From the Two serologically distinct type I interferons
(IFNs), designated ChIFN1 and ChIFN2, are known in the chicken. ChIFN1
is encoded by a family of 10 or more genes, whereas ChIFN2 is encoded
by a single gene. We show here that ChIFN1 and ChIFN2 transcripts are
both strongly induced by Newcastle disease virus in primary chicken
macrophages. By contrast, oral administration of the imidazoquinoline S-28463, which selectively induces IFN- Interferons (IFNs)1 are
cytokines that exhibit antiviral activity. IFN- The IFN system of birds has only recently been characterized at the
molecular level. A cDNA for a virus-induced chicken IFN was first
described by Sekellick et al. (9). Recombinant protein generated from this cDNA exhibited some biochemical features that are typical for type I IFN (10, 11). Genomic DNA analysis revealed that
the chicken contains several genes that code for two serologically
distinct forms of type I IFN, designated ChIFN1 and ChIFN2 (12). ChIFN1
is encoded by a family of 10 or more intronless genes, whereas ChIFN2
is encoded by a single gene that also lacks introns. The various ChIFN1
gene products are >98% identical, whereas ChIFN2 is only ~57%
identical to ChIFN1 (12). Although type I IFNs of chicken and mammals
appear to have related structures, their primary sequences show <30%
similarity. Consequently, it could not be decided on this basis which
of the two chicken IFNs represents the avian equivalent of mammalian
IFN- The mechanisms that lead to transcriptional activation of mammalian
IFN- Some nonviral IFN inducers were reported to be effective in cells of
various mammals (23) and chicken (24), of which the imidazoquinoline
S-28463 is the most powerful agent known to date (23). Oral as well as
parenteral applications of imidazoquinolines are effective in
vivo (23). Imidazoquinolines were found to strongly induce the
synthesis of IFN- In this study, we examined the expression of the ChIFN1 and
ChIFN2 genes in response to viral and nonviral inducers and
characterized their promoters. Both IFNs were induced equally well by
virus in primary macrophages. However, preferential expression of
ChIFN1 was observed in lymphoid organs of chickens treated
with the imidazoquinoline S-28463, suggesting that this IFN represents
the avian homolog of IFN- Cell Culture--
The chicken fibroblast cell line CEC-32 (27)
and the chicken macrophage cell line HD-11 (28) were maintained in
Dulbecco's modified Eagle's medium supplemented with 8% fetal calf
serum and 2% chicken serum. The duck embryo cell line DEC141 (ATCC
CCL141) was maintained in Dulbecco's modified Eagle's medium
supplemented with 5% fetal calf serum and 5% chicken serum.
Abteilung Virologie,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
in mammals, led to a rapid
accumulation of ChIFN1 (but not ChIFN2) transcripts in adult chicken
spleen and thymus. The 5'-upstream region of the ChIFN2 gene contains a NF-
B consensus motif flanked by a sequence element that could serve as a binding site for transcription factor IRF-1, reminiscent of mammalian IFN-
promoters, and it mediated powerful virus inducibility in a duck fibroblast cell line when cloned in front
of a promoterless luciferase reporter gene. The 5'-upstream region of
the cloned ChIFN1 gene contains two putative binding sites
for IRF-1, but lacks NF-
B-binding sites, and it did not respond well
to virus in transfected cells. Thus, the promoters of
ChIFN1 and ChIFN2 genes not only exhibited
differential responses to nonviral inducers in vivo, but
also differed in structure and response to virus in transfected cells.
These findings indicate that ChIFN2 represents the avian homolog of
mammalian IFN-
, whereas ChIFN1 seems to correspond to mammalian
IFN-
.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, IFN-
, IFN-
,
IFN-
, and IFN-
, which all signal through the same cell-surface
receptor complex, are collectively designated type I IFNs. IFN-
,
which employs a different receptor complex for signaling, is referred
to as type II IFN. In probably all mammals, it is coded for by a single
intron-containing gene whose expression is induced by antigens and
mitogens (1). A typical feature of type I IFN genes is that they lack
introns (2). IFN-
, IFN-
, and IFN-
genes are induced in
response to virus in many cell types (3), whereas IFN-
and IFN-
genes are induced in response to developmental signals in trophoblasts of pigs and ruminants, respectively (4, 5). IFN-
is encoded by a
gene family in mammals (6), whereas IFN-
is encoded by a single gene
in humans and several other species (2), but by a small gene family in
cattle (7). Based on amino acid comparisons, it was calculated that
IFN-
and IFN-
have arisen by a gene duplication event that
occurred at least 250 million years ago (2), probably before the
mammals-birds/reptiles divergence. Genes for IFN-
are found in
humans and several other mammals, but not, for example, in dog and
rodents (8), indicating that IFN-
is phylogenetically much younger
than IFN-
and IFN-
.
and IFN-
. A cDNA for chicken IFN-
has been cloned
only recently (13, 14).
and IFN-
genes in response to virus have been studied in
great detail, and the important regulatory sequences and transcription factors were characterized (15-17). The virus response elements in the
promoters of the human IFN-
1 gene (18) and the mouse IFN-
4 gene
(19) have binding sites for IRF-1 and a virus-induced DNA-binding
protein designated TG factor (18) or
F1-binding protein (20).
Cooperation of these two factors seems to be required for efficient
virus-mediated induction of IFN-
genes (18, 20). The promoter of the
human IFN-
gene contains two distinct positive and one negative
regulatory domain (21). Virus induction requires cooperative
interaction between the two positive regulatory domains, which
represent binding sites for IRF-1 or a related transcription factor and
NF-
B, respectively (22). Thus, although some aspects of the
regulation of mammalian IFN-
and IFN-
genes are conserved, the
selective binding of unique transcription factors seems to determine
specificity. The involvement of either NF-
B or the TG factor appears
to be responsible for the cell type-specific selective induction of
IFN-
and IFN-
genes in response to different viruses.
, but not IFN-
or IFN-
, as evident from the
fact that the antiviral activity induced by this drug could be
neutralized very effectively with antisera against IFN-
(25). In
human peripheral blood mononuclear cells, monocytes are mainly
responsible for IFN synthesis in response to imidazoquinolines (26). An
antiviral activity was present in the serum and spleen cell
supernatants of chickens treated with an imidazoquinoline that was
neutralized by antiserum raised against recombinant ChIFN1 (24). No
information is available on the signaling pathways leading to IFN gene
activation in mammalian or avian cells after treatment with
imidazoquinolines.
. The presence of a NF-
B consensus motif
in the promoter of the ChIFN2 (but not ChIFN1)
gene further supports the concept that ChIFN2 is the avian homolog of
mammalian IFN-
.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Infections with Newcastle Disease Virus-- NDV (strain H53) was propagated in 10-day-old embryonated chicken eggs. Allantoic fluids harvested 2 days after virus inoculation usually contained ~5 × 108 infectious units of virus/ml. For IFN induction experiments, virus stocks were diluted 10-fold in serum-free culture medium and treated with UV light as described (30). 10 ml of this material were used to infect the cells in a 90-mm dish.
Interferon Induction in Chickens-- White Leghorn chickens were treated orally with 2 mg of S-28463/kg of body weight. 2 h after drug application, the animals were killed, and RNA was extracted from various organs.
Construction of Plasmids--
The ChIFN1 promoter
constructs were generated by PCR from a plasmid that contained a 2.8-kb
KpnI fragment of chromosomal chicken DNA that contained the
5'-noncoding region and the open reading frame of the
ChIFN1-2 gene (12). PCR was performed with an
oligonucleotide primer corresponding to plasmid sequence and with
primer 1 (5'-GACAGAGATCTGGTGGGACTCGCG-3'), which is complementary to
nucleotides 13 to
1 of the ChIFN1-2 upstream sequence
(see Fig. 3A) and which contains a new BglII restriction site. An aliquot of the amplified product was digested with
BglII, which also cuts in the plasmid multicloning site, and
the resulting 1.8-kb fragment was cloned in the correct orientation upstream of the luciferase gene of the plasmid pGL-2basic, generating construct
-1.8kbChIFN1-2. Another aliquot of the same PCR product was digested with EcoRV and BglII, and the
resulting 0.7-kb fragment was ligated between the SmaI and
BglII sites of pGL-2basic, generating construct
-699ChIFN1-2.
RNA Isolation and Northern Blot Analysis--
Total RNA from
cultured cells was prepared by the acid/guanidium
isothiocyanate/phenol/chloroform method (31). For extraction of total
RNA from organs, the tissues were snap-frozen in liquid nitrogen and
stored at 70 °C. Frozen tissue (samples of 1 g) were thawed
and homogenized in guanidium isothiocyanate buffer before RNA was
extracted as described above. RNAs were separated by electrophoresis
through 1.2% agarose gels containing 4% formaldehyde, transferred
onto nitrocellulose membranes, and hybridized to radiolabeled DNA
probes as described above.
RNase Protection Assay--
For synthesis of radiolabeled
antisense RNA, pChIFN2-RPA was linearized with XbaI and
transcribed with T7 polymerase in the presence of
[-32P]UTP. In vitro transcription and RNase
treatment were carried out using the RiboQuantTM kit (Pharmingen, San
Diego, CA). The radiolabeled antisense RNA was hybridized overnight to
10 µg of total RNA at 56 °C. After RNase treatment, the sample was
denatured and separated by electrophoresis through a denaturing 5%
polyacrylamide gel. The size of the protected RNA fragments was
estimated using appropriate DNA length standards.
Transfections and Luciferase Assays-- DEC141 cells were plated in 6-well dishes and transfected with 5-µg samples of the various luciferase reporter constructs by the calcium phosphate method according to standard procedures (32). At ~30 h post-transfection, the cultures were infected with NDV for 6 or 15 h, before cell lysates were prepared, and the luciferase activity was determined by standard procedures (33).
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RESULTS |
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Virus Infection Induces ChIFN1 and ChIFN2 Genes in Primary Macrophages-- To determine whether the two previously described families of chicken type I IFN genes (12) could both be induced in response to virus infection, we exposed the fibroblast cell line CEC-32, primary blood monocyte-derived macrophages, and the macrophage cell line HD-11 to UV-inactivated NDV. Total RNA was extracted from the various cell cultures at 6 h post-infection and analyzed by Northern blotting for the presence of IFN transcripts. Under the high stringency washing conditions used, hybridization of ChIFN1 and ChIFN2 cDNA probes was mainly to their respective transcripts, and cross-hybridization was minimal (data not shown). High concentrations of ChIFN1 and ChIFN2 RNAs were present in the virus-induced primary macrophages (Fig. 1). By contrast, neither ChIFN1 nor ChIFN2 transcripts could be detected in the two permanent cell lines under these conditions. We further failed to detect ChIFN1 or ChIFN2 transcripts in NDV-treated primary chicken embryo cells (data not shown).
|
The Synthetic Cytokine Inducer S-28463 Activates ChIFN1 (but Not
ChIFN2) Genes in Vivo--
Oral administration of the imidazoquinoline
S-28463 induces IFN- and other cytokines in mammals (23). We
therefore tested whether administration of this substance would induce
both gene families that encode type I IFN in chickens. High levels of
ChIFN1 transcripts were detected in the spleens and thymuses of two
chickens treated for 2 h with 2 mg of S-28463/kg of body weight,
whereas the corresponding organs of the untreated control chicken did not contain detectable amounts of this RNA (Fig.
2). ChIFN1 RNA was also found in the
liver of one drug-treated animal, whereas a second treated animal and
the control animal did not contain detectable amounts of ChIFN1
transcripts in this organ. The bursae of all three chickens lacked
ChIFN1 RNA. A time course experiment showed that the response to
S-28463 was very rapid and transient: ChIFN1 transcripts appeared in
the spleens of treated animals within 1 h, reached a maximum after
~2 h, and were no longer detectable at 4 h post drug application
(data not shown). Interestingly, we failed to detect transcripts for
ChIFN2 in the various organs of drug-treated chickens (Fig. 2),
indicating that at least one member of the ChIFN1 gene
family responded much better to stimulation with S-28463 than the
ChIFN2 gene. Neither ChIFN1 nor ChIFN2 transcripts were
detected in primary blood monocyte-derived macrophages that were
treated with 3 µg/ml S-28463 for 3 or 6 h (data not shown). Since the IFN-
genes are selectively induced by imidazoquinolines in
mammals (25), this result suggested that the ChIFN1 genes, rather than the ChIFN2 gene, might represent the avian
counterparts of mammalian IFN-
.
|
The Upstream Region of the ChIFN2 Gene Contains a Powerful Virus
Response Element--
To further study the regulation of the chicken
IFN genes, we sequenced the upstream regions of one member of the
ChIFN1 gene family (ChIFN1-2) (Fig.
3A) and of the single
ChIFN2 gene (Fig. 3B). Both upstream sequences
lacked classical TATA boxes and most other regulatory elements
frequently found in promoters of mammalian genes. About 160 and 120 bp
upstream of the beginning of the ChIFN1 open reading frame,
we identified two sequence elements that might represent binding sites
for transcription factor IRF-1 (Fig. 3A and Ref. 34),
previously found to be part of the virus response elements of the
mammalian IFN- and IFN-
gene promoters (18). About 130 bp
upstream of the beginning of the ChIFN2 open reading frame,
we identified a consensus binding motif for the transcription factor
NF-
B (Fig. 3B), which plays a critical role in the virus response of mammalian IFN-
genes, but not IFN-
genes (22). Immediately upstream of the putative NF-
B-binding site of the ChIFN2 promoter, we found a sequence element that resembles
the binding site for mammalian transcription factors of the IRF family (Fig. 3B).
|
Functional Characterization of the ChIFN2 Promoter-- To characterize the ChIFN2 promoter in more detail, we first determined the transcriptional start site of this gene. An RNase protection experiment was performed with RNA from virus-induced primary chicken macrophages and radiolabeled in vitro synthesized antisense RNA derived from a DNA fragment carrying the ChIFN2 upstream region (see "Materials and Methods"). We found that ChIFN2 transcripts have heterogeneous 5'-ends (Fig. 4). From their lengths, we calculated that transcription from the ChIFN2 gene initiates at six major sites located 71, 67, 63, 47, 44, and 41 nucleotides upstream of the open reading frame, the most upstream of which was defined as position +1 (Figs. Fig. 3B and 4).
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DISCUSSION |
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Based on coding sequence comparisons, it could previously not be
decided which of the two serologically distinct type I IFNs of chickens
was the true homolog of IFN-, IFN-
, or IFN-
(12). It was also
unclear whether the ChIFN2 gene was inducible by virus or
whether it would not respond to this stimulus, like IFN-
of ruminants (5) and IFN-
of pigs (4). The fact that ChIFN2 is encoded
by a single gene like human and murine IFN-
was not sufficient
evidence for a final assignment, as IFN-
is encoded by a family of
genes in bovines and other species (7). We now found that both
ChIFN gene families are strongly induced by NDV in primary
macrophages, indicating that they cannot be the chicken homologs of
mammalian IFN-
or IFN-
. High levels of ChIFN1 (but not ChIFN2)
transcripts were present in lymphoid organs of chickens treated with
the imidazoquinoline S-28463. In human peripheral blood mononuclear
cells, this drug induces an antiviral activity that can be neutralized
almost completely with antiserum to IFN-
(25). Since such antisera
are usually highly specific and do not cross-react with IFN-
or
IFN-
(35), it appears that imidazoquinolines selectively induce
IFN-
. Our results therefore suggest that ChIFN1 is the chicken
homolog of mammalian IFN-
. We further found that the promoter of the
ChIFN2 gene contains a NF-
B consensus motif, whereas the
ChIFN1 promoter lacks such a motif. This difference in
promoter structure is reminiscent of mammalian IFN genes, where NF-
B
motifs are exclusively found in IFN-
promoters (21). Taken together,
these observations provide strong evidence that ChIFN2 represents the
avian IFN-
homolog, whereas ChIFN1 seems to be the homolog of
mammalian IFN-
. This conclusion is compatible with phylogenetic
analyses of mammalian type I IFN genes, which suggested that the gene
duplication event that gave rise to IFN-
and IFN-
occurred before
the mammals-birds/reptiles divergence (2).
The ChIFN2 promoter contains a NF-B-binding site located
some 60 nucleotides upstream of the transcriptional start site, which
is preceded by a sequence motif that resembles the binding site of
mammalian transcription factors of the IRF family. A promoter fragment
containing these putative regulatory elements was sufficient to mediate
virus inducibility, whereas constructs lacking either the IRF motif or
both motifs no longer responded to virus. NF-
B-binding sites are
present at corresponding positions in the promoters of human and murine
IFN-
genes, where they constitute the positive regulatory domain II
(22, 36). Binding sites for IRF-1 or related transcription factors (21)
are present immediately upstream of the NF-
B site, where they
constitute the positive regulatory domain I in human and murine IFN-
promoters. Taken together, these observations suggest that conserved
signal pathways are responsible for the activation of the
ChIFN2 and mammalian IFN-
promoters. A more refined
analysis of the ChIFN2 promoter and binding studies with
purified transcription factors are required to confirm this
concept.
Except for two IRF-binding sites, the promoter region of the cloned
ChIFN1 gene contains no sequence motifs with obvious
similarity to virus response elements of mammalian IFN genes. Although,
as stated above, the lack of a NF-B site indicates that this gene is
not homologous to mammalian IFN-
, we have no clear evidence that the
promoter of this gene is organized like that of mammalian IFN-
genes. In particular, we failed to detect a binding motif for the TG
factor (18) or the
F1·B complex (19). As our experiments showed
that the isolated ChIFN1-2 promoter responded rather poorly to virus in transfected cells, it was formally possible that we cloned
the 5'-upstream region of a poorly expressed member of the
ChIFN1 gene family that lacks an important regulatory
element. This seems unlikely because the promoter region of another
gene of the ChIFN1 family (ChIFN1-1 (12)) showed
that the 400 nucleotides located upstream of the open reading frame
were identical to the ChIFN1-2 sequence shown in Fig.
3A, except for two nucleotide exchanges. In particular, the
two putative IRF-binding sites were present at corresponding positions
in both promoters.3 Thus, the
poor activity of the cloned ChIFN1 promoters might instead
be explained by assuming that the duck cell line used for the
transfection experiments lacks a transcription factor required for
effective regulation of the ChIFN1 genes. In the mammalian
system, it is well known that the response of the IFN-
and IFN-
promoters to virus is not uniform, but rather varies considerably
between cell lines (37). Future experiments using other cell lines
should allow optimal conditions to be defined for virus induction of
the isolated ChIFN1 promoters.
In human peripheral blood mononuclear cells treated with S-28463, the
monocyte population was found to be the principal producer of IFN-
(26). In a previous study (24), spleen cells and blood lymphocytes from
imidazoquinoline-treated chickens were found to secrete high amounts of
IFN. In agreement with these results, we now found that ChIFN1
transcripts were abundantly present in the spleens and thymuses of
drug-treated chickens. Since S-28463 failed to induce ChIFN transcripts
in primary macrophages, it seems that IFN present in the serum of
imidazoquinoline-treated chickens mainly originates from lymphoid
organs.
A surprising result of our study was that we failed to induce the
chicken IFN genes by Newcastle disease virus in fibroblasts and in a
macrophage cell line under conditions that strongly induced ChIFN1 and
ChIFN2 transcripts in primary macrophages. Although the molecular basis
for nonresponsiveness remains elusive, our findings might explain why
efficient production of virus-induced chicken IFN in fibroblasts was
successful in the hands of only a few experts after careful
conditioning of the cell cultures (38, 39). The elaborate conditioning
might have induced essential transcription factors that are not
constitutively present in most chicken cells. Thus, in agreement with
previous findings in the mammalian system (40), our results suggest
that primary macrophages are the most effective producers of
virus-induced IFNs. Our principal finding that mammalian IFN- and
IFN-
probably have their true counterparts in birds supports
previous notions (41, 42) that most elements of the mammalian IFN
system are conserved in birds, although the primary sequences of the
critical protein components are quite dissimilar.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mark Tomai (3M Pharmaceuticals) for providing the imidazoquinoline S-28463 and Annette Ohnemus for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y14968 and Y14969.
¶ To whom correspondence should be addressed: Dept. of Virology, University of Freiburg, Hermann-Herder-Str. 11, D-79008 Freiburg, Germany. Tel.: 49-761-203-6579; Fax: 49-761-203-6562; E-mail: staeheli{at}ukl.uni-freiburg.de.
1 The abbreviations used are: IFNs, interferons; NDV, Newcastle disease virus; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s).
2 U. Schultz, unpublished results.
3 C. Sick, unpublished results.
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REFERENCES |
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