(Received for publication, December 31, 1995; and in revised form, January 11, 1996)
From the
Southern blot analysis and screening of a genomic phage
library with the previously cloned chicken interferon (IFN) cDNA
indicated that the chicken genome contains at least 10 IFN genes. A
particularly strongly hybridizing phage clone that we analyzed in more
detail carried a head to tail arrangement of three intron-less IFN
genes that differed from each other and from the cloned chicken IFN
cDNA by only a few base changes. The primary translation products of
these three IFN genes consist of 193 amino acids, and the mature
proteins are composed of 162 amino acids. All three genes of this IFN
family, designated IFN1, yielded active chicken IFN when
expressed individually in transfected COS7 cells. A weakly hybridizing
phage clone contained an additional intron-less chicken IFN gene,
designated IFN2, whose product was 57% identical to chicken
IFN1. Southern blot analysis suggested that the chicken genome contains
a single IFN2 gene. The primary translation product of IFN2 consists of 203 amino acids, and the mature protein is
composed of 176 amino acids. Purified recombinant chicken IFN2 from Escherichia coli had a specific antiviral activity of about
10
units/mg, which was about 20-fold lower than that of
chicken IFN1 purified in parallel. The antiviral activity of chicken
IFN2 from E. coli or from transfected COS7 cells could not be
neutralized by antiserum to recombinant chicken IFN1. Thus, like
mammals, the chicken has a large number of type I IFN genes that code
for at least two serologically distinct antiviral activities.
In mammals, cytokines with antiviral activity are classified as
type I and type II interferons (IFNs). ()Type I IFN includes
-,
-,
-, and
-IFNs, which have related structures
and use a common receptor. The former three are synthesized in response
to virus infection(1, 2) , whereas
-IFN is
synthesized in response to developmental stimuli in the trophoblast of
ruminants (3) and humans(4) . Mature
- and
-IFNs of humans consist of 172 amino acids, whereas
- and
-IFNs are composed of 165 or 166 amino acids(2) . Antisera
prepared against
-IFN or
-IFN showed a high degree of subtype
specificity and did not neutralize the antiviral activity of
-IFN(5) . All mammalian type I IFNs are coded for by
intron-less genes(2) . The
- and
-IFNs are encoded
by gene families with as many as 20 closely related
members(2) . In the various species, the
-IFNs are coded
for by single genes (e.g. mouse) or by gene families (e.g. cattle). Type II IFN or IFN-
is synthesized by antigen- or
mitogen-stimulated T cells(1) . It is encoded by a single gene
with introns, has pleiotropic regulatory effects on cells of the immune
system(6, 7) , and is the principal
macrophage-activating factor of mammals(8, 9) .
In
contrast to mammals, the IFNs of birds are poorly characterized. The
first cDNA for a chicken IFN was isolated only recently(10) .
Its sequence similarity to mammalian IFNs is marginal, but conservation
of cysteine residues and inducibility by virus indicate that it
represents a type I IFN. This notion was supported by the finding that
recombinant chicken IFN is a potent antiviral agent that lacks other
biological activities associated with IFN- of
mammals(11) . Antibodies to the cloned chicken IFN neutralized
the bulk of antiviral activity in preparations of partially purified
chicken IFN from various natural sources(11) , suggesting that
a single serotype of IFN is predominately induced under experimental
conditions. These results were compatible with the assumption that the
chicken has a single gene for type I IFN (10) . However,
Southern blot analysis now suggested the presence of several IFN genes
in chicken. A more detailed analysis showed that the chicken genome
contains a family of at least 10 IFN genes, now designated IFN1, which all appear to code for one serotype of chicken
IFN. A second serotype of ChIFN is encoded by a single gene, designated IFN2, that shows limited sequence conservation.
Figure 2:
Restriction mapping of three IFN1 genes on a single phage (8/18) containing genomic chicken
DNA. The sites for BamHI (B), NcoI (N), KpnI (K), and XbaI (X) are shown. Orientations and positions of the open reading
frames for IFN1-1, IFN1-2, and IFN1-3 are
indicated by arrows. The sequences of the three IFN1 genes were deposited with EMBL/GenBank(TM), accession numbers
X92476, X92477, and X92478.
DNA of the phage clone 1/4 was digested with combinations of various restriction enzymes and analyzed for IFN-related sequences by Southern blotting. A hybridizing 2.3-kb EcoRI-BglII fragment, later shown to contain the complete coding region of the IFN2 gene, was subcloned into pSP65. PCR was used to generate the IFN2 expression constructs. Primer 1 (5`-CGACGGAATTCCCAGCAGAACACAAGTCCC-3`) corresponded to nucleotide positions 103-121 of the cloned DNA fragment and introduced an additional EcoRI restriction site. Primer 2 (5`-GTGCACTCGAGACAGTCACTGGGTGTTGAG-3`) was reverse complementary to nucleotide positions 749-728 and provided an new XhoI restriction site. The PCR product of these primers was digested with EcoRI and XhoI and cloned in the corresponding sites of the eukaryotic expression vector pcDNAI. Primer 3 (5`-GCGTACATATGTGCAACCATCTTCGTCACCAGG-3`) corresponded to nucleotide positions 212-233 of the cloned DNA fragment and introduced an additional NdeI restriction site. Primer 4 (5`-TCACGTAGGATCCAGTCACTGGGTGTTGAG-3`) corresponded to nucleotide positions 745-728 of the cloned DNA fragment and introduced an additional BamHI restriction site. The PCR product of this primer combination was digested with NdeI and BamHI and cloned into the corresponding sites of the bacterial expression vector pET-3a.
Expression of the pET constructs in E. coli was performed as described(13) . Purification of recombinant ChIFN1 and ChIFN2 was done by a method that includes solubilization of recombinant proteins in guanidine hydrochloride and binding to nickel-chelate agarose, a protocol originally established for the purification of recombinant duck IFN(14) .
Figure 1: Genomic Southern blots of chicken DNA probed with IFN1 or FN2. DNA (20 µg/lane) was restricted with BamHI, HindIII, or PstI, electrophoresed through a 1% agarose gel, blotted onto a nylon membrane, and hybridized to radiolabeled IFN1 (A) or IFN2 (B) probes. To estimate the sizes of the IFN gene families in the chicken, various amounts of plasmid DNA containing the genes for ChIFN1 or ChIFN2 (corresponding to 0.6, 2, 6, and 18 gene equivalents) were analyzed in parallel, and the intensities of the resulting signals were compared with those obtained with chromosomal DNA. The gel positions of various DNA size markers are indicated.
To learn more about the IFN1 gene family, we
decided to perform a more detailed analysis of one particular
phage that yielded a very strong hybridization signal, suggesting that
it contained more than one IFN1 gene. Restriction analysis and
partial sequencing confirmed that this
phage contained a 14.5-kb
fragment of chicken DNA that harbored three intron-less genes,
designated IFN1-1, IFN1-2, and IFN1-3 (Fig. 2). Their sequences were almost identical to that of
the previously cloned chicken IFN cDNA(10) , except for an A to
G transition at position 248 in IFN1-1 and IFN1-2,
and a C to T transition at positions 202 and 227 in IFN1-3.
All three IFN1 genes coded for putative precursor proteins of
193 amino acids that may be processed to mature proteins of 162 amino
acid residues. ChIFN1-1 and ChIFN1-2 have identical amino acid
sequences but differ from the originally cloned ChIFN by an Asp to Ser
change at position 34. ChIFN1-3 differs from the originally cloned
ChIFN by a Leu to Phe change at position 19 and a Pro to Leu change at
position 27. To determine whether these natural variants of ChIFN1 are
biologically active, we cloned appropriate fragments of the phage DNA
carrying the individual IFN1 genes into an eukaryotic
expression vector, transfected the resulting constructs into COS7
cells, and tested the supernatants for chicken IFN activity. At 72 h
post-transfection, the supernatants of transfected cells contained
between 20,000 and 50,000 units/ml of antiviral activity, indicating
that all IFN1 constructs coded for active IFN. Rabbit antiserum to
purified recombinant ChIFN (11) efficiently neutralized the
antiviral activities of the various COS7 cell supernatants (data not
shown), suggesting that the various IFN1 genes coded for a
single serotype of chicken IFN.
Figure 3: The intron-less IFN2 gene and its flanking sequences. Shown is the sequence of 900 nucleotides of the chicken genome located immediately downstream of an EcoRI site. The deduced amino acid sequence of ChIFN2 is shown in the single letter code. The putative signal peptide is underlined; potential N-glycosylation sites are indicated with asterisk. The sequence of the IFN2 gene was deposited with EMBL/GenBank(TM), accession number X92479.
The IFN2 gene codes for a polypeptide
of 203 amino acids (Fig. 3), whose N terminus lacks charged
amino acids, suggesting that it may function as a signal peptide. An
alignment of the ChIFN2 sequence with the prototype sequence of ChIFN1 (10) and the product of a recently cloned duck IFN gene (14) is shown in Fig. 4. This comparison indicated that
the cysteine residue at position 28 is the N-terminal amino acid of
mature ChIFN2. Secreted ChIFN2 thus seems to be 14 residues longer than
ChIFN1: it is composed of 176 amino acids and has a calculated
molecular mass of 20,372 Da. Sequence conservation between ChIFN1,
ChIFN2, and duck IFN is pronounced in most regions, except for the
signal peptides and the C termini. When the first 150 amino acids of
the mature proteins are considered, ChIFN2 is 57% identical to ChIFN1
and 61% identical to duck IFN. The C-terminal 26 amino acids of ChIFN2
show no obvious similarity to either the C-terminal 12 amino acids of
ChIFN1 or the 11 C-terminal amino acids of duck IFN (Fig. 4).
Remarkably, the four cysteine residues (marked by asterisks in Fig. 4), which are highly conserved in all -,
-, and
-IFNs of mammals, are conserved in both chicken IFNs as well as in
duck IFN.
Figure 4:
Comparison of the amino acid sequences of
ChIFN1 (top), ChIFN2 (middle), and duck IFN (bottom). The cysteine residues believed to mark the N termini
of the mature proteins are labeled number 1. Amino acids of
the putative signal peptides are numbered with S prefixes. The four
cysteine residues conserved in all mammalian -,
-, and
-IFNs are highlighted by asterisks.
To
determine whether ChIFN1 and ChIFN2 represent two distinct serotypes of
chicken IFN, we evaluated the cross-neutralizing potential of a rabbit
antiserum that we had prepared against E. coli-produced
ChIFN1(13) . CEC-32 cells were incubated with 100 units/ml of
the various IFNs and 0.5% of either rabbit antiserum or preimmune
serum, before the cultures were challenged with VSV. In the presence of
preimmune serum, ChIFN1 as well as ChIFN2 were very effective and
reduced the VSV yields by almost 10-fold (Fig. 5).
In the presence of antiserum, the activity of ChIFN1 was neutralized
quite effectively. By contrast, the antiserum did not significantly
reduce the antiviral activity of ChIFN2 from E. coli or from
transfected COS7 cells (Fig. 5), suggesting that it represents a
novel serotype of chicken IFN.
Figure 5:
The antiviral activity of ChIFN2 cannot be
neutralized by rabbit antiserum to ChIFN1. CEC-32 cells were incubated
for 15 h with 100 units/ml of either ChIFN1 from E. coli,
ChIFN2 from E. coli, or ChIFN2 from COS7 cells in the presence (black bars) of 0.5% rabbit antiserum to recombinant ChIFN1.
Parallel cultures were treated with the various IFNs in the presence of
0.5% preimmune serum (stippled bars). All cell cultures were
then infected with VSV. The virus titers in the culture supernatants at
24 h post-infection were determined on mouse 3T3 cells by the
TCID method.
We have shown here that the chicken contains at least two serologically distinct subtypes of IFN. The first subtype is coded for by a gene family of more than 10 members, while the second subtype seems to be encoded by a single gene. All these genes lack introns, like the mammalian genes for the various type I IFNs. Thus, although the primary structures of avian IFNs are poorly conserved, the presence of a gene family rather than a single gene and the lack of introns are highly conserved features of type I IFNs.
The search for additional
subfamilies of chicken IFNs is complicated by the fact that the IFN1 gene family is quite large. Southern blot analysis
suggested the presence of about 10 IFN1 genes. However, the
high frequency by which positive phages were identified in a
chicken genomic library suggested the presence of as many as 100 IFN
genes in the chicken genome. It is possible that the latter high
frequency is an artifact of the phage library. Alternatively, the
library screen may indeed have revealed the true complexity of the
chicken IFN superfamily. We assumed that the complex pattern of weak
hybridization signals on the Southern blot (Fig. 1A)
resulted from single IFN1 genes with altered restriction sites
in their flanking regions. However, we cannot exclude the alternative
possibility that these signals resulted from weak cross-reactivity to a
novel IFN gene family.
One cross-reactive phage that we
characterized contained a novel chicken IFN gene that we designated IFN2. Southern blotting experiments suggested that it is a
single copy gene. Functional studies showed that IFN2 codes
for a chicken IFN that escapes neutralization by antibodies to ChIFN1,
a finding that may be explained by the fact that the amino acid
sequence of ChIFN2 is only 57% identical to that of ChIFN1.
Interestingly, mature ChIFN2 is 14 amino acids longer than ChIFN1. In
mammals, ``long'' variants of
-IFN with extra amino
acids at the C terminus are known as
- and
-IFNs (2) . Their sequences are about 60% identical to
-IFNs,
and their activities cannot be neutralized by antisera that neutralize
-IFNs (5) . Although this suggests that ChIFN2 represents
the avian homolog of mammalian
- or
-IFNs, we believe that
the familiar terms
-,
-,
-, and
-IFN, which refer
to subtypes of mammalian IFNs with specific biological properties,
should not be used at present for the chicken IFN system. Detailed
studies on the induction of IFN2 in response to virus or
developmental stimuli will be required to determine whether this
assumption is correct.
An unexpected result of our studies was that
the specific antiviral activity of purified recombinant ChIFN2 was
about 20 times lower than that of ChIFN1. Evidence that this difference
was not an artifact of the purification procedure came from experiments
with supernatants of COS7 cells that were transfected with expression
constructs for ChIFN1 or ChIFN2. Because the two constructs were
identical except for the IFN coding regions, it seems reasonable to
assume that similar amounts of recombinant protein were produced in the
two cultures. Nonetheless, the supernatants of ChIFN1-producing
cultures contained about 20-fold more antiviral activity than
supernatants of ChIFN2-producing cultures, strongly suggesting that
this difference reflects a true biological difference of the two ChIFN
subtypes. This result further indicated that the low specific activity
of E. coli-produced ChIFN2 cannot be explained by simply
assuming that ChIFN2 needs glycosylation for full activity. This
situation found here for ChIFNs is reminiscent to that described for
the various -IFN subtypes of humans and mice, whose individual
specific activities were also found to differ
significantly(15, 16) .