Download Atlantic Salmon Interferon Genes: Cloning, Sequence Analysis

JOURNAL OF INTERFERON & CYTOKINE RESEARCH 23:601–612 (2003)
© Mary Ann Liebert, Inc.
Atlantic Salmon Interferon Genes: Cloning, Sequence
Analysis, Expression, and Biological Activity
BØRRE ROBERTSEN, VERONICA BERGAN, TORUNN RØKENES, RANNVEIG LARSEN,
and ARTUR ALBUQUERQUE
ABSTRACT
In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon,
called SasaIFN-a1 (829 bp) and SasaIFN-a2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN
appears to have five a-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-a sequences, and 17%–18% sequence identity with mammalian IFN-b and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the
SasaIFN-a1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells
against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C)
induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.
INTRODUCTION
V
of farmed Atlantic
salmon in Norway and other countries. More knowledge
about innate antiviral mechanisms of Atlantic salmon is important for a better understanding of its ability to resist viral infections. It is well established that type I interferons (IFN) mediate a major first line of defense against viral infection of
higher vertebrates, but little is known about the IFN system of
fish. IFNs are cytokines that induce cells into an antiviral state.
Type I IFNs are induced by viruses in several cell types, preferentially by double-stranded (ds) RNA intermediates produced
during viral replication.(1) Mammalian type I IFNs constitute a
multigene family grouped into IFN-a, IFN-b, IFN-d, IFN-t,
and IFN-v.(1) Type I IFNs are produced by most cell types, although IFN-a and IFN-v are primarily produced by leukocytes
and IFN-b by fibroblasts.(1–3) IFN-d and IFN-t are produced
by trophoblasts of pig and ruminants, respectively. In mammals, IFN-a is a family of 14–20 closely related genes, whereas
IFN-b is encoded by one gene except in ungulates, which have
5 IFN-b genes.(1) Chicken possesses two distinct type I IFNs,
one with a gene family of at least 10 members and one encoded
by a single gene.(4–6) Mammalian type I IFNs constitute a clusIRAL DISEASES CAUSE HIGH LOSSES
ter of genes lacking introns, in contrast to type II IFN (IFN-g),
which is encoded by a single intron-containing gene.(7) IFN-g
is produced by T lymphocytes and natural killer (NK) cells in
response to mitogens, antigens, or interleukin-12 (IL-12) and is
nonhomologous to type I IFNs.(7) Type I IFNs can also be distinguished from IFN-g by their stability at pH 2.(2) Although
mammalian type I IFN vary in size (143–172 amino acids) and
number of disulfide bonds (0–2), crystal structure determinations show that they share a common three-dimensional structure composed of 5 a-helices.(8–11)
The antiviral action of type I IFNs is exerted through binding to the IFN-a/b receptor, which is composed of subunits
IFNAR-1 and IFNAR-2.(12) Binding triggers signal transduction through the Jak-Stat pathway and results in induction of
transcription of IFN-stimulated gene products, several of which
encode proteins that inhibit viral replication.(3) Examples of
IFN-induced antiviral proteins are Mx protein, protein kinase
PKR and 2-5 oligoadenylate synthetase.
One IFN gene has been cloned recently from zebrafish,(13)
but IFN genes from salmonids have not yet been published.
Atlantic salmon macrophages stimulated with poly(I)poly(C) produce type I IFN-like activity, which inhibits replication of infectious pancreatic necrosis virus (IPNV) and in-
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway.
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duces Mx protein in salmonid cell lines.(14,15) IFN-like activity also has been demonstrated in rainbow trout cells after
infection with viruses,(16–18) and the molecular mass of the
IFN was estimated to be 18 kDa and 94–100 kDa.(16,19) Sequence information about IFN from salmonids is of interest
not only for studies of virus-host interactions in these economically important fish species but also with respect to evolutionary studies of IFN.
In the present paper, we describe cDNA cloning, phylogenetic sequence analysis, and expression of two IFN genes from
the head kidney of Atlantic salmon. Antiviral activity of salmon
IFN is demonstrated against IPNV, which is one of the economically most important salmon pathogens. We also show that
Atlantic salmon IFN I genes possess introns.
MATERIALS AND METHODS
Fish
Atlantic salmon (Salmo salar) were obtained from the
Aquaculture Research Station (Tromsø, Norway) where they
were kept in 600 l tanks. For Northern blotting, fish weighing approximately 500 g kept in salt water at 8–9°C were injected intraperitoneally (i. p.) with 2.5 ml saline containing 2
mg/ml poly(I)-poly(C) (Amersham Pharmacia Biotec, Uppsala, Sweden) or with saline alone. Head kidney was harvested
14 h post-injection. For RACE cloning and RT-PCR of IFN,
presmolts weighing 30–40 g kept in fresh water at 10°C were
injected i.p. with either 0.2 ml saline containing 2 mg/ml
poly(I)-poly(C) or 0.2 ml saline. Head kidneys from three control fish and three poly(I)-poly(C)-treated fish were harvested
at 12, 24, and 48 h postinjection. In all experiments, organs
were preserved in RNA Later™ (Ambion, Austin, TX) at
220°C before isolation of RNA. Prior to treatment, the fish
were anesthetised with 0.005% ethyl p-aminobenzoate
(Sigma, St Louis, MO).
TABLE 1.
Primer No.
1a
2
3b
4b
5
6
7
8
16
18
19
21
24
29
aR
PRIMERS USED
FOR
CLONING
Orientation
Forward
Reverse
Forward
Forward
Reverse
Forward
Reverse
Forward
Forward
Reverse
Forward
Reverse
Forward
Reverse
5 a/g, Y 5 c/t, and I 5 inosine.
site is underlined
b PstI
AND
Cell culture and virus
TO cells originate from Atlantic salmon head kidney and
were obtained from Dr. Heidrun Wergeland (University of
Bergen, Norway).(20) TO cells were cultured in Eagle’s minimal essential medium (MEM) supplemented with 100 mg/ml
streptomycin, 100 U/ml penicillin, 2 mM L -glutamine, 1%
nonessential amino acids, and 5% fetal bovine serum (FBS) in
5% CO2 at 20°C. Chinook salmon embryo cells (CHSE-214)
were cultured as TO cells except with 7.5% FBS. Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
antibiotics and 10% FBS in 5% CO2 at 37°C. IPNV serotype
Sp was propagated in CHSE-214 cells. The cell-free supernatants contained 3.1 3 107 50% tissue culture infective doses
of virus per milliliter calculated according to the method of
Reed and Muench.(21) CHSE-214 and IPNV were obtained from
Dr. Ann Inger Sommer (Norwegian Institute of Fisheries and
Aquaculture Ltd, Tromsø, Norway).
Primers
Table 1 shows a list of the primers used for cloning and RTPCR analysis of Atlantic salmon IFN.
RNA isolation
Total RNA was isolated using Trizol® Reagent (Invitrogen,
Groningen, The Netherlands) following the manufacturer’s instructions. Poly(A)1 mRNA was purified from total RNA using the OligoTex™ mRNA isolation kit (Qiagen, Hilden, Germany).
Suppressive subtractive hybridization (SSH)
cDNA library
TO cells were grown to subconfluency in 150-cm3 culture
flasks containing 40 ml medium. The IFN system of the cells
was stimulated by transfection of poly(I)-poly(C) as follows.
EXPRESSION ANALYSES
OF
ATLANTIC SALMON IFN
Sequence (59 to 39)
aatacagtgcgcYgaRgcItggga
ttgcagatgacgttttgtctctt
actgcaggaagactacggaacaataattcggact
actgcagggaaaactaacagcgaaacaaacagcta
actgacagggtcccacatgat
atagatgatggcgaggttgaggac
tcaacttcttgaagtagcgtttcag
cagagcgtgtgtcattgctgtga
actaacagcgaaacaaacagctatttac
aagactggggcatcctgctttg
aacctgagaagagactgaaacgct
actttataaactggtaagggcgtagc
cattgctgtgactggatccgacaccaactac
agtagcgtttcagtctcttctcaggtttca
ATLANTIC SALMON IFN
Fugene (60 ml) (Roche, Indianapolis, IN) alone or complexed
with 4 mg poly(I)-poly(C) was diluted in 2 ml serum-free MEM.
The transfection mixture was added to the cells, which were incubated for 8, 12, 16, or 22 h. Poly(A)1 mRNA was then purified from the cells as described. SSH of cDNA was carried
out using the PCR-Select cDNA Subtraction kit from Clontech
(Palo Alto, CA) using 2 mg mRNA for the cDNA synthesis.
Driver cDNA was generated by reverse transcription of pooled
mRNA from cells treated with Fugene alone, and tester cDNA
was generated of pooled mRNA of cells transfected with
poly(I)-poly(C) in Fugene. The resulting subtracted cDNA was
used as a template in the initial PCR amplification of IFN.
Initial PCR cloning
The degenerate forward primer No. 1 (Table 1) was designed
based on conserved regions in vertebrate IFN sequences, including a putative zebrafish IFN EST sequence (accession No.
wz32679). Amplification of cDNA by a standard PCR was performed using the subtracted cDNA library as a template and
primer No. 1 and the PCR primer 1 supplied with the cDNA
subtraction kit from Clontech. PCR products were subcloned
into the pCR4 TOPO vector (Invitrogen), sequenced, and analyzed by BLASTX for similarity with sequences deposited in
the GenBank. Several cDNA fragments were obtained, which
showed similarity with mammalian IFN-a sequences.
RACE cloning of full-length IFN cDNA
A cDNA library was generated from pooled samples of mRNA
from poly(I)-poly(C)-stimulated fish using the Marathon cDNA
amplification kit from Clontech. The reverse primer No. 2 (Table
1) was designed based on the salmon IFN cDNA sequences obtained from the SSH library. The 59-end of putative IFN cDNA
was amplified using the RACE cDNA library as a template with
primer No. 2 and the universal primer AP1 supplied with the
RACE kit. Touchdown PCR was performed following the manufacturer’s recommendations. Products were subcloned into the
pGEM T-Easy vector (Promega, Mannheim, Germany), sequenced, and analyzed for similarity to vertebrate IFN sequences.
Based on differences in the 59-untranslated region (59-UTR) of
these clones, two forward primers, No. 3 and No. 4 (Table 1),
were designed to amplify full-length cDNA of the putative Atlantic salmon IFNs by PCR. Both primers were supplied with a
PstI cut site for easier subcloning and analysis of the direction
of the PCR product. Using the RACE cDNA library as a template, primer No. 3 or primer No. 4 and the universal primer AP1
supplied with the RACE kit were included in separate touchdown
PCR reactions. Amplified products from each reaction were subcloned into the pGEM T-Easy vector. Of 20 clones picked, 6 different clones were sequenced completely. Two clones, named
SasaIFN-a1 and SasaIFN-a2, were of full length and were investigated further in this work.
Northern blot analysis
mRNA (3 mg) from head kidney of saline-treated and
poly(I)-poly(C)-treated fish was loaded into each well of a 1%
glyoxal-based agarose gel (Ambion) and subjected to electrophoresis. Northern blotting was performed by the downward
transfer method, using NorthernMax Transfer Buffer (Ambion),
603
onto a positively charged nylon membrane. The mRNA was
immobilized by UV cross-linking. Prehybridization and hybridization were performed at 52°C in ULTRAHyb hybridization buffer (Ambion). A DIG-labeled 444-bp IFN probe corresponding to nucleotides 553–996 was amplified from the
SasaIFN-a2 vector using primers No. 2 and No. 8 (Table 1) as
described in the PCR DIG Probe Synthesis Kit (Roche).
Hybridization was carried out overnight with the DIG-labeled IFN probe at 10 ng/ml. The membrane was washed once
in 2 3 SSC containing 0.1% SDS for 10 min at ambient temperature and twice in 0.1 3 SSC, 0.1% SDS, at 52°C for 15
min. Hybridizing bands were detected with the DIG Luminescent Detection Kit (Roche).
Semiquantitative RT-PCR assay of IFN
TO cells were grown to subconfluency in 6–well culture
plates containing 3 ml medium. Total RNA was extracted from
untreated cells and from cells incubated with 1 mg/ml poly(I)poly(C) for 12, 24, or 48 h. Two wells were harvested for each
time point, and the experiment was repeated three times. Total
RNA was extracted from the head kidneys of control and
poly(I)-poly(C)-treated fish as described. RNA (1 mg) from
cells or head kidneys was reverse transcribed into cDNA in a
reaction mixture containing 0.5 mg random hexamers, 0.5 mM
dNTPs, 20 U RNasin, and 200 U M-MLV reverse transcriptase
(all Promega) in 20 ml reaction buffer (50 mM Tris-HCl, 75
mM KCl, 3 mM MgCl2, 10 mM DTT, pH 8.3). The synthesis
was performed at 42°C for 60 min. PCR was performed using
10% of the resulting cDNA as a template. A 220-bp IFN fragment was amplified by PCR using primer No. 6 and primer No.
7 (Table 1) at the following conditions: 96°C for 2 min, 30 cycles of 96°C for 20 sec, 58°C for 20 sec, 72°C for 20 sec. To
confirm that all the samples contained an equal amount of
cDNA, a 344-bp actin fragment was amplified by PCR using
10% of the cDNA as template and forward primer 59-cactcaaccccaaagccaacagg-39 and reverse primer 59-aaagtccagcgccacgtagcacag-39 at the following conditions: 96°C for 2 min,
20 cycles of 96°C for 20 sec, 68°C for 20 sec, 72°C for 30 sec.
Identification of introns in IFN sequences from
genomic DNA
Total DNA was purified from full blood of three individual
fish using the QIAamp DNA blood Midi kit (Qiagen). The DNA
was used as template in standard PCR reactions used to identify introns in genomic IFN sequences from Atlantic salmon.
The primer pairs used were Nos. 16 and 18, Nos. 19 and 21,
and Nos. 24 and 29, respectively.
Antiviral assay
The SasaIFN-a1 gene was amplified by a standard PCR using primers No. 3 and No. 4 (Table 1). The product was subcloned into the eukaryotic TA expression vector pCR3.1 (Invitrogen), sequenced, and selected for insert in forward or
reverse orientation with respect to the cytomegalovirus (CMV)
promoter. Subconfluent HEK293 cells grown in 10 cm culture
plates were transfected with 20 mg plasmid for 4 h using the
calcium-phosphate coprecipitation method. The cells were
given fresh medium and incubated for 48 h. Cell supernatants
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ROBERTSEN ET AL.
A
FIG. 1. Nucleotide and amino acid sequences of the SasaIFN-a1 (A) and SasaIFN-a2 (B) precursors. The putative ORF is
shown in capital letters along with the translated sequence. The predicted signal protein is underlined. Cysteines in the mature
protein are circeled. The potential N-glycosylation site of the mature protein is boxed. In the 39-UTR of the nucleotide sequences,
putative motifs associated with mRNA instability are shown in bold letters, and the polyadenylation site is underlined.
were harvested, adjusted to pH 2 with HCl, and incubated for
24 h at 20°C. The supernatants were neutralized with NaOH
and assayed for antiviral activity by the cytopathic effect (CPE)
reduction assay.(22) The HEK293 cell supernatants were 2-fold
serially diluted in cell medium, and 100 ml of each dilution was
added to quadruplicate wells of subconfluent TO or CHSE-214
cells grown in 96-well culture plates. After 24 h, the cell supernatants were removed, and the cells were infected with IPNV
(moi 5 0.1) in 100 ml MEM. Virus was absorbed for 1.5 h before 100 ml medium with serum was added. After 72 h, when
complete destruction of unstimulated infected cells occurred,
all cells were washed with phosphate-buffered saline (PBS),
fixed, and stained by incubation with 1% (w/v) crystal violet
in 20% ethanol for 10 min. The cells were then washed three
times with distilled water and air dried. The stain was dissolved
by addition of 100 ml 50% ethanol containing 0.05 M sodium
citrate and 0.05 M citric acid, and the absorbance was read at
550 nm. The mean dye absorbance of lysed control cells was
subtracted from the mean absorbance of noninfected control
cells and infected stimulated cells. One unit of IFN activity was
calculated as the reciprocal dilution of the HEK293 supernatant
causing 50% reduction in absorbance at 550 nm.
Immunoblot analysis
CHSE-214 cells were seeded in 24-well culture plates and
incubated for 24 h with 2-fold dilutions of supernatants from
HEK293 cells transfected with the SasaIFN-a1 gene. The cells
were washed once in PBS and lysed in 50 ml SDS-gel sample
buffer. The extracts were boiled for 5 min, and 15 ml extract
per lane was subjected to SDS-10% PAGE. (23) Blotting, blocking, and antibody incubation were performed as described.(14)
A polyclonal rabbit antiserum generated against the rainbow
trout Mx3 protein diluted 1:2000 was used to detect Mx protein. The antiserum was generated against a fragment of the
rainbow trout Mx3 protein as described.(24) The plasmid used
to express the rainbow trout Mx3 fragment was a gift from JoAnn C. Leong (Department of Microbiology, Oregon State University, Corvallis, OR). Horseradish peroxidase (HRP)-conjugated goat antirabbit antibody (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) diluted 1:10,000 was used as secondary antibody. Detection was performed using the SuperSignal West
Pico Chemiluminiscent Substrate (Pierce, Rockford, IL). After
Mx protein detection, membranes were stripped and immunostained for actin using a polyclonal antiactin antibody (Sigma)
diluted 1:500 as the primary antibody.(14)
GenBank accession numbers
The cloned salmon IFN cDNAs have GenBank accession
numbers AY216594 (SasaIFN-a1) and AY216595 (SasaIFNa2). Accession numbers for sequences used in the creation of
alignment and phylogenetic analysis are X92476 (chicken IFNa1), X92479 (chicken IFN-b), X84764 (duck IFN), P05003
ATLANTIC SALMON IFN
605
B
FIG. 1.
(horse IFN-a1), AAA30949 (horse IFN-v2), P01563 (human
IFN-a2b), M25460 (human IFN-b), P37290 (human IFN-d),
X58822 (human IFN-v1), NP_034634 (mouse IFN-a4),
X14455 (mouse IFN-b), Z22706 (pig IFN-d), I46398 (sheep
IFN-a), P56828 (sheep IFN-t), AAL76919 (woodchuck IFNa7), AAM95448 (zebrafish IFN cDNA), AJ544820 (zebrafish
IFN gene).
RESULTS
Cloning and sequencing of Atlantic salmon IFN genes
The cDNA cloning of Atlantic salmon IFN was based on induction of IFN mRNA by poly(I)-poly(C), which is a dsRNA
with well-known IFN-inducing properties. SSH was used to
generate cDNA from Atlantic salmon TO cells highly enriched
in genes induced by poly(I)-poly(C). TO cells are derived from
the head kidney of Atlantic salmon.(20) PCR amplification of
IFN-like cDNA fragments from TO cells was accomplished us-
Continued.
ing the subtracted cDNA as a template and a degenerate forward primer designed from the conserved vertebrate type I IFN
sequence motif YSACAW at the C-terminal. The reverse primer
was one supplied by the SSH kit. These sequences allowed design of a specific gene primer that could be used to amplify the
59-end of Atlantic salmon IFN by RACE cloning. Two 59-end
IFN products were obtained using cDNA from head kidney of
poly(I)-poly(C)-stimulated fish. Based on these sequences, new
forward primers were designed that could amplify the whole
open reading frames (ORFs) of Atlantic salmon IFN by 39RACE cloning. Two putative IFN cDNAs were amplified, 829
bp and 1290 bp in length. BLASTX analysis of the sequences
showed lowest E-value for a putative zebrafish IFN precursor
(2e-25), followed by woodchuck IFN-a7 precursor (2e-07),
mouse IFN-a4 precursor (8e-7), and other mammalian type I
IFNs. Figure 1 shows the nucleotide sequences and the predicted translation of the two cDNAs. The most likely ORF of
both cDNAs contained 528 nucleotides starting at position 37
in the small cDNA clone and position 502 in the large cDNA.
Both translated ORFs gave a 175-amino acid peptide with a
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ROBERTSEN ET AL.
FIG. 2. ClustalX alignment of the deduced signal protein (a) and mature protein (b) of SasaIFN-a1 and other vertebrate IFN
sequences. Amino acids identical to the SasaIFN-a1 sequence are shown shadowed and in bold letters. Dashes indicate gaps.
The helixes A, B, C, D, and E are underlined in the SasaIFN-a1 sequence and in sequences of IFNs for which the crystal structure has been solved.
molecular mass of 20.8 kDa, which represents IFN precursor
molecules of Atlantic salmon. The two clones showed 98% nucleotide sequence identity and 95% amino acid sequence identity.
The hydrophobic 23 amino sequence at the amino-terminal
end was identified as the signal peptide sequence using the SignalP program version 1.1 (www.cbs.dtu.dk/services/SignalP/).(25) This suggested that the mature Atlantic salmon IFNs
contain 152 amino acids, representing a molecular mass of 18.2
kDa. The putative mature salmon IFNs have a cysteine as the
first amino acid, similar to the mammalian IFN-a, and were
named SasaIFN-a1 (small cDNA) and SasaIFN-a2 (large
cDNA). The cysteine content (5 residues) of the signal sequences is notably higher than in other IFN signal sequences,
whereas the mature proteins contain only 2 cysteine residues.
The salmon IFNs have an isoelectric point of 9.2. An N-linked
glycosylation motif (NYSA) is present at amino acid 124.
A polyadenylation signal is present 10–12 nt upstream of the
poly(A) tail in both cDNAs. Four mRNA destabilization elements ATTTA occur in the 39-UTR of the cDNAs (Fig. 1). Two
of them are present in overlapping copies of the sequence
UUAUUUAU reported in the 39-UTR of human IFN-b mRNA
and other cytokines.(26,27)
Alignment of salmon IFN with other vertebrate
type I IFNs
An alignment of the SasaIFN-a1 precursor protein with 24
different members of vertebrate type I IFNs was created using
the CLUSTALX program.(28) Figure 2 shows the alignment
with 10 selected members from different classes of IFN, with
the signal sequences in Figure 2a and the mature proteins in
Figure 2b. The signal sequence of the salmon IFN showed
hardly any homology with IFN signal sequences of higher vertebrates (0–9% identity), pig IFN-d being the exception with
19% identity (Table 2). In contrast, salmon and zebrafish signal sequences showed marked homology (32% identity). Analysis of secondary structure using the PHD program(29,30) at the
Protein Predict server (www.embl-heidelberg.de/predictprotein/) suggested that Atlantic salmon IFN possesses 5 alpha helixes (A, B, C, D, and E), which overlap with the helixes of
other type I IFNs (Fig. 2b). The most conserved amino acids
ATLANTIC SALMON IFN
607
TABLE 2. COMPARISON OF SASAIFN-a1 AMINO ACID
SEQUENCE WITH OTHER TYPE I IFN SEQUENCES (% IDENTITY )
IFN type
SasaIFN-a2
Zebrafish IFN
Mouse IFN-a4
Human IFN-a2b
Human IFN-v1
Sheep IFN-t
Mouse IFN-b
Pig IFN-d
Chicken IFN-a1
Chicken IFN-b
Signal peptide
Mature protein
96
32
9
9
5
0
5
19
4
0
97
45
29
28
26
23
17
18
18
17
appeared to occur in and adjacent to the helixes. The salmon
IFN showed a high degree of similarity with the zebrafish IFN,
including the same pattern of cysteines in the mature protein.
The alignment confirmed that the most conserved region of type
I IFNs is in the C-terminal region from amino acid 125 to 136
of salmon IFN. The long loop region between helix A and B
of the fish IFNs shows very little homology with AB loops from
other IFNs. The CD loop of salmon IFN contains a larger gap
than other IFNs, including the zebrafish IFN.
The Atlantic salmon IFNs showed 45% identity with the zebrafish IFN with respect to both amino acid and nucleotide sequence. As shown in Table 2, salmon IFN showed highest sequence identity with the IFN-a sequences (#29%) and lowest
identity with mouse and human IFN-b (17%) and the chicken
IFN sequences (17%–18%). In the region from amino acid 125
to amino acid 136, the salmon IFN showed about 60% sequence
identity with mammalian IFN-a.
To analyze more rigorously the degree of evolutionary relatedness between type I IFN of fish and higher vertebrates, we
performed a phylogenetic analysis of the sequences using the
PAUP program(31) based on the CLUSTAL X alignment. The
phylogenetic tree generated by the Neighbor-Joining method
(Fig. 3), indicates that the fish IFNs form a cluster separate from
the other vertebrate type I IFNs and are not more related to the
IFN-a cluster than the IFN-b or the chicken IFNs. The tree was
bootstrapped 1000 times and showed 100% support for the
grouping of the fish sequences. The same conclusion was drawn
using the Parsimony method of PAUP.
Biological activity of SasaIFN-a1
To study whether the SasaIFN-a1 gene encoded a biologically active IFN, it was subcloned into a eukaryotic expression
vector pCR3.1 in forward and reverse orientation from the CMV
promoter. The constructs were transfected into human HEK293
cells. Media supernatants were harvested 48 h later, acid treated
FIG. 3. Unrooted phylogenetic tree showing the relationships between the Atlantic salmon SasaIFN-a1 amino acid sequence
and other known type I IFN sequences. The tree was constructed by the Neighbour-Joining method using the CLUSTALX and
PAUP programs.
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ROBERTSEN ET AL.
TABLE 3. IFN ACTIVITY IN SUPERNATANTS OF HEK293
CELLS TRANSFECTED WITH THE SASAIFN-a1 GENE
IFN activity (U/ml)
Test cells
Gene orientation
CHSE-214
CHSE-214
TO
TO
Forward
Reverse
Forward
Reverse
a ND,
Experiment 1 Experiment 2
5560
NDa
120400
ND
3060
ND
36900
ND
no detectable activity.
over night, and then assayed for antiviral activity against IPNV
in Atlantic salmon TO cells or Chinook salmon CHSE-214 cells
using the CPE reduction method. As shown in Table 3, supernatants from HEK293 cells transfected with the SasaIFN-a1
gene in the correct orientation showed relatively high levels of
antiviral activity (36,000 and 120,000 U/ml) when tested on TO
cells and 10-fold lower activity when tested on CHSE-214 cells.
In contrast, HEK293 cells that were transfected with the SasaIFN-a1 gene in the reverse orientation gave no IFN activity
in either cell system. This demonstrates that the SasaIFN-a1
cDNA translates into a protein, which is cleaved and secreted
as an active IFN by human cells. CHSE-214 cells transfected
with SasaIFN-a1 also produced IFN activity, but at much lower
titers due to lower transfection rates (data not shown).
As shown in Figure 4, supernatants of HEK293 cells transfected with the SasaIFN-a1 gene in the forward orientation of
the CMV promoter induced Mx protein in the CHSE-214 cells
after 24 h of stimulation. In contrast, supernatants from
HEK293 cells transfected with the SasaIFN-a1 gene in the reverse orientation did not induce Mx protein in the cells. Similar results were obtained with TO cells (data not shown).
FIG. 5. Northern blot analysis of salmon IFN mRNA in head
kidney of Atlantic salmon treated with poly(I)-poly(C). Organs
were harvested and extracted for RNA 14 h after injection of
poly(I)-poly(C). IFN-specific mRNA was detected by hybridization with a DIG-labeled cDNA probe. Lanes 1 and 2,
mRNA from two nontreated fish; lanes 3, 4, and 5, mRNA from
three different poly(I)-poly(C)-treated fish.
were, however, not able to detect IFN transcripts by Northern
blotting when total RNA from stimulated fish or TO cells was
electrophoresed. This indicated that IFN mRNA was present at
relatively low levels even in the stimulated fish cells. Semiquantitative RT-PCR analyses, therefore, had to be used for
time course studies of induction. These measurements showed
highest level IFN transcripts in the head kidney of the fish 12
h after injection of poly(I)-poly(C) and diminishing levels 24
and 48 h later (Fig. 6 top). TO cells stimulated with poly(I)-
Induction of type IFN salmon genes
Northern blot analysis of mRNA showed that two IFN transcripts of about 850 nt and 1200 nt were expressed in head kidney of Atlantic salmon 14 h after injection of poly(I)-poly(C)
(Fig. 5). Transcripts were not detected in untreated fish. We
FIG. 4. Induction of Mx protein by SasaIFN-a1 in CHSE214 cells. Supernatants from HEK293 cells transfected with SasaIFN-a1 in forward and reverse (control) orientation of the
CMV promoter were 2-fold serially diluted in cell medium and
incubated with CHSE-214 cells for 24 h. Extracts of the cells
were subjected to immunoblot analysis with polyclonal antibodies against Mx protein and actin.
FIG. 6. RT-PCR analysis of IFN transcripts in head kidney
(top) and TO cells (bottom) of Atlantic salmon. TO cells were
incubated with poly(I)-poly(C) for 0, 12, 24, and 48 h and extracted for RNA. Atlantic salmon presmolts were injected with
poly(I)-poly(C), and head kidneys were harvested and extracted
for RNA 0, 12, 24, and 48 h later. RNA samples were reverse
transcribed, and the cDNA was subjected to PCR with IFN-specific primers or actin primers as described. The intensity of the
220-bp IFN product and the 344-bp actin product are illustrated.
ATLANTIC SALMON IFN
poly(C) showed detectable IFN transcripts after 12 h and highest levels of IFN transcripts after 24 h of stimulation (Fig. 6
bottom). IFN transcripts were present at low or undetectable
levels in control samples of cells or head kidney.
Atlantic salmon IFN genes contain introns
PCR of genomic DNA with primers designed from the
salmon IFN cDNA sequences revealed that the salmon IFN
genes contained introns. Primers No. 16 and No. 18 (Table 1)
amplified a 493-bp genomic region containing a 294-bp intron
flanked by salmon IFN cDNA sequences (GenBank accession
number AY327544). The first sequence contained the start
codon of salmon IFN and was assumed to be part of exon I. As
shown in Figure 7, primers 24 and 29 amplified a 1048-bp product that contained 3 introns, 271, 126, and 311 bp in length,
flanked by exon 1 and exon 4. This sequence must originate
from another gene than that amplified by primers No. 16 and
No. 18 because of the difference in intron 1 size. The combined
exon sequence of the 1048-bp product was 16 and 19 nt different from the corresponding cDNA sequences of SasaIFN-a1
and SasaIFN-a2. This indicates the presence of a third salmon
IFN gene or pseudogene. Primers No. 19 and No. 21 amplified
a 525-bp genomic region containing a 329-bp intron flanked by
two salmon IFN sequences assumed to be exon 4 and exon 5
(GenBank accession number AY327545). PCR of genomic
DNA using various primers from the 39-UTR indicated that no
more introns were present in the terminal part of the IFN genes.
These data suggest that the salmon IFN genes contain 5 exons
and 4 introns (Fig. 8). All introns possess the classic 59GT and
39AT intron splice motifs (Fig. 7). The first exon contained the
59-UTR, the 23 amino acid signal sequence, and the 22 first
amino acids of the mature protein. Exons 2, 3, and 4 encoded
25, 59, and 26 amino acids, whereas exon 5 encoded 29 amino
acids, the translation stop codon, and the 39-UTR.
609
DISCUSSION
We describe cloning of two IFN genes from Atlantic salmon
that both encode precursor proteins containing 175 amino acids,
which are putatively cleaved into 152 amino acid mature proteins called SasaIFN-a1 and SasaIFN-a2. Several lines of evidence suggest that the cloned cDNAs encode type I IFNs. First,
the translated amino acid sequences showed significant homology with mammalian IFN-a but no homology with IFN-g
or non-IFN proteins. Second, human HEK293 cells transfected
with the SasaIFN-a1 cDNA produced and secreted high titers
of acid-stable antiviral activity, which protected salmonid cells
against IPNV. In addition, the recombinant SasaIFN-a1 induced Mx protein in the fish cells. Mx protein is a major antiviral protein induced by type I IFN in mammals(3,32) and has
recently been cloned from Atlantic salmon.(33) Third, transcripts
of the IFN genes were not expressed constitutively but were induced in both live fish and TO cells by the dsRNA poly(I)poly(C), which is a well-known inducer of type I IFN. Moreover, the presence of several putative mRNA destabilization
elements (ATTTA) in the 39-UTR of the IFN cDNAs is a typical feature of cytokines, including IFN. (26,27)
Surprisingly, the Atlantic salmon IFN genes were found to
contain 5 exons and 4 introns (Fig. 8). The gene structure has
to be confirmed by cloning of full-length genomic salmon IFN
sequences but is similar to the gene structure of a zebrafish IFN
gene deposited in the gene bank (AJ544820), which also contains 5 exons. The sizes of IFN exons are similar in the two
species, but the introns are generally smaller in salmon than in
zebrafish (Fig. 8). The presence of introns in the fish IFN genes
is in contrast to the typical intron lacking feature of type I IFN
of mammals and birds. Loss of introns has, however, not been
uncommon during evolution of vertebrates.(34) For example, introns in the coding region of the recombination activating gene
rag 1 have been identified in teleosts but not in higher verte-
FIG. 7. Partial genomic IFN sequence from Atlantic salmon. The nucleotide sequence was amplified from genomic DNA by
PCR using primers No. 24 and No. 29 (Table 1) and corresponds to position 98–437 in the cDNA sequence of SasaIFN-a1. Exon
sequences are shown in capital letters and introns in lowercase italic letters.
610
FIG. 8. Schematic diagram depicting the gene structure of
putative Atlantic salmon IFN gene compared with zebrafish IFN
gene (AJ544820). Values represent nucleotide numbers. Unshaded boxes represents exons, and shaded boxes represent
UTRs. Lines represent introns and are drawn one fifth.
brates.(35) It is thus possible that type I IFNs have lost introns
during the evolution of higher vertebrates. Analysis of the zebrafish and pufferfish genomes may answer whether fish also
possess intronless type I IFN genes. Genes of a novel human
type I IFN designated IFN-l have recently been discovered on
chromosome 19, which also contain 5 exons and 4 introns.(36)
However, its relationship to the fish IFNs is uncertain because
IFN-l did not appear in either BLASTX or BLASTP analysis
of salmon IFN sequences, and CLUSTAL X alignment of
SasaIFN-a1 and IFN-l1 showed only 16% sequence identity.
The gene structure of fish IFN is clearly different from that of
IFN-g from higher vertebrates, which have 3 introns and 4 exons with sizes different from the fish IFN exons.(37)
The functional properties of SasaIFN-a1 are in agreement
with those of type I IFN-like activity produced by Atlantic
salmon macrophages stimulated with poly(I)-poly(C).(14,15)
However, with the cloned salmon IFN, more IFN activity with
a defined content is available for further studies of virus-fish
interactions. Activity in supernatants from HEK293 cells transfected with the salmon IFN gene was at least 200-fold higher
than in supernatants from stimulated macrophages. The observation that IFN transcripts were induced more rapidly in live
fish than in TO cells in response to poly(I)-poly(C) suggests
that some cell populations in the fish, perhaps leukocytes, are
more reactive to poly(I)-poly(C) than are TO cells. The low
abundance of IFN transcripts in the head kidney of the poly(I)poly(C) fish support that salmon IFN mRNA is short-lived, as
suggested by the presence of the ATTTA elements in the 39UTR of the mRNA.
The difference in cDNA size and sequence of SasaIFN-a1
and SasaIFN-a2 suggests that they represent distinct type I IFN
genes. The expression of the two IFN genes in the head kidney
was supported by Northern blot analysis of mRNA from
poly(I)-poly(C)-treated Atlantic salmon, which showed two
IFN transcripts with sizes similar to the cloned IFN cDNAs.
The head kidney is the major hematopoietic organ of Atlantic
salmon. The two IFNs may thus be produced by leukocytes. An
important question is whether nonhematopoietic cells produce
type I IFNs different from SasaIFN-a1 and SasaIFN-a2. In
mammals, IFN-a are primarily produced by hematopoietic
cells, whereas IFN-b is produced by fibroblasts.(1)
The genome of salmonids is believed to be in part
tetraploid.(38) This adds complexity to the organization of
ROBERTSEN ET AL.
salmon genes and may explain why some genes are present in
duplicate. At present, we do not know if SasaIFN-a1 and SasaIFN-a2 are clustered on the same chromosome or if they are
present on different chromosomal locations. Development of
multigene families appears to be a common feature of type I
IFNs of higher vertebrates.(39) In contrast, IFN-g of birds and
mammals is the product of only one gene. The type I IFN genes
of human are clustered, IFN-a have multiple copies, whereas
IFN-b has one gene copy.(40) The diversification of IFN-a
genes is suggested to have occurred after the separation of the
main mammalian orders by duplication followed by gene conversion.(39) The genome of Atlantic salmon most probably encodes additional transcribed genes or pseudogenes, as indicated
by sequence in Figure 7. Preliminary results indicate that truncated versions of salmon IFN are expressed (unpublished observations). The number of salmon IFN genes has yet to be determined, however.
The putative salmon IFNs have a molecular mass in agreement with the 18 kDa determined for the low molecular mass
IFN activity produced by rainbow trout gonad cells.(19) The
salmon IFNs were notably shorter than the zebrafish IFN, which
has a putative length of 163 amino acids. The salmon and zebrafish IFNs contain the same number and positions of cysteines and show 45% sequence identity. IFN-a from different
mammals show similar degree of homology. In contrast to zebrafish IFN, the salmon IFNs possess a glycosylation motif.
The degree of glycosylation varies among vertebrate type I
IFNs, from none in most IFN-a to 3 in mouse I IFN-b and 4
in the chicken IFNs. Thus, glycosylation of IFN does not appear to be conserved during evolution. Like the zebrafish IFN,
the salmon IFNs show no resemblance with a previously published sequence for flatfish IFN,(41) but the latter is apparently
more similar to bacteriophage Mu genes than IFN genes(6) and
should, therefore, not be classified as an IFN.
The sequence similarity suggested that the salmon IFNs are
more related to mammalian IFN-a than IFN-b or type I IFNs
from birds. Phylogenetic analysis demonstrated, however, that
the salmon and zebrafish IFNs form a group separated from
mammalian IFN-a and IFN-b and the chicken type I IFNs. This
supports phylogenetic analyses, which suggests that IFN-a and
IFN-b appeared at later stage in evolution. In fact, the mammalian IFN-a and IFN-b genes are proposed to have originated
by duplication of a progenitor after the divergence of birds about
250 million years ago.(39) Although chicken IFN-a and IFN-b
apparently are not orthologs of mammalian IFN-a and IFN-b,
they appear to be functional homologs.(4,42) IFN-a and IFN-b
show only 30%–45% sequence identity in both mammals and
chicken. Whether Atlantic salmon or other fish species possess such diverse type I IFN molecules remains to be determined.
The signal sequence of the salmon IFNs showed hardly any
homology with signal sequences of mammalian IFNs, which
demonstrates that the degree of conservation in IFN structure
is much larger than the conservation of signal sequences.
Clearly, however, even human cells possess the ability to cleave
and export the Atlantic salmon IFN precursor. This indicates
that the protease(s) cleaving the preprotein and the export mechanisms have broad specificity. Still, there appears to be some
conservation of cleavage and export mechanisms for type I IFN
within the teleosts because the signal sequences of Atlantic
ATLANTIC SALMON IFN
salmon and zebrafish IFNs showed a marked degree of similarity.
The presence of a 5 a-helix structure typical of type I IFNs
suggests that SasaIFN-a1 has a similar conformation to other
type I IFNs despite a relatively low percent sequence identity.
The IFNs are members of the a-helical cytokine family, which
adopts a common up-up-down-down 4-helix-bundle topology.(9) The mature Atlantic salmon IFN is one of the shortest
IFNs yet detected. The shortness of the molecule is due to an
8 amino acid gap that occurs between helices C and D. Whether
this is due to deletion(s) in the ancestral IFN gene or an insertion(s) in the higher vertebrate IFN genes is uncertain at present. The alignment revealed that some amino acid positions appear to be conserved in all type I IFNs and may be important
for the stabilization and activity of the IFNs. These are Leu-19,
Trp-75, Leu-94, Leu-120, Trp-130, and Arg-134 in the salmon
IFN. Several other amino acids in the salmon IFN sequence are
present in most, but not all, of the other IFN sequences and
may, therefore, also have their origin in an ancestral IFN. In
particular, both salmon and zebrafish IFN possess Cys-1 and
Cys-97, which are present and thought to form a disulfide bond
in most type I IFNs except mammalian IFN-b.(9–11) On the other
hand, both salmon and zebrafish IFN lack the two other disulfide-forming cysteines (positions 29 and 138 of human IFNa2b), which are present in most other type I IFNs except mammalian IFN-b. This indicates that the ancestral vertebrate IFN
possessed only one pair of cysteines and that the other pair appeared at a later stage but before divergence of the birds.
The type I IFNs in vertebrates serve several important functions besides antiviral activity. Examples of accessory functions
are activation of NK cell cytotoxic activity, antiproliferatory effects on cells, in vivo induction of memory CD8 T cell proliferation, upregulation of class I MHC expression, and inhibition
of IL-12 expression.(43) Moreover, different IFNs exert regulatory effects on each other’s expression.(44) Accessory functions
of type I IFNs in fish are likely to be present but have not yet
been demonstrated.
ACKNOWLEDGMENTS
We thank Aud Øvervatn and Geir Bjørkøy for their assistance with transfection of HEK293 cells. This work was supported by the Research Council of Norway (grants 134168/120
and 151938/150).
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Address reprint requests or correspondence to:
Dr. Børre Robertsen
Norwegian College of Fishery Science
University of Tromsø
N-9037 Tromsø
Norway
Tel: 147 77 64 61 30
Fax: 147 77 64 60 20
E-mail: [email protected]
Received 14 March 2003/Accepted 9 July 2003
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.