . Thus, although vaccinia encodes at least two ORFs with discontinuous homology to TNF receptors (Howard et at, 1991 ; Fig, 6, this paper) the presence of frameshifts and stop codons makes expression of bona fide TNF-binding protein by

UPTON ET AL .

380

TABLE 1 OBSERVATIONS OF MYXOMA AND MVXOMA T2 - MINUS INFECTED RABBITS

Day

vMYX-lac (wild type)

vMYX-T2gpt (T2 mutant)

0

Two adult (3 kg) female NZW rabbits inoculated id in two sites with 5 X 10 2 PFU/site myxoma virus . Primary lesions at inoculation sites, approx . 1 .5 cm, soft, flat; no other symptoms . Primary lesions approx . 3 cm, soft in center, flat, hemorrhagic ; multiple secondary lesions ; swollen eyes ; moderate bacterial infection of nasal and conjunctival mucosa .

Eight adult (3 kg) female NZW rabbits inoculated id in two sites with 5 x 10' PFU/site vMYX-T2gpt virus . Primary lesions at inoculation sites, approx . 0 .5-1 .0 cm, hard, raised; no other symptoms . Primary lesions 1-2 cm, hard, raised, slightly hemorrhagic and flattening in center . Some secondary lesions ; eyes somewhat swollen ; slight to moderate bacterial infection of nasal and conjuctival mucosa . Primary lesions approx. 2 cm, raised, hard, necrotic ; multiple secondary tumors on 5096 of animals; mild to severe bacterial infections . Three animals sacrificed due to increased severity of symptoms . Five rabbits recovering : primary and secondary lesions drying up and scabbing over ; bacterial infections moderate to absent. Five rabbits recovering : all lesions scabbed over ; all animals recovered from bacterial infections . Five rabbits completely recovered : small scars remain from primary and secondary lesions ; no further signs of infection remain

4 7

10

Primary lesions approx. 4 cm, necrotic, flat ; multiple secondary lesions ; moderate to severe bacterial infections . Animals sacrificed due to increasing severity of symptoms .

14 21 31

these strains of vaccinia unlikely . It would be of interest to know if a more virulent relative of vaccinia, such as variola virus, might retain these particular genes in intact form and thereby contribute to the invasive and progressive nature of smallpox in humans . The apparent acquisition of cellular receptor analogs by poxviruses represents a new strategy for viral virulence . Kotwal and Moss (1988b) have proposed the term "virokine" to describe secreted viral proteins which either mimic cellular lymphokines or regulators of immune response pathways . We propose the term "viroceptor" to describe viral analogues of immune receptor molecules where the function is to intercept and short circuit antiviral immune response pathways dependent on the normal functioning of the cognate cytokine . Note that there is no formal requirement that a viroceptor be secreted, and thus be a member of the secreted virokine family, because a membrane-bound viroceptor could also function to intercept a lymphokine so long as it was not coupled to the appropriate signal transduction apparatus which transmits the lymphokine signal . Given the current knowledge of multiple antiviral pathways in vertebrate hosts, we would predict the existence in the most pathogenic poxviruses of multiple viroceptors, each targeted for a different pathway dependent on lymphokine-receptor interaction . For example, the recent discovery of a vaccinia analog for the II-1 receptor (Smith and Chan, 1991) suggests that more viroceptors remain to be discovered in poxviruses capable of compromising immune function . Indeed, it is likely that the highly virulent sys-

temicc poxviruses, such as myxoma, have been particularly adapted to target and intercept many, if not all, of the host antiviral pathways and that analysis of other secreted proteins will shed light on the relative efficacies of these different pathways in clearing other viral infections as well . ACKNOWLEDGMENTS We thank C . Smith (Immunex) for helpful discussions on homologies between TNFRI and T2, G . Smith and E. Paoletti for communicating sequence information of the vaccinia TNFR homologues prior to publication, A . Opgenorth for vMYX-lac and vMYX-GF-AM-11, C . Macaulay for plasmid p7 .5gptA, Perry D'Obrenan and Rita Whitford for DNA sequencing, and Shari Kasinec and Vicki Luxton for help with the manuscript . G .M . is a Medical Scientist of the Alberta Heritage Foundation for Medical Research . This work was supported by the National Cancer Institute of Canada . REFERENCES D . G ., and WALLACH, D . (1985) . Increase of vulnerability to lymphotoxin in cells infected by vesicular stomatitis virus and its further augmentation by interferon . Cell Immunol. 92, 218-225 . ASOH, K ., WATANABE, Y ., MIZOGUCHI, H ., MAWATARI, M ., ONO, M ., KoHNO, K ., and KuwANo, M . (1989) . Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF-7 cell line and its TNF-resistant variant . Biochem. Biophys. Res. Comm. 162(2), 794-801 . BALKINILL, F. R. (1989) . Tumor necrosis factor. Br. Med. Bull. 45(2), 389-400 . BEUTLER, B ., and CERAMI, A . (1988) . The history, properties, and biological effects of cachectin . Biochemistry 27(20), 7575-7582 . BEUTLER, B ., and CERAMI, A . (1989). The biology of cachectin/TNFa ADREKA, D ., NOVICK, D ., HAHN, T ., FISCHER,



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Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence.

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for gr...
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