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Volume 270, Number 7, Issue of February 17, 1995 pp. 3031-3038
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Myxoma Virus-soluble Interferon- Receptor Homolog, M-T7, Inhibits Interferon- in a Species-specific Manner (*)

(Received for publication, September 2, 1994; and in revised form, November 22, 1994)

Karen Mossman (§), ,  Chris Upton (¶), ,  Grant McFadden (**)

From the Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The myxoma virus M-T7 protein contains significant sequence similarity to the ligand binding domain of the mammalian interferon- receptors, and functions as a soluble homolog which can bind and inhibit the biological activities of rabbit interferon- (Upton, C., Mossman, K., and McFadden, G.(1992) Science 258: 1369-1372). M-T7, the most abundantly secreted protein from myxoma virus-infected cells, was shown to be expressed in significant biological amounts as a typical poxvirus early gene product, efficiently secreted at early times of infection to levels that exceed 5 times 10^7 molecules/cell, and function as a stable inhibitory protein in infected cell supernatants until late times of infection. M-T7 was specific in binding and inhibiting rabbit interferon-, and did not bind either human or murine interferon-. Scatchard analysis of rabbit interferon- binding curves yielded a single high affinity binding site on M-T7, with a K of 1.2 times 10M, which is comparable to the affinity between soluble forms of cellular interferon- receptors and their cognate ligands. In comparison, rabbit interferon- was shown to bind its cellular receptor with a K of 5.9 times 10M, again comparable to the affinity of membrane bound forms of other mammalian interferon- receptors for interferon-. Thus, the myxoma virus M-T7 protein is a functional soluble interferon- receptor homolog which binds and inhibits interferon- with high affinity in a species-specific manner.

INTRODUCTION

Interferon- (IFN-) (^1)is a potent immunomodulatory cytokine primarily produced by activated T lymphocytes and natural killer cells(1) . While IFN- was discovered by virtue of its anti-viral activities, it also serves critical functions as an immunoregulator in the presence and absence of pathogenic challenge(2, 3) . IFN- exerts pleiotropic effects on the immune system through ligand-dependent activation of the IFN- receptor (IFN-R)(3) . The human and murine IFN-Rs have been extensively characterized, and are known to bind IFN- with high affinity in a species specific manner (4, 5) . The known mammalian IFN-Rs are composed of two subunits, denoted alpha and beta. The IFN-R alpha chain possesses an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain devoid of any obvious kinase or phosphatase motifs, but containing two important sequences involved in internalization of the receptor-ligand complex and signal induction (reviewed in (3) ). The IFN-R beta chain is a type 1 transmembrane protein which confers species specificity to the IFN-R(6, 7) .

The importance of IFN- within the immune system is exemplified by the observation that IFN- is the primary cytokine mediator of innate resistance to both viral and non-viral pathogens(8, 9) . IFN- functions to combat viral infections by inducing anti-viral pathways and by modulating cellular immune responses to viral challenge. While IFN- does not itself directly inhibit viral multiplication, it induces the synthesis of a variety of effector proteins which function in inducing an anti-viral state(10, 11) . For example, PKR, a double-stranded RNA-dependent protein kinase, and 2-5 A synthase are enzymes induced by all interferons, including IFN-(12, 13) . IFN- also modulates the cellular immune response to reduce overall viral multiplication and spread by a variety of mechanisms. IFN- is a potent macrophage activation factor, resulting in the elaboration of a variety of macrophage derived cytocidal compounds, along with the production of cytolytic and pro-inflammatory cytokines, reactive nitrogen intermediates, and nitric oxide synthase, which produces the toxic compound nitric oxide(14, 15, 16, 17, 18, 19, 20) . Aside from the activation of macrophages, IFN- possesses the ability to enhance the expression of major histocompatibility complex class I and II glycoproteins, resulting in an increase of viral antigen presentation(21, 22, 23) . Furthermore, IFN- induces the secretion of the ligand binding domain of the low density lipoprotein receptor, which interferes with assembly and budding of certain enveloped viruses(24) .

To overcome the anti-viral and immunoregulatory effects of IFN-, many viruses have evolved both extracellular and intracellular anti-interferon strategies(25, 26, 27, 28, 29) . Poxviruses, a family of large, double-stranded DNA viruses which replicate within the cytoplasm of host cells(30) , were the first viruses found capable of interrupting the extracellular ligand-dependent triggering of the IFN-Rs, thus preventing signal transduction from an extracellular location (31) . In particular, the Leporipoxvirus myxoma virus was found to express a soluble IFN-R homolog, denoted M-T7, which has the ability to bind and inhibit the anti-viral activities of rabbit IFN-(31) . Here, we investigate the secretion and inhibitory properties of M-T7, the myxoma soluble IFN-R homolog, in an attempt to further understand the role this viral protein plays in combating IFN- during the establishment of a virus infection.

MATERIALS AND METHODS

Viruses and Cells

vMyxlac, the myxoma virus (strain Lausanne) derivative, has been previously described(32) . Vesicular stomatitis virus (VSV) was a generous gift of D. Tovell. RK13 cells, a rabbit kidney cell line, and BGMK cells, a primate cell line, were maintained in Dulbecco's Minimal Essential medium with 10% newborn calf serum. Mouse L929 cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum. All cells were supplied by the ATCC.

Purification of Myxoma M-T7 Protein

BGMK cells were infected with vMyxlac virus at a multiplicity of infection (m.o.i.) of 3. After extensive washing with phosphate-buffered saline (PBS) to remove unabsorbed virus and serum, serum-free medium was added, harvested 4 h later (early supernatant), replenished with serum-free medium, and re-harvested 16 h post-infection (late supernatant). Early and late supernatant samples were pooled and subjected to ammonium sulfate fractionation, and proteins that precipitated between 30 and 50% ammonium sulfate, which contained the bulk of M-T7, were collected. Precipitated proteins were resuspended in 20 mM Bis-Tris, pH 6.0, loaded on a Q Sepharose Fast Flow column (Pharmacia Biotech Inc.), and eluted with a 0-300 mM sodium chloride gradient, in which M-T7 eluted in a broad peak between 150 and 180 mM sodium chloride. Fractions containing the M-T7 protein were identified by SDS-PAGE, pooled, precipitated with 50% ammonium sulfate, and desalted using an Econopac 10-DG column with 20 mM Hepes, pH 7.9, and 1 mM EDTA buffer. The resulting M-T7 protein preparations were stored at 4 °C.

Analysis of Peptide Sequences

The Protein Identification Resource (PIR) (Release 38) and GenBank (Release 79) data bases were used to obtain the peptide sequences of the mammalian IFN-Rs and the poxviral IFN-R homologs. Peptide sequences were compared using Bestfit, PileUp, and LineUp programs (Genetics Computer Group, Madison, WI). All computer analyses were performed at the Molecular Mechanisms in Growth Control Computer Facility, University of Alberta.

Analysis of M-T7 Transcription

BGMK cells were infected with vMyxlac virus at an m.o.i. of 30, and RNA from infected cells was extracted at various time points post-infection using guanidine thiocyanate, as described previously(33) . For Northern blotting analysis, 3 µg of purified viral RNA was prepared in MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA) containing 50% deionized formamide and 20% formaldehyde. Samples were incubated at 55 °C for 15 min, cooled on ice, and loaded onto a 1% agarose gel containing 1 times MOPS buffer, 2% formaldehyde, and 0.5 µg/ml ethidium bromide. RNA was transferred to nitrocellulose via capillary action. For a probe, random priming was performed using an 880-bp M-T7 PCR product with [alpha-P]dCTP (Amersham Corp.) and random primer (Boehringer Mannheim). Results were visualized with autoradiography.

For primer extension analysis, a 22-base oligonucleotide primer, (5`-AAGTCGTAGGACGTAAGGCGTA-3`), was end-labeled using [-P]ATP (ICN) and T4 kinase (Life Technologies, Inc.). The primer was constructed to complement the M-T7 coding sequence, with its 3` end residing 52 bases downstream of the initiating ATG. Primer extensions were performed as described in Sambrook et al.(34) using 5 µg of viral RNA, and Superscript reverse transcriptase (Life Technologies, Inc.). DNA sequencing was performed by adding one of four ddNTP mixtures (for example, ddATP: 1 mM each dGTP, dTTP, dCTP, 0.5 mM dATP, 0.125 mM ddATP) to annealed RNA/primer, and incubating at 55 °C for 45 min. All primer extension reactions were subjected to sequencing gel analysis and visualized with autoradiography.

Radiolabeling of Secreted Viral Proteins

BGMK cells (6 times 10^5/sample) were infected with vMyxlac virus at an m.o.i. of 30. At 1.5 h (for labeling of early viral proteins) or 8 h (for labeling of late viral proteins) post-infection, cells were washed with PBS, pulsed for 30 min with 200 µCi of [S]Cys/Met (Translabel, ICN) in 400 µl of cysteine/methionine-deficient medium (Life Technologies, Inc.), washed three times to remove unincorporated label, and then placed in complete medium. To detect labeled viral proteins secreted within the indicated 1-h time frame, complete medium was replaced with serum-free medium for 1 h prior to harvesting of labeled supernatants, with the exception of the first time point of 2.5 h post-infection, in which case serum-free medium was added for 30 min after labeling. To detect accumulated labeled secreted viral proteins, cells were infected and labeled as above, and serum-free medium was added to washed cells following labeling. Cellular supernatants containing labeled, secreted viral proteins were harvested at various time points post-infection, centrifuged at 5000 times g to remove cellular debris, subjected to SDS-PAGE analysis, and visualized by autoradiography.

Quantitation of M-T7 Protein during Myxoma Virus Infection

Following infection of BGMK cells with vMyxlac virus (m.o.i. of 30), monolayers were washed to remove unabsorbed virus, overlaid with serum-free medium, and supernatants containing secreted proteins were harvested at various times post-infection. Supernatants were centrifuged at 5000 times g to remove cellular debris, concentrated using Amicon Centriprep, subjected to SDS-PAGE analysis, and visualized by silver staining. To quantitate the amount of M-T7 produced during myxoma virus infection, M-T7 bands were scanned using a Chromoscan3 densitometer and quantitated using purified M-T7 standards. Inhibitory units were defined as the amount of M-T7 (nanograms) required to inhibit 1 unit of rabbit IFN-, based on 50% inhibition in a VSV inhibition bioassay.

Bioassay in Tissue Culture Cells

60-mm dishes seeded to confluency with RK13 cells, BGMK cells, or L929 cells were incubated overnight in medium containing purified rabbit, human, or murine IFN- (Genentech), respectively, with increasing amounts of M-T7 protein. The next day, medium was removed, and monolayers were infected with 100 plaque-forming units of VSV for 1 h, then overlaid with 1% low melting point agarose in complete medium. The number of VSV plaques were scored 24-48 h post-infection and compared with the uninhibited control, in which cells were preincubated with only control medium.

Competition Binding Assays

Rabbit, human, and murine IFN- were labeled in vitro with protein kinase A and [-P]ATP, as described previously(35) , with the exception that 5 µg of IFN- were phosphorylated at 30 °C for 30 min. For competition binding assays, 2 ng of P-labeled IFN- was incubated simultaneously with increasing amounts of unlabeled competitor IFN- and 500 ng of M-T7 protein. Complexes formed at room temperature within 1 h, at which time the cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC, from Sigma), in 100 mM potassium phosphate, pH 7.5, was added to 20 mM. The reaction was quenched 15 min later by the addition of Tris-HCl, pH 7.5, to 0.1 M, and the resulting protein complexes were analyzed by SDS-PAGE and visualized by autoradiography.

Solid Phase M-T7 Binding Assay

Falcon immunoplates were coated overnight with 50 ng of M-T7 protein in 50 µl of PBS at 4 °C. The next day, wells were blocked at room temperature for 2 h with 5% skim milk powder in PBS containing 0.1% Tween 20, followed by binding of P-labeled IFN- in blocking buffer for 1 h. Following three washes with PBS, the wells were removed and counted by Cerenkov counting. To determine the specific binding of IFN- to M-T7, binding in the presence of 100-fold excess cold IFN- was subtracted from total binding. Nonspecific binding routinely represented less than 5% of total binding. All assays were performed in triplicate. Binding of P-labeled IFN- to M-T7 was analyzed by the method of Scatchard(36) .

Binding of P-Labeled Rabbit IFN- to RK13 Cells

Binding of rabbit IFN- to RK13 cells in monolayer culture was performed essentially as previously described(37) . Briefly, 1 times 10^4 RK13 cells seeded in 24-well dishes were washed with ice-cold serum-free medium, followed by the addition of increasing amounts of P-labeled IFN- in 100 µl of serum-free medium, either alone or in the presence of 100-fold excess of unlabeled IFN-. The cultures were incubated at 4 °C with gentle rocking for 1 h, at which time the monolayers were washed three times with PBS to remove unbound P-labeled IFN-. Cells were removed with 0.15 M sodium chloride and 0.015 M sodium citrate, and the radioactivity in the samples was determined by Cerenkov counting. All assays were performed in triplicate. P-labeled IFN- binding to RK13 cells was analyzed by the method of Scatchard(36) .

RESULTS

Comparison of Poxviral IFN- Receptor Homologs

The Leporipoxvirus myxoma virus was recently shown to encode and express a 37-kDa soluble protein, designated M-T7, containing significant homology to the ligand binding domain of the human and murine IFN- receptors(31) . Shope fibroma virus (SFV), a Leporipoxvirus closely related to myxoma virus, also encodes a similar IFN- receptor homolog, designated S-T7(31) . As the genomic sequence information from a variety of other poxviruses accumulates(38, 39, 40, 41, 42, 43) , additional putative viral IFN- receptor homologs have been discovered (reviewed in (44) ). Included are the Orthopoxvirus vaccinia virus (B8R) and variola virus (B8R in strain Bangladesh-1975 and B9R in India-1967) (38, 41, 43) proteins, and the Suipoxvirus swinepox virus C6L protein(40) . Fig. 1illustrates the amino acid alignment of the five known poxviral IFN- receptor homologs with the two known mammalian IFN- receptors. Although the overall identity scores between the poxviral proteins and the ligand binding domain of the mammalian IFN-Rs is low (20-25%), it is important to note the conservation of cysteine residues, which for the mammalian IFN-Rs have been shown to be essential for ligand binding by stabilizing disulfide bonds(45, 46) . As highlighted in Fig. 1, the myxoma M-T7, SFV S-T7, and swinepox C6L proteins contain all 8 conserved cysteine residues, while the vaccinia and variola B8R proteins have the first 2 cysteine residues replaced with tyrosine residues, while a single cysteine residue is situated at a unique site four amino acids NH(2)-terminal to cysteine 3 (Fig. 1). Note that all of the poxviral proteins lack transmembrane domains and are predicted to be secreted proteins. Thus, the poxviral IFN- binding proteins are uniformly shorter than their mammalian receptor homologs and possess neither hydrophobic membrane-spanning domains nor cytoplasmic signaling domains required to initiate an IFN- signal transduction event.

Figure 1: Alignment of soluble poxviral IFN- receptor homologs with the cellular mammalian IFN- receptors. Amino acid sequence alignment of myxoma virus M-T7 (31) with the peptide sequences of four putative poxviral IFN-R homologs, Shope fibroma virus S-T 7(63) , swinepox virus SPV C6L(40) , vaccinia virus B8R (VV-B8R)(64) , and variola virus Bangladesh-1975 B8R (VAR-B8R)(41) , and the cellular murine (MuIFNR) (5) , and human (HuIFNR) (4) IFN-Rs. Boxes indicate amino acid identity among all proteins, while the asterisk (*) denotes amino acids which are conserved in at least four of the seven proteins. The disulfide-forming cysteine residues conserved between the mammalian and viral proteins are both boxed and numbered 1-8. The arrow indicates the location of the NH(2)-terminal residue, determined by sequencing, of the mature secreted myxoma M-T7 protein, while the predicted transmembrane domains of the mammalian IFN-Rs are underlined. The full-length of the human and murine IFN-Rs are 489 and 477 amino acids, respectively. Accession numbers (EMBL Data Library) for the receptors are myxoma (M81919), swinepox (L22013), vaccinia strain Copenhagen (M35027), variola strain Bangladesh 1975 (L22579), human (A31555), and murine (M26711).


M-T7 Is Expressed from a Relatively Stable mRNA from an Early Poxviral Promoter

The myxoma 37-kDa M-T7 protein was previously observed to be the major viral protein detected in medium harvested from infected cells at both early and late times of infection(31) . Since some poxviral proteins, such as the 7.5-kDa polypeptide of vaccinia, are expressed constitutively during infection due to the presence of overlapping early and late promoters(47) , it was of interest to examine the kinetics of M-T7 expression. Northern blotting analysis, using a radiolabeled M-T7 probe, demonstrates that a single M-T7 mRNA transcript of approximately 1.1 kb from myxoma virus-infected cells peaks at early times (Fig. 2, lanes 2 and 3), but is still detectable at late times (lanes 4 and 5). Primer extension analysis confirms the results of the Northern blot analysis, i.e. a defined extended product can be clearly detected with RNA collected at early times post-infection (Fig. 3a, lanes 3 and 4), but is still observed at late times post-infection (lanes 5 and 6). By combining the primer extension reaction with dideoxy sequencing, we mapped the 5`-initiation sites of the M-T7 transcript to a sequence motif with all the elements of a strong poxviral early promoter (Fig. 3, b and c). In contrast, the highly conserved TAAAT motif characteristically observed in most poxviral late promoters (48) is absent. Vaccinia virus early promoters (Fig. 3c) typically include a 16-base pair (bp) critical region, followed by a T-rich 11-bp spacer and a 7-bp initiation region, where transcript start sites are clustered at purine residues(49) . The M-T7 critical region is highly homologous to the critical region experimentally engineered in vaccinia virus (49) to be substantially stronger than natural early promoters (Fig. 3c). Transcription of M-T7 predominantly initiates at a single purine (adenine) 10 nucleotides upstream of the ATG, although three surrounding bases are also used in initiation, to a lesser extent (refer to Fig. 3, b and c). Furthermore, no evidence of a 5`-poly(A) leader, characteristic of poxviral late mRNAs (48) , was detected when primer extensions were performed with mRNA collected at early or late times (data not shown). Thus, the abundance of the M-T7 protein secreted throughout myxoma virus infection is not due to constitutive transcription, but rather because M-T7 mRNA is expressed early as a relatively stable viral message that is still detectable at late times of infection.

Figure 2: Northern blot analysis of M-T7 transcripts from myxoma virus infected cells. RNA from mock infected BGMK cells (lane 1) and myxoma virus (multiplicity of infection = 30) infected cells harvested at the indicated times post-infection (lanes 2-5) was subjected to Northern blot analysis using a radiolabeled M-T7 probe, as described under ``Materials and Methods.'' RNA marker sizes are indicated to the left.


Figure 3: Primer extension analysis of the 5` end of the M-T7 gene. a, hybridization of a P-end-labeled oligonucleotide to RNA from mock infected cells (lane 2) and myxoma virus infected cells harvested at the indicated times post-infection (lanes 2-6). Size markers at the left are HinfII cut X174. b, dideoxy sequencing reaction using the same oligonucleotide primer as in A, using RNA harvested 4 h post-infection. Lane 2 is a control extension reaction in which no dideoxynucleotide is used, while the dideoxynucleotide used in each remaining reaction is indicated above the figure (lanes 3-6). Sequence of the extended product (non-coding strand) is illustrated on the right, with the asterisk (*) indicating the intensity of the run-off positions. The boxed nucleotides correspond to the ATG of the coding strand. c, promoter region upstream of the M-T7 start site (see text for details). Procedures are described under ``Materials and Methods.'' The vaccinia consensus motifs are from Davison and Moss(49) .


M-T7 Is Efficiently Secreted as a Stable Soluble Protein

Since M-T7 is expressed from a typical early poxviral gene, and yet is the major secreted protein observed in supernatants harvested at both early and late times of a myxoma virus infection (31) , we next studied the kinetics of protein secretion from myxoma virus-infected cells. Both the kinetics of M-T7 secretion and the stability of the secreted M-T7 protein were assayed by infecting cells with myxoma virus, pulse labeling with [S]Cys/Met, and then following the fate of the labeled M-T7 protein. To determine the kinetics of M-T7 protein secretion during myxoma virus infection, we labeled proteins 1.5-2 h post-infection, when M-T7 mRNA transcripts are abundant (refer to Fig. 2), then followed the secretion of proteins translated within this labeling period. Fig. 4a demonstrates that the transit time for M-T7 is greater than 30 min (lane 2), but less than 4 h (lane 4). Within 4 h of translation, almost all of the M-T7 has been secreted (compare lanes 3 and 4). Cellular supernatants from myxoma virus infected cells which had been labeled with [S]Cys/Met at 8 h post-infection, washed, and then harvested 2 h later, confirmed that M-T7 is secreted at early times, and although M-T7 mRNA is still readily detectable at these times (Fig. 2), secreted protein is not (Fig. 4a, lane 7). Analysis of the levels of total accumulated proteins following myxoma virus infection (Fig. 4b) demonstrates no significant reduction in the levels of intact M-T7 protein, indicating that the soluble mature form of M-T7 is particularly stable. Thus, we conclude that M-T7 is expressed as an early gene product which is secreted within 4 h of translation, but persists as a stable soluble protein well into late times of infection. Indeed, we have been unable to detect any significant degradation of mature M-T7 protein even after several days at 37 °C in unfractionated supernatants (data not shown).

Figure 4: Kinetics of viral protein secretion from myxoma virus infected cells. a, S-labeled proteins secreted from mock infected cells (lane 1) and myxoma virus-infected cells (lanes 2-7), were measured as outlined under ``Materials and Methods.'' At various times post-infection, proteins secreted within a 1-h time frame ending at the indicated times were harvested. All samples were labeled with [S]Cys/Met for 30 min at 1.5 h post-infection, with the exception of lane 7, where labeling was initiated 8 h post-infection. b, accumulation of S-labeled proteins secreted from mock infected cells (lane 1) and myxoma virus-infected cells at the indicated times post-infection (lanes 2-5), as outlined under ``Materials and Methods.'' The location of the myxoma M-T7 protein is indicated to the right, while the location of size markers is indicated to the left.


Quantitation of M-T7 Protein from Infected Cells

In order for M-T7 to play a significant role in the inhibition of host derived IFN-, sufficient amounts of the soluble IFN-R homolog to outcompete cellular receptors must be secreted by myxoma virus during infection. Quantitation of M-T7 secreted at various times during myxoma virus infection demonstrated that, indeed, the amounts of M-T7 secreted vastly exceeds the number of cellular IFN-Rs at the cell surface (Table 1). In accordance with the kinetic studies outlined above, the majority of M-T7 is secreted by 4 h post-infection, with a slight increase in accumulation seen until 8 h post-infection, at which time the total amount of M-T7 slowly decreases. While the amount of IFN- produced locally during viral infection has yet to be determined, the amount of M-T7 produced per cell is significant when compared to the level of IFN-Rs expressed on cell membranes. IFN- receptive cells are known to express 200-25,000 IFN-Rs per cell (3) , which even at the maximal level of expression is three orders of magnitude less than the amount of M-T7 secreted by 4 h post-infection. Furthermore, quantitation of the inhibitory potential of M-T7 demonstrated that 10^6 cells secrete sufficient M-T7 to neutralize 2000-3000 units of rabbit IFN- (Table 1). Thus, biologically significant amounts of M-T7 are produced during a myxoma virus infection.

M-T7 Is a Specific Inhibitor of Rabbit IFN-

Previously, we showed that M-T7 was extremely efficient in abrogating the antiviral state induced by rabbit IFN- on RK13 cells(31) . To determine the specificity of M-T7 binding, we pretreated rabbit, murine, and primate cell lines with sufficient cognate IFN- to induce the antiviral state, either in the presence or absence of M-T7 protein. Following pretreatment, cells were infected with VSV, and virus yield was calculated as the percentage of plaque formation compared to cells pretreated with neither IFN- nor M-T7. As shown in Table 2, as the molar ratio of M-T7 incubated with rabbit IFN- on RK-13 cells is increased, the extent of induction of the antiviral state becomes progressively inhibited, thus permitting replication of the VSV to 100% of uninhibited controls. However, with primate and murine cells pretreated with human and murine IFN-, respectively, increasing amounts of M-T7 did not decrease induction of the antiviral state, and the VSV yield in each case remained essentially unchanged. Thus, M-T7 is species-specific and can prevent the induction of the antiviral state induced by rabbit IFN-, but not human or murine IFN-.

To further confirm that the species-specific ability of myxoma M-T7 protein to inhibit the biological activity of IFN-, a series of competition binding assays, as assessed by the chemical cross-linking of M-T7 to radiolabeled IFN-, were performed. The 37-kDa M-T7 protein readily binds to P-labeled rabbit IFN-, forming a complex which migrates with an apparent molecular mass of 48-50 kDa. As shown in Fig. 5a, lane 6, M-T7bulletIFN- complexes and small amounts of higher molecular mass complexes are observed in these cross-linking assays, along with IFN- monomers, dimers, and trimers, which are commonly observed in cross-linking assays involving radiolabeled IFN-(50) . However, with the addition of increasing amounts of unlabeled competitor rabbit IFN-, only the intensity of the M-T7bulletIFN- complex decreases, and at higher cold competitor ratios IFN- trimers, which co-migrate with M-T7bulletIFN- heterodimers, become more prominent. If unlabeled human or murine IFN- are substituted as the cold competitor (Fig. 5, b and c, respectively), the amount of the labeled M-T7bulletIFN- complex does not decrease, even at 500 times molar excess. We have also been unable to detect significant amounts of cross-linked M-T7bulletIFN- complex when murine or human IFN- is used as the radiolabeled ligand (not shown). Thus, human and murine IFN- cannot compete with rabbit IFN- for binding to M-T7, further contributing to the observation that M-T7 has evolved to specifically inhibit rabbit IFN-.

Figure 5: Competition studies involving chemical cross-linking of P-labeled rabbit IFN- to the myxoma M-T7 protein. Competition assays of P-labeled rabbit IFN- to M-T7 were performed as described under ``Materials and Methods'' in the presence of increasing molar excess of unlabeled rabbit IFN- (Panel a), human IFN- (Panel b), and murine IFN- (Panel c). The fold excess of unlabeled IFN- used as competitor is indicated at the top. The locations of monomer (M) and dimer (D) forms of the labeled IFN-, as well as the heterodimeric complex formed between rabbit IFN- and M-T7, are indicated on the right. Higher molecular mass bands of complexes containing IFN- are also visible. IFN- trimers co-migrate with M-T7/IFN- heterodimers(50) . The locations of SDS-PAGE markers are shown on the left.


Scatchard Analysis of M-T7 Binding to Rabbit IFN-

To quantitate the affinity of M-T7 for rabbit IFN-, solid phase binding assays were performed, followed by Scatchard analysis. Saturable binding was observed between M-T7 and P-labeled rabbit IFN-, while no binding could be observed with either radiolabeled human or murine IFN- (Fig. 6a). Scatchard analysis of the binding data yielded a linear plot, consistent with a single affinity binding site, with a dissociation constant (K(d)) of 1.2 times 10M and correlation coefficient of -0.93 (Fig. 6b). Since the K(d) of binding of rabbit IFN- with the cognate rabbit cellular receptor had not previously been reported, binding of IFN- to intact rabbit kidney (RK-13) cells was also measured. Saturable binding was observed with rabbit IFN-, but not with human or murine IFN- (Fig. 7a). Scatchard analysis of the binding curve for rabbit IFN- gave a linear plot, with a K(d) of 5.9 times 10M and correlation coefficient of -0.97 (Fig. 7b).

Figure 6: Solid phase equilibrium binding analysis of P-labeled IFN- to M-T7. a, solid phase binding analysis of the myxoma M-T7 protein with P-labeled rabbit IFN- (bullet), [P] human IFN- (circle), and P-labeled murine IFN- (+), as outlined under ``Materials and Methods.'' b, Scatchard analysis of the binding curve of P-labeled rabbit IFN- and M-T7.


Figure 7: Equilibrium binding analysis of P-labeled IFN- to the rabbit IFN- cellular receptor. a, solid phase binding analysis of P-labeled rabbit IFN- (bullet), P-labeled human IFN- (circle), and P-labeled murine IFN- (times) to the rabbit IFN- receptor on intact rabbit RK13 cells, as outlined under ``Materials and Methods.'' b, Scatchard analysis of the binding curve of P-labeled rabbit IFN- to the RK13 cellular receptor.


DISCUSSION

IFN- is a critical regulator of the immune system, particularly during the response to pathogenic challenge(3) . The importance of IFN- in the response to poxvirus infections has been amply demonstrated(19, 20, 51, 52, 53, 54, 55) , and thus mechanisms to combat the anti-viral effects of IFN- would clearly be advantageous for virus survival in vertebrate hosts. Myxoma virus, a Leporipoxvirus, was the first virus shown to have evolved a strategy to overcome the affects of the IFN- ligand prior to receptor engagement(31) , by elaborating a bona fide soluble homolog of the mammalian IFN-Rs, named M-T7(31) . Recently, soluble IFNR homologs have also been described in a variety of other poxviruses, including SFV, vaccinia virus, variola virus, and swinepox virus(31, 38, 40, 42, 43) . While the poxviral IFNR homologs all contain significant homology to the mammalian IFNR ligand binding domain, the overall extent of homology with the entire sequence of the two known mammalian receptors is relatively low. Interestingly, the percent identity between IFNR homologs of different poxviral genera (ortho-, lepori- and sui-poxviruses) is equally low. However, conservation of the eight cysteine residues believed critical for forming stabilizing disulfide bonds (46, 56) is observed, although the vaccinia and variola homologs are noticeably missing the first two of the eight conserved cysteine residues (Fig. 1)(44) . It will be useful to ascertain if these two orthopoxvirus homologs have altered ligand binding properties compared with other members of the receptor family.

The mammalian IFN-Rs, through cooperation of the ligand binding and signal transducing accessory components, have been shown to possess strict species specificity for both ligand binding and ligand-dependent signaling. Since myxoma virus is known to have evolved in the tapeti, or South American brush rabbit, it was of interest to deduce whether the myxoma M-T7-soluble receptor homolog might possess the same strict species specificity toward ligand binding. Here we show that M-T7 is highly specific in binding to and abrogating the anti-viral effects of rabbit IFN-, and cannot bind or inhibit human or murine IFN-. Similar results have been found with the myxoma virus tumor necrosis factor receptor homolog(57) , which inhibits rabbit tumor necrosis factor alpha in a species specific manner, similar to that of M-T7. This observation suggests that different poxvirus IFN- receptor homologs will have ligand specificities that reflect the vertebrate host(s) in which each virus has uniquely evolved. Furthermore, the ligand binding properties of soluble poxviral IFN-R homologs would be predicted to be similar to their mammalian counterparts. We show here that M-T7 binds its ligand with a K(d) comparable to the soluble mammalian receptors. Moreover, upon characterization of the rabbit RK13 high affinity IFN- cellular receptor, which has a K(d) value similar to the human and murine cellular IFN-Rs, we demonstrate that M-T7 binding to its ligand, as with mammalian soluble receptor binding, is an order of magnitude lower than that observed with the intact cellular receptor complexes(46, 56) .

Analysis of the interaction of IFN- with secreted versions of the mammalian IFN-Rs, described above, has demonstrated that the extracellular ligand binding domain is sufficient for IFN- binding (46) . Naturally occurring and engineered forms of soluble IFN-Rs possess immunomodulatory properties consistent with IFN- inhibition, presumably by binding and effectively sequestering the ligand away from its membrane bound receptor(46, 56, 58, 59) . Viral IFN-R homologs presumably abrogate the effects of IFN- in the same fashion. In order for myxoma virus to successfully inhibit IFN-, sufficient amounts of M-T7 must be secreted during viral infection. Indeed, we found that biologically significant amounts of M-T7 were expressed, with respect to both the amount of M-T7 produced and its inhibitory capacity, confirming the original observation that M-T7 is a bona fide IFN-R homolog.

By virtue of the amount of M-T7 expressed during myxoma virus infection, it is feasible to suggest that this abundance is a reflection of the relative importance of IFN- as an anti-viral cytokine. M-T7 is expressed as a typical early poxvirus gene product, and is efficiently secreted and extremely stable as an extracellular protein. We suspect that selection pressures on myxoma virus for replication in rabbits has resulted in efficient shuttling of high amounts of M-T7 through cellular secretory pathways, but further experiments are required in order to determine whether this represents an accelerated secretion profile or simply a strict absence of any Golgi/endoplasmic reticulum retention signals in the M-T7 protein itself.

Aside from IFN-R homologs, poxviruses encode proteins with homologies to a number of different cytokine receptors(29, 38, 41, 60, 61) . The origin of these viral cytokine receptor genes remains unproven, but it is likely that either an ancestral poxvirus acquired a progenitor cytokine binding function which subsequently evolved to mimic the ligand binding specificity of the host, or that individual poxviruses have acquired cytokine binding genes independently from their respective hosts. Myxoma virus is one of the few poxviruses for which the evolutionary host, the South American brush rabbit (Sylvilagus brasiliensis), has been clearly established (62) . However, the exact evolutionary relationship between M-T7 and the rabbit IFN-R must await the cloning and sequencing of the rabbit receptor(s). In conclusion, this work strongly suggests that careful analysis of the interaction between the poxviral IFN-Rs and IFN- ligands from a variety of vertebrate hosts will shed light on the evolutionary origins of poxviruses whose natural histories are still obscure.

FOOTNOTES

*
This work was funded by the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by studentships from Medical Research Council and Alberta Heritage Foundation for Medical Research.

Current address: Dept. of Microbiology and Biochemistry, University of Victoria, P.O. Box 3055, Victoria, British Columbia, V8W 3P6, Canada.

**
Medical Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence and reprint requests should be addressed. Tel.: 403-492-2080; Fax: 403-492-9556.

(^1)
Abbreviations used are IFN-, interferon-; IFN-R, interferon- receptor; VSV, vesicular stomatitis virus; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; SFV, Shope fibroma virus; bp, base pair; MOPS, 3-(N-morpholino)propanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Dr. C. Spencer for helpful discussions and advice concerning RNA analysis, Dr. I. Martin and Dr. F. Boess for invaluable help with Scatchard analysis, and R. Maranchuk for excellent technical assistance.

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