(Received for publication, September 2, 1994; and in revised form, November 22, 1994)
From the
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
10
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
10
M, 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
10
M, 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.
Interferon- (IFN-
) (
)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
and
. The IFN-
R
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
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.
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.
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
-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).
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) .
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.
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-T7
IFN-
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-T7
IFN-
complex decreases, and at higher cold competitor
ratios IFN-
trimers, which co-migrate with M-T7
IFN-
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-T7
IFN-
complex does not decrease, even at 500
molar excess. We have also been unable to detect significant amounts of
cross-linked M-T7
IFN-
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.
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-
(
), [
P] human IFN-
(
), 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-
(
),
P-labeled human IFN-
(
), and
P-labeled murine IFN-
(
) 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.
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 IFN
R 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 IFN
R homologs all contain significant homology
to the mammalian IFN
R 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
IFN
R 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
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
comparable to the soluble
mammalian receptors. Moreover, upon characterization of the rabbit RK13
high affinity IFN-
cellular receptor, which has a K
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.