Abbreviations 2-AP, 2-aminopurine; EMEM, Eagle’s minimal essential medium; IFN, interferon; IPNV, infectious pancreatic necrosis virus; IRF, interferon regulatory factor; IRF-E, interfer
Trang 1contain two main regulatory regions
Veronica Bergan, Silje Steinsvik, Hao Xu, Øyvind Kileng and Børre Robertsen
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Norway
The type I interferon (IFN) system plays a critical role
in the innate immune defense against viruses in
verte-brates Virus-infected cells synthesize and secrete type I
interferons (IFN-a⁄ b), which circulate in the body and
protect other cells from viral infection The antiviral
action is caused by binding of IFN-a⁄ b to the type I
IFN receptor resulting in activation of transcription
of several hundred IFN-stimulated genes, some of
which encode proteins that inhibit viral replication
The antiviral properties of at least three type I
IFN-induced proteins are well established These comprise dsRNA-activated protein kinase R (PKR), 2¢,5¢-oligo-adenylate synthetase and Mx proteins [1]
Although the structures of the IFN-a and IFN-b promoters from human and mouse have been long known, the mechanisms involved in viral induction of type I IFNs have only recently been uncovered [2,3]
Of great importance has been the discovery of IFN super-producing blood cells called plasmacytoid dendritic cells (pDCs) and the realization that the
Keywords
Atlantic salmon; interferon promoter;
interferon regulatory factor; nuclear factor
kappa B (NFjB); poly(I:C)
Correspondence
B Robertsen, Department of Marine
Biotechnology, Norwegian College of
Fishery Science, University of Tromsø,
N-9037 Tromsø, Norway
Fax: +47 776 45110
Tel: +47 776 44487
E-mail: borre.robertsen@nfh.uit.no
(Received 24 April 2006, revised 9 June
2006, accepted 15 June 2006)
doi:10.1111/j.1742-4658.2006.05382.x
Recognition of viral nucleic acids by vertebrate host cells results in the syn-thesis and secretion of type I interferons (IFN-a⁄ b), which induce an anti-viral state in neighboring cells We have cloned the genes and promoters of two type I IFNs from Atlantic salmon Both genes have the potential to encode IFN transcripts with either a short or a long 5¢-untranslated region, apparently controlled by two distinct promoter regions, PR-I and PR-II, respectively PR-I is located within 116 nucleotides upstream of the short transcript and contains a TATA-box, two interferon regulatory factor (IRF)-binding motifs, and a putative nuclear factor kappa B (NFjB)-bind-ing motif PR-II is located 469–677 nucleotides upstream of the short tran-script and contains three or four IRF-binding motifs and a putative ATF-2⁄ c-Jun element Complete and truncated versions of the promoters were cloned in front of a luciferase reporter gene and analyzed for promo-ter activity in salmonid cells Constructs containing PR-I were highly induced after treatment with the dsRNA poly(I:C), and promoter activity appeared to be dependent on NFjB In contrast, constructs containing exclusively PR-II showed poor poly(I:C)-inducible activity PR-I is thus the main control region for IFN-a⁄ b synthesis in salmon Two pathogenic RNA viruses, infectious pancreatic necrosis virus and infectious salmon anemia virus, were tested for their ability to stimulate the minimal PR-I, but only the latter was able to induce promoter activity The established IFN promoter-luciferase assay will be useful in studies of host–virus inter-actions in Atlantic salmon, as many viruses are known to encode proteins that prevent IFN synthesis by inhibition of promoter activation
Abbreviations
2-AP, 2-aminopurine; EMEM, Eagle’s minimal essential medium; IFN, interferon; IPNV, infectious pancreatic necrosis virus; IRF, interferon regulatory factor; IRF-E, interferon regulatory factor binding element; ISAV, infectious salmon anemia virus; LPS, lipopolysaccharide; NFjB, nuclear factor kappa B; pDCs, plasmacytoid dendritic cells; PDTC, pyrrolidine dithiocarbamate; poly(I:C), polyinosinic polycytidylic acid; PKR, dsRNA-activated protein kinase; PR, promoter region; PRD, positive regulatory domain; TLR, toll-like receptor.
Trang 2mechanism of virus-mediated induction of IFNs is
dif-ferent in pDCs and other body cells [4,5] Most
nucle-ated cells of the body produce IFN-a⁄ b in response to
recognition of dsRNA intermediates produced during
viral replication The main sensors of dsRNA are two
intracellular RNA helicases (RIG-I and MDA5) [6–9],
which, on binding of dsRNA, interact with the
mitoch-ondrial protein MAVS (also called IPS-1) [10,11] This
interaction leads to transcriptional induction of the
IFN-b gene through the co-ordinated activation of the
transcription factors interferon regulatory factor 3
(IRF-3), nuclear factor kappa B (NFjB) and
ATF-2⁄ c-Jun heterodimer [2] Infected cells secrete mainly
IFN-b in the initial phase of infection, but switch to
IFN-a as a result of induction of IRF-7 synthesis
dur-ing the subsequent amplification phase of the IFN
response [12,13] pDCs are specialized IFN producers
and represent a major source of IFN-a in humans
through activation of IRF-7 [14] In pDCs, the main
sensors of viral infection are Toll-like receptors (TLRs)
expressed on the surface or in endosomes that
recog-nize viral RNA or DNA Human pDCs mostly express
TLRs, which recognize ssRNA (TLR7 and TLR8) or
dsCpG-rich DNA (TLR9) [15] Recognition of viral
nucleic acids by TLRs activates IRF-7, which
tran-scriptionally activates multiple IFN-a genes [16,17] A
major difference between pDCs and other cell types is
their capacity to constitutively produce relatively high
concentrations of IRF-7 [18]
Virus-induced expression of IFN-a and IFN-b genes
is mediated by regulatory sequences located within
200 bp upstream of the transcription start site of their
promoters [19] The IFN-b promoter contains four
positive regulatory domains (PRDs), which bind IRFs:
mainly IRF-3 and IRF-7 (PRDI and PRDIII), NFjB
(PRDII) and ATF-2⁄ c-Jun (PRDIV) [20] The
promot-ers of IFN-a genes all contain DNA elements binding
IRF members, notably IRF-3, IRF-5 and IRF-7, but
they do not contain NFjB or ATF-2⁄ c-Jun binding
sites [21]
In mammals and birds, IFN-a⁄ b genes are encoded
by intron-lacking genes whereas IFN-k genes possess a
4-intron⁄ 5-exon structure [22,23] Recently, type I IFN
genes of teleost fish were shown to possess a gene
structure similar to IFN-k genes, although their
pro-tein sequences are more similar to IFN-a than IFN-k
[24–28] At present, little is known about the
regula-tion of fish IFN genes, although the promoter of the
zebrafish type I IFN gene was recently reported to
contain one IRF-binding site and one NFjB-binding
site [28] The present work shows that type I IFN
genes of Atlantic salmon show a rather unique
organ-ization of the promoter in comparison with mammals,
birds and zebrafish Atlantic salmon stimulated with the dsRNA polyinosinic polycytidylic acid [poly(I:C)] produces an IFN transcript with a short 5¢-UTR called SasaIFN-a1, and another IFN transcript with a long 5¢-UTR called SasaIFN-a2 In this work, we cloned two different Atlantic salmon IFN genes from genomic DNA that encode putative transcripts similar in sequence to SasaIFN-a1 and SasaIFN-a2 Surprisingly both genes apparently have the potential to produce both a short and long transcript because of the loca-tion of two separate promoter regions, one of which is present in the 5¢-UTR of the long transcript To per-form functional analysis of the Atlantic salmon IFN promoter region, we fused the complete and truncated versions of the promoter region to a luciferase reporter gene and transfected it into Chinook salmon embryo (CHSE-214) or Atlantic salmon head kidney TO cells Promoter activity was measured after stimulation with poly(I:C) or virus infection
Results
Cloning of full-length type I IFN genes from genomic DNA
A genome walking approach was used to clone a 1281-nucleotide sequence upstream of the SasaIFN-a1 tran-scription start site This allowed design of primers that amplified genomic IFN sequences that expanded from )1281 of the promoter region (PR) to the polyA signal
by PCR Two full-length IFN genes, designated Sasa-IFN-A1 (A1 for short) and SasaIFN-A2 (A2), were identified in two different BAC clones (GenBank acces-sion nos DQ354152 and DQ354153) A summary of nucleotide data on the A1 and A2 gene is shown in Table 1 Both genes possessed the five-exon⁄ four-intron structure found previously in fish type I IFN genes, although the intron sizes were somewhat different from those originally found in DNA from Atlantic salmon [26] The joined exon sequences of A1 and SasaIFN-a1 cDNA are completely identical, and the joined exon sequences of A2 and SasaIFN-a2 cDNA have only three nucleotide differences, possibly because they represent different alleles (Table 2) In contrast, A1 diverges from SasaIFN-a2, with 10 mismatches, and A2 and SasaIFN-a1 diverge by 12 mismatches This strongly suggests that A1 encodes the SasaIFN-a1 transcript and A2 the SasaIFN-a2 transcript Overall differences in A1 and A2, including differences in pro-moter and intron regions (deletions, insertions and sub-stitutions), confirm that they represent two different genes rather than allele variants (Table 1) Two pseudo-genes were also identified in the screening of BAC
Trang 3clones, one having a premature stop codon (accession
no DQ354154) and one that appeared to be interrupted
by a transposase gene (accession no DQ354155) The
pseudogenes were not investigated any further in this
work
Analysis of the promoter regions
The alignment of the 765-bp sequence regions
upstream of the ORFs are very similar in the two
genes except for 10 nucleotide substitutions and two
insertions⁄ deletions (Fig 1) This was surprising
because SasaIFN-a1 was originally identified as a short
transcript (829 nucleotides) and SasaIFN-a2 as a long
transcript (1290 nucleotides) We thus expected that
the A1 gene would encode a short transcript and A2 a
long transcript The present data indicate, however,
that both genes have the potential to encode both
tran-scripts
A total of six (in A1) or seven (in A2) IRF-binding elements (IRF-E) were identified in the 765-nucleotide region upstream of the putative transcription start site
of SasaIFN-a1 (Fig 1) The motifs conform to the GAAA(G/C)GAAA(T/C) consensus sequence [29] and were located at positions )63, )116, )376, )503, )545, )639, and )669 relative to the putative Sasa-IFN-a1 transcription start site Interestingly, the IRF-E sequences at positions )116 and )545 were identical and probably bind the same IRF(s) In addi-tion, we found two potential NFjB-binding sites, one
in close proximity to the SasaIFN-a1 transcriptional start site ()80) and one more distant ()720) that appeared to be truncated in the A2 promoter An ATF-2⁄ c-Jun element, which is essential for activity of the human IFN-b promoter, was found in the distal promoter region in close proximity to the IRF-E at position )557 Moreover, an atypical TATA-box was located at position )42 in both genes, and two CCAAT-boxes at positions )296 and )579 in the A1 gene
In summary, both genes appear to possess two major regulatory regions:
(a) promoter region I (PR-I) located within 116 nucle-otides upstream of the short transcript, containing a noncanonical TATA-box, two IRF-binding motifs and
a putative NFjB-binding motif;
(b) promoter region II (PR-II), located 469–677 nucle-otides upstream of the short transcript, containing three to four IRF-binding motifs and an ATF-2⁄ c-Jun element
The putative salmon IFN promoters thus seem to have a unique feature, as PR-I controls the synthesis
of a transcript with a short 5¢-UTR and PR-II controls the synthesis of a transcript with a long 5¢-UTR Accordingly, the 5¢-UTR of the long transcript in fact contains PR-I
Activity of the A1 and A2 promoters
on poly(I:C) induction
To study the activity of the promoters, we cloned full-length and deleted versions of the promoters in front of a promoterless luciferase reporter gene (Fig 2A) From the A1 gene the following constructs were made: pA1()135), pA1()202) and pA1()333) containing only PR-I; and pA1()747) and pA1()1281) containing both PR-I and PR-II From the A2 gene, the construct pA2()275) containing only PR-II was made The constructs were transfected into CHSE-214 cells or Atlantic salmon TO cells along with
a constitutively expressed b-gal standard (pJatLacZ) and then stimulated with poly(I:C) to induce IFN
Table 1 Comparison of the Atlantic salmon genomic A1 and A2
IFN sequences.
Sequence compared
with SasaIFN-a1
Number of nucleotides
a Putative 5¢-UTR of A2 was 501 nucleotides based on similarities
to SasaIFN-a2, but for comparison reasons the 5¢-UTR of A1 and
A2 was set to the same b Only partial 3¢-UTR of A1 was cloned
and compared.
Table 2 Comparison of salmon IFN exon sequences to verify
link-age between genomic and cDNA clones Percentlink-age similarity is
shown in the upper triangle, and number of nucleotide differences
in the lower triangle The most likely match is highlighted.
Pairwise percentage identity
SasaIFN-a1 SasaIFN-a2 Genomic A1 Genomic A2
Nucleotide differences
Trang 4promoter activity Figure 2 shows the luciferase
activity from the different constructs relative to b-gal
measurements for poly(I:C)-stimulated or untreated
CHSE-214 (Fig 2B) or TO cells (Fig 2C) All
con-structs were induced by poly(I:C) in both cell types
The overall higher promoter activity observed in
CHSE-214 cells compared with TO cells is probably
due to the fact that poly(I:C) was transfected
into CHSE-214 cells, whereas it was applied
extra-cellularly to TO cells In CHSE-214 cells, the level of
induction was highest for pA1()1281), pA1()747)
and pA1()202), and lowest for pA2()275) This
indicates that PR-I is most important for poly(I:C)
induction in these cells In TO cells, all constructs
showed similar levels of relative luciferase activity
after stimulation with poly(I:C) The main difference
from CHSE-214 cells was that pA1()1281), pA1()747)
and pA2()275) all showed relatively high basal
luciferase activity Accordingly, the level of
induc-tion was highest for pA1()333), pA1()202) and
pA1()135) The minimal promoter showing highest inducibility in TO cells was thus pA1()135), contain-ing only PR-I
The highly inducible minimal promoter construct, pA1()202), and the full-length construct, pA1()1281), were next compared for poly(I:C) induction in a time course study in CHSE-214 and TO cells (Fig 3) In CHSE-214 cells, both promoter constructs were hardly induced at all at 12 h, but showed increasing luciferase activity at 24 h and 48 h after poly(I:C) treatment (Fig 3) At 48 h, the minimal IFN promoter was induced more than 50-fold, whereas the full-length promoter was induced only 13-fold (Fig 3A) The minimal promoter construct showed similar time kinet-ics in TO cells, whereas the pA1()1281) construct showed hardly any induction at any of the time points (Fig 3B)
A dose–response curve for poly(I:C) induction of the minimal promoter construct was established As little as 50 ngÆmL)1 was sufficient to induce the
Fig 1 Promoter regions of SasaIFN-a1 (A1) and SasaIFN-a2 (A2) genes Potential tran-scription factor binding sites and translation start codon are boxed The two putative transcription start sites are indicated by bent arrows Bold lowercase letters or dashes indicate nucleotides in A2 which are differ-ent from A1 IRF-core binding motifs that match the GAAANN consensus are highligh-ted with bold letters Putative promoter regions (PR-I and PR-II) are shaded in grey.
Trang 5promoter significantly (14-fold), and 500 ngÆmL)1
poly(I:C) was sufficient to give maximal induction
(50-fold) of the promoter (Fig 4)
The optimal conditions to study the salmon inter-feron promoter were to use CHSE-214 cells and transfect them with the minimal promoter construct,
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Fig 2 Analysis of IFN promoter activity in CHSE-214 and TO cells (A) Salmon IFN promoter–luciferase constructs including the posi-tions of IRF-E, NFjB, and ATF-2 ⁄ c-Jun sites relative to the tran-scription start site (+1) pA1 constructs are from the putative SasaIFN-a1 promoter, and pA2 is the putative SasaIFN-a2 promo-ter (B) CHSE-214 or (C) TO cells were transiently transfected with the promoter constructs plus a b-gal internal control vector in 24-well plates At 24 h after transfection, triplicate wells of cells were treated with 1 lgÆmL)1 poly(I:C) (and Fugene) or left untreated Luciferase and b-gal activities were measured 48 h after the stimulus using the dual-light luciferase kit Luciferase activity is expressed relative to b-gal (mean ± SD from three wells).
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pA1(1.2) pA1(-202)
pGL3basic
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CHSE cells
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B
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pGL3basic
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24 h
48 h
TO cells
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Fig 3 Induction of the minimal IFN promoter ( )202) and the full-length IFN promoter region ( )1.2kb) over time in (A) CHSE-214 and (B) TO cells Reporter vectors: pGL3basic; absent promoter, pA1( )202); minimal IFN promoter, pA1()1.2kb); full-length IFN upstream region Cells were transiently transfected with the repor-ter constructs plus a b-gal inrepor-ternal control vector in 24-well plates.
At 24 h after transfection, triplicate wells of cells were treated with
1 lgÆmL)1 poly(I:C) (and Fugene) or left untreated (control) Lucif-erase and b-gal activities were measured 12, 24 and 48 h after the stimulus using the dual-light luciferase kit Fold induction is lucif-erase activity expressed relative to b-gal of poly(I:C)-treated cells divided by nontreated control cells (mean ± SD from three wells).
Trang 6pA1()202), trigger the promoter with at least
500 ngÆmL)1 poly(I:C) (complexed with the Fugene
transfection reagent), and read the luciferase values at
48 h after poly(I:C) treatment
Effect of LPS and virus infection on the salmon
IFN promoter
As the salmon IFN promoter contained a putative
NFjB-binding motif, we wanted to test if
lipopolysac-charide (LPS) was able to induce the IFN promoter
However, 50 lgÆmL)1 LPS did not increase luciferase
activity from the minimal IFN promoter in neither
CHSE-214 (Fig 5) or TO (not shown) cells The cells
were also treated with poly(dG:dC) (complexed to
Fu-gene), to study whether dsDNA triggered the minimal
IFN promoter, but this was not the case (Fig 5)
As viruses are known to induce IFN production
through dsRNA intermediates, the effect of virus
infec-tion on the IFN promoter was examined For this
pur-pose, we used the two most common viral pathogens
of Atlantic salmon, the aquatic birnavirus infectious
pancreatic necrosis virus (IPNV) and the
orthomyxo-virus infectious salmon anemia orthomyxo-virus (ISAV) No
increase in promoter activity was detected 48 h after
treatment of CHSE-214 cells with multiplicity of
infec-tion (moi) 5 of live IPNV (Fig 5) Strong cytopathic
effects occurred in the cells 72 h after infection for
IPNV As CHSE-214 cells are nonpermissive to most
ISAV strains, we used TO cells to study the effect of
ISAV infection on the IFN promoter ISAV is strongly detectable in these cells 48 h after infection and produ-ces cytopathic effects after a period of 4–7 days [30] ISAV (moi 5) was able to induce the minimal IFN promoter 4–5-fold at 48 and 72 h, and more than nine-fold 96 h after infection in TO cells (Fig 6) Approxi-mately 10–20% of the cells showed a cytopathic effect
at 96 h These results show that, in nonimmune cells, ISAV is able to turn on the IFN promoter, although
0 5 10 15 20 25 30 35
Untreated Poly(I:C) Poly(dG:dC) LPS IPNV
Fig 5 Effect of different stimulants on the minimal IFN promoter
in CHSE-214 cells Cells were transiently transfected with the pA1( )202) construct plus a b-gal internal control vector in 24-well plates At 24 h after transfection, triplicate wells of cells were trea-ted with 1 lgÆmL)1 poly(I:C) (and Fugene), 1 lgÆmL)1poly(dG:dC) (and Fugene), 50 lgÆmL)1LPS, moi 5 of IPNV, or left untreated Lu-ciferase and b-gal activities were measured 48 h after the stimulus using the dual-light luciferase kit Fold induction is luciferase activity expressed relative to b-gal of stimulated cells divided by nontreated control cells (mean ± SD from three wells).
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ng/ml Poly(I:C) Fig 4 Dose–response of poly(I:C) induction on the minimal IFN
promoter ( )202) CHSE-214 cells were transiently transfected with
the pA1( )202) construct plus a b-gal internal control vector in 24
well plates At 24 h after transfection, triplicate wells of cells were
treated with different concentrations (5000–0 ngÆmL)1) of poly(I:C)
(and Fugene) or left untreated Luciferase and b-gal activities were
measured 48 h after the stimulus using the dual-light luciferase kit.
Fold induction is luciferase activity expressed relative to b-gal of
poly(I:C)-treated cells divided by nontreated control cells (mean ±
SD from three wells).
0 2 4 6 8 10 12 14
Time (h) Fig 6 Effect of ISAV infection on the minimal IFN promoter in TO cells Cells were transiently transfected with the pA1( )202) con-struct plus a b-gal internal control vector in 24-well plates At 24 h after transfection, triplicate wells of cells were treated with moi 5
of ISAV or left untreated Luciferase and b-gal activities were meas-ured at 12, 24, 48, 72 and 96 h after the stimulus using the dual-light luciferase kit Fold induction is mean luciferase activity expressed relative to b-gal of stimulated cells divided by nontreated control cells (mean ± SD from three wells).
Trang 7at a very late stage of infection, whereas IPNV is
apparently unable to trigger the IFN promoter
Effect of 2-aminopurine (2-AP) and pyrrolidine
dithiocarbomate (PDTC) on the salmon IFN
promoter
The NFjB inhibitor, PDTC, and the kinase inhibitor,
2-AP, were used to study the involvement of NFjB in
the poly(I:C)-induced activation of the IFN promoter
As shown in Fig 7, PDTC produced 90% inhibition
of poly(I:C)-induced promoter activity at 1 lm and
70% inhibition at 0.01 lm, which suggests that
NFjB is indeed involved in the poly(I:C)-induced
acti-vation of the salmon IFN promoter About 55%
inhi-bition of promoter activity was observed with 0.01 and
0.1 mm 2-AP, which indicates that PKR or another
2-AP-sensitive kinase is involved in activation of
salmon IFN promoter
Long IFN transcripts are produced at very low
levels in TO cells
Northern blot studies have previously shown that
tran-scripts with both short and long 5¢-UTRs are produced
in head kidney of poly(I:C)-treated Atlantic salmon
[26] To examine whether both transcripts were
pro-duced in cultured TO cells after poly(I:C) induction,
a quantitative RT-PCR assay was designed Primers
were designed from conserved regions within the ORF
to detect total IFN transcripts, and within the 5¢-UTR
of SasaIFN-a2 to detect long IFN transcripts Total IFN transcripts were gradually increased over time in response to poly(I:C) stimulation, starting from basal levels of about 4· 104 transcript copies at 0 h, and reaching almost 108 copies at 24 h (Fig 8A) Tran-scripts with a long 5¢-UTR were also produced in TO cells (Fig 8B), but at very low levels ranging from 14 copies at 0 h to 5044 24 h after stimulation; 2000–
20 000 times below the total IFN transcript quantity Total IFN was thus induced about 2300-fold at 24 h compared with time point 0 h, whereas IFN2 was induced only 360-fold This confirms that the proximal PR-I is most activated upon poly(I:C) induction, at least in nonimmune cells, producing high amounts of short IFN transcripts, while the distal PR-II is poorly induced in TO cells
Discussion
In this work we have identified the genes encoding the two previously reported cDNAs SasaIFN-a1 and
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PDTC
PDTC
2-AP
2-AP
2-AP
Fig 7 Effect of the kinase inhibitor 2-AP and the NFjB inhibitor
PDTC on poly(I:C)-induced expression of the minimal IFN promoter.
CHSE-214 cells were transiently transfected with the pA1( )202)
construct and a b-gal internal control vector in 24-well plates At
24 h after transfection, triplicate wells of cells were treated with
different concentrations of inhibitors followed by poly(I:C) (and
Fugene) treatment or not (control) Luciferase and b-gal activities
were measured 48 h after the stimulus using the dual-light
lucif-erase kit Fold induction is luciflucif-erase activity expressed relative to
b-gal of poly(I:C)-treated cells divided by nontreated control cells
(mean ± SD from three wells).
B
7.00E+03 6.00E+03 5.00E+03 4.00E+03 3.00E+03 2.00E+03 1.00E+03 0.00E+00
4
IFN2
Time (h)
A
1.20E+08
1.00E+08
8.00E+07
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0.00E+00
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Total IFN
Time (h)
Fig 8 Quantitative real-time PCR of total IFN transcripts (A) and long IFN2 transcripts (B) in TO cells at different time points (hours) after stimulation with poly(I:C) (and Fugene).
Trang 8SasaIFN-a2 [26], and studied their promoters The
genes were previously thought to be distinguished by
their ability to produce different length transcripts
However, the present data suggest that both genes
have the potential to encode transcripts with either a
short or a long 5¢-UTR This can apparently be
explained by the presence of two main regulatory
regions in both genes: (a) a proximal promoter region
(PR-I) which includes position )202 to +26 from the
SasaIFN-A1 transcription start site, which controls
synthesis of a short transcript; (b) a distal region
(PR-II) corresponding to position )747 to )413, which
gives rise to a long transcript (Fig 1) Luciferase
reporter gene assays in two different salmonid cell lines
showed that PR-I was strongly induced by the
syn-thetic dsRNA, poly(I:C), whereas PR-II of the A2
gene was hardly induced at all (Fig 2A,B) This sug-gests that PR-I is the main control region
PR-I contains a putative NFjB-binding element flanked by two IRF-Es and is thus most similar to the human and mouse IFN-b and chicken IFN2 promoters (Fig 9) In contrast with IFN-b promoters, PR-I lacks
an ATF-2⁄ c-Jun element Comparison of IFN promo-ter sequences from different vertebrate species suggests that the essential IRF-Es responsible for virus-induced expression are located within the 170-nucleotide region upstream from the ORF, and they all match either the IRF-1⁄ 2 (AANNGAAA), the IRF-3 (G⁄CGAAANN)
or the IRF-7 (T⁄CGAAANN) consensus-binding motif (Table 3) [31–33] IRF-7 has recently been shown to be the most important IRF controlling type I IFN expres-sion, although IRF-3 also contributes substantially
Salmon IFNA1 Zebrafish IFN -800
-800
-360
-800
-700
-750
-900
Human IFNB Human IFNA1
Human IFNA4 Chicken IFN1-2
Chicken IFN2
ATF-2/c-Jun
NF κB IRF-E
TATA-box
Fig 9 Distribution of transcription factor binding sites in IFN promoters from selec-ted species.
Table 3 Comparison of proximal interferon regulatory elements within selected type I IFN promoters The IRF binding cores are shaded grey.
Trang 9[16,34] The role of IRFs in induction of fish IFNs is
as yet unknown In salmonids, only IRF-1 and IRF-2
have been cloned [35]
One of the main differences in mammalian IFN
pro-moters is the presence or absence of an NFjB-binding
element The putative NFjB-binding sequence in PR-I
(5¢-GGGAAATTCT-3¢) is only one nucleotide
differ-ent from the NFjB-binding site in the human IFN-b
promoter (5¢-GGGAAATTCC-3¢) The NFjB element
in the IFN-b promoter is believed to be essential for
an immediate early response to virus infection [36,37]
IFN-a promoters lack the NFjB element and are
usu-ally activated at a later time point, except for mouse
IFNA4, which also shows early expression in response
to virus infection [38] The structure of PR-I thus
sug-gests that it controls an early response to virus
infec-tion in Atlantic salmon A role for NFjB in the
activation of PR-I was further supported by two
dif-ferent inhibitor experiments First, dose-dependent
inhibition of promoter activity by the NFjB inhibitor
PDTC was shown (Fig 7) PDTC is a metal chelator
with antioxidant properties which specifically inhibits
NFjB-induced pathways [39] Secondly, partial
inhibi-tion of promoter activity by 2-AP (Fig 7), indicates a
role for PKR, a kinase known to be involved in NFjB
activation [40,41] Although fish PKR has yet to be
reported, PKR-like sequences are present in the
Gen-Bank On the other hand, it cannot be excluded that
2-AP inhibits another kinase in the IFN signaling
pathway
The zebrafish IFN promoter, which is the only other
fish IFN promoter characterized so far, is claimed to
contain an NFjB element, but the putative binding
site does not conform with the NFjB consensus [28]
However, the salmon and zebrafish IFN promoters
both contain two IRF-Es at similar position and
orien-tation (Fig 9) The IRF-E located at position )96 in
salmon and at position)115 in zebrafish differ by only
one nucleotide substitution and they are both present
in antisense orientation The second IRF-E, at)149 in
salmon and )151 in zebrafish, differs in three
nucleo-tide positions (Table 3) Some species seem to have
IRF-Es and ATF-2⁄ c-Jun elements in the distal region
from the major transcription site, but only the salmon
IFN promoters, and perhaps also human IFNA1
pro-moter, have the unique PR-I and PR-II organization
(Fig 9) The PR-II has a somewhat different structure
in the two salmon IFN genes (Fig 1) Both contain
three identical IRF-Es and an ATF-2⁄ c-Jun site
How-ever, only PR-II of A1 contains an NFjB element and
a CCAAT-box Furthermore, PR-II of A2 contains an
additional IRF-E Whether the PR-IIs of the two
genes are regulated differently is not yet known
Promoter constructs that have both PR-I and PR-II or only PR-II showed a basal expression independent of poly(I:C) induction (Fig 2C) Basal expression is, how-ever, a phenomenon often seen in promoter–reporter assays containing long upstream regions from the tran-scriptional start site The leakiness of transcription is probably due to lack of negative regulatory structures such as chromatin packing and⁄ or methylation Basal expression has also been observed for the zebrafish IFN promoter distal 5¢-flanking regions ()2.2 to )0.7 kb) in similar experiments [28]
PR-II probably controls synthesis of a transcript with a long 5¢-UTR Northern blotting showed that both transcripts are present in head kidney of poly(I:C)-treated Atlantic salmon, although the inten-sity of the long transcript was about half of the short transcript [26] In TO cells, however, the long tran-script was estimated to constitute only 10-3)10-4 of the total IFN mRNA (Fig 8) The long transcript thus appears to be mainly produced in cells of lymphoid tissues
The function of the long transcripts is interesting since both transcripts from one gene are believed to produce identical proteins Long 5¢-UTRs are often associated with genes related to cell growth and differ-ent types of cellular stress [42,43] Most of these genes are poorly translated because of complex secondary structure within the long 5¢-UTR or they contain small upstream ORFs that are translated before the major ORF [44] Recently, promoter activity was found in long 5¢-UTR sequences of genes that were believed to have internal ribosome entry sites (IRES) as a mechan-ism for translation [45–47] These alternative promot-ers were thought to be activated by certain types of stressors to speed up transcription to smaller and more efficiently translated mRNA; especially for genes that were required in small amounts and which could be toxic if over-produced [44] This strengthens the idea that the long 5¢-UTR may have a negative regulatory function in salmon IFN production In fact, alternative promoter options usually have regulatory functions or are associated with specific cell type expression [48] As salmonids have a tetraploid origin, the organization of the IFN promoter in the two regions may be import-ant for regulation of the expression levels of IFNs, to prevent overproduction from the many IFN loci in the salmon genome
The alternative promoter found in the 5¢-UTR of salmon IFN genes suggests an answer to another ques-tion on the evoluques-tion of the intronless IFN genes of birds and mammals The intronless type I IFNs of higher vertebrates most probably originated from a retro-transposition event involving the transcript of an
Trang 10ancestral intron-containing IFN gene This does,
how-ever, not immediately explain the origin of the IFN
promoters The present observation of a promoter in
the 5¢-UTR of salmon IFN transcripts suggests that
the promoter of higher vertebrate IFN-a⁄ b also
origin-ates from the same retroposition event that created the
first intronless IFN gene in vertebrates
dsRNA is thought to activate the NFjB pathway by
binding to TLR3, RIG-I⁄ MDA5 or PKR [6,41,49]
NFjB is involved in many different cellular stress
responses [50] LPS is a well-known inducer of NFjB
activation, but is also thought to be central to
TLR4-mediated activation of IRF-3 to induce the IFN-b gene
in mice [51,52] The minimal IFN promoter was not
triggered by LPS treatment in either CHSE-214 cells
(Fig 5) or TO cells (data not shown) This indicates
that LPS alone is not sufficient to give an IFN
response or that the cell types used lack cell surface
receptors for LPS such as TLR4 Results suggest that
NFjB has a role in the activation of the salmon IFN
promoter, but it cannot act alone to initiate
transcrip-tion
A hallmark of mammalian IFN-a⁄ b is their rapid
induction by virus infection mainly because of the
recognition of viral dsRNA products [20] Although
the dsRNA poly(I:C) strongly activated the salmon
IFN promoter, infection with neither IPNV nor ISAV
resulted in convincing activation of the promoter
(Figs 5 and 6) This suggests that both viruses have
developed mechanisms to avoid or inhibit the IFN
promoter in the early critical phases of infection For
IPNV, this may explain why the virus does not trigger
Mx protein production in cultured cells [30,53] IPNV
is very sensitive to IFN treatment, and the antiviral
mechanism is at least partly mediated by the Atlantic
salmon Mx1 protein [54] The chicken birnavirus,
infectious bursal disease virus, has also been shown to
inhibit transcription of IFN genes [55], which suggests
a common immunosuppressive mechanism of this
fam-ily of dsRNA viruses ISAV, on the other hand, was
able to induce the salmon IFN promoter 96 h after
infection (Fig 6) However, a previous report has
shown that 5 moi of ISAV resulted in peak Mx protein
expression 24–48 h after infection [30] This indicates
that ISAV may stimulate the Mx promoter
independ-ent of IFN ISAV belongs to the same family as
influ-enza viruses, Orthomyxoviridae The NS1 protein of
influenza virus is a well-known IFN antagonist which
is believed to act upstream of the IFN promoter,
through either NFjB or IRF-3 [56–58] The ISAV NS
protein is thought to be encoded by segment 7, and
may also represent a candidate antagonist of the
salmon IFN promoter [59] Taken together, our results
suggest that ISAV and IPNV are successful fish viruses that have developed strategies to hinder IFN produc-tion, at least in monocellular systems lacking signals from the multicellular lymphoid system However, the establishment of a salmon IFN promoter reporter assay gives the opportunity to search for viral proteins that antagonize IFN production
Experimental procedures
Cloning of genomic IFN sequences
Atlantic salmon genomic DNA was purified from full blood
of one individual fish by proteinase K digestion and
DNA library was prepared using the Universal Genome-Walker kit (Clontech Laboratories Inc., Mountain View,
CA, USA) Nested-PCR of GenomeWalker library DNA using the primer pairs BR23 and AP1 first and BR22 and AP2 second was performed according to the kit manual (primer details are listed in Table 4) The major PCR prod-ucts were sequenced and new primers were designed to clone full-length IFN genes from an Atlantic salmon BAC library purchased from BACPAC Resources (http://bacpac chori.org/salmon214.htm) The genomic sequence for Sasa-IFNa1 was obtained from the 409K8 BAC clone using the
The sequences were obtained with long distance PCR using the Dynazyme EXT PCR kit (Finnzymes Oy, Espoo, Finland) and cloned into the pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA, USA)
Promoter constructs
Various constructs of the promoter region of SasaIFN-A1 and SasaIFN-A2 were PCR-cloned into the pGL3-basic vector (Promega, Southampton, UK) using the primers specified in Table 4 The full-length luciferase construct,
b-galactosi-dase control vector (pJatLacZ) was a gift from J Jørgensen (Norwegian College of Fishery Science, Tromsø, Norway), and contains the LacZ gene under the control of a rat b-actin promoter [61]
Cells and viruses
TO cells originate from Atlantic salmon head kidney [62] and were obtained from H Wergeland (University of