JOURNAL OF INTERFERON RESEARCH 11:341-350 (1991)Mary Ann Liebert, Inc., Publishers

The Porcine Family of Interferon-co: Cloning, StructuralAnalysis, and Functional Studies of Five Related Genes

DOMINIQUE MEGE, FRANÇOIS LEFEVRE, and CLAUDE LABONNARDIERE

ABSTRACT

We report the molecular characterization of the porcine IFN-w multigenic family which appears to comprisesix to seven loci. Five of these loci were cloned and sequenced, revealing two pseudogenes (ujPoIFN-oj, and -w2)and three genes with an intact reading frame (PoIFN-w3, -oj4, and -oj5) that could encode preproteins of179-190 amino acids including a putative signal peptide of 23 residues. By comparison of porcine IFN-w codingsequences to those of known mammalian counterparts, it appeared that porcine sequences contain an in-framefive-codon deletion (between positions 113 and 117) in a region of relatively high sequence variability. In vitrotranscription and translation of the three potentially functional reading frames gave rise to proteins withantiviral activity, showing that the porcine-specific deletion does not abolish the biological activity. Compara-tive analysis of flanking sequences revealed unique features of the 3' untranslated region of IFN-w4 gene: Itcontained a consensus estrogen-responsive element (ERE) in the vicinity of an extensive A-T-rich sequenceknown to serve a specific regulatory role in the expression of many genes involved in the inflammatoryresponse. This finding suggests that the expression of the functional members of the porcine IFN-w family maybe mediated through different mechanisms. The expression of these genes was studied by Northern blotanalysis of mRNAs from pig conceptus (days 14-20). Five bands of poly(A)+ RNAs hybridized with an IFN-wprobe provided the stringency was low, suggesting that a distinct family of IFN-w-related genes were expressedby porcine trophoblastic cells during early gestation.

INTRODUCTION

IN SEVERAL MAMMALIAN SPECIES, Type I interferon (IFN)genes form a complex group of clustered intronless genes,

which appear to fall into three distinct families (a, to, and ß) on

the basis of sequence homology. The IFN-w family (also re-

ferred to as IFN-a,,*) has been found in human, bovine,1" andequine'2' species by screening cDNA or genomic libraries withIFN-ct-specific probes under low-stringency conditions. Theanalysis of cDNA libraries prepared from virus-induced cul-tures of human leukocytes'" or human lymphoblastoid cellline'1' indicates that human (Hu) IFN-to is virally induced andcan therefore be considered as a fraction of the IFN mixturesecreted by stimulated leukocytes. While the human, bovine,and porcine families of IFN-a genes appear to be similar insize,'4'51 the size of the IFN-co multigenic family considerably

varies among different species. Thus, at least 5 and 30 membersare estimated in human and bovine genomes, respectivelywhereas these genes seem to be absent in the canine genome.161IFN-w preproteins are 195 amino acids long with a putativesignal peptide of 23 residues; they contain six additional aminoacids at their carboxyl termini, as compared to IFN-a proteins.The differences in sequence and antigenicity'71 between IFN-aand -co suggest different biological activities and a differentialregulation. Although IFN-w is a component of natural mixturesof IFN produced by leukocytes after viral infection,""' little isknown about the properties, receptor interactions, or functionalspecialization of this class of IFNs.

It was recently shown that the trophoblast of ovine and bo-vine conceptuses (embryo + annexes) in early gestation syn-thesizes proteins (denoted OTP-1 or trophoblastin, and BTP-1,respectively), presenting obvious structural and functional re-

I.N.R.A., Laboratoire de Virologie et d'Immunologie Moléculaires, Centre de Jouy, 78350 Jouy-en-Josas. France.*The use of the IFN-w terminology is consistent with the recommendation of the Nomenclature Committee of the International Society of

Interferon Research (1990).

341

342 MEGE ET AL.

latedness to IFN-co. In both species, these proteins were shownto mediate maternal recognition of pregnancy by maintainingthe function of the corpus luteum.{9l0> Considering that theproduction of IFN by the developing conceptus does not appearto be a phenomenon restricted to ruminants,' '1-13) these not yetcharacterized embryonic IFNs might play an important regula-tory role in the maternal immune response to conceptus anti-gens or in the cellular embryo differentiation in several nonru-

minant mammals.In the pig, a transient IFN-like antiviral activity has been

detected in uterine flushings and conceptus culture mediumfrom day 12 through day 20,'l4l6) at a time when conceptusesundergo rapid and considerable elongation and come into con-

tact with the uterine endometrium. Recent results demonstratedthe presence of at least two antigenically distinct antiviral pro-teins in the uterine lumen at this period; one of them was clearlyidentified as an IFN-y."7' The other antiviral component was

probably related to Type I IFN, as suggested by its antigeniccross-reactivity with human leukocyte IFN (HuIFN-Le)."416'However, it was not neutralized by an antiserum to OTP-1."41To identify this Type I-like porcine embryonic IFN, it seemedof interest to isolate different probes corresponding to varioussubtypes of porcine Type I IFN. The isolation of most porcineIFN-ci) genes and pseudogenes is described in this paper. Thenucleotide sequences are analyzed and the functionality of threegenes is demonstrated. Preliminary data on the expression ofIFN-co-related mRNA in pig trophoblast are also given.

MATERIALS AND METHODS

Isolation ofmRNAsfrom Conceptus Tissues: Animal rearingand conceptus collection have been described previously."61Total RNA was isolated by the guanidium isothiocyanate-ce-sium chloride method from porcine conceptuses obtained be-tween days 14 and 20 of pregnancy."8' Poly(A)+ RNA was

obtained by a single chromatography on oligo(dT)-cellulose.

Southern and Northern blot Analysis: Pig liver genomicDNA was prepared according to standard methods"1" and di-gested to completion with five different restriction endonu-cleases, then electrophoresed through a 0.8% agarose gel andsubsequently transferred to a nitrocellulose filter.'I9) Prehybrid-ization and hybridization were performed for 18 h at 37°C un-

der stringent conditions in 5 x SSC ( I x SSC is 0.15 M NaCl,0.015 M sodium citrate), 50% deionized formamide, 5x Den-hardt's (lOOx Denhardt's is 2% wt/vol of each Ficoll, polyvi-nylpyrrolidone, and bovine serum albumin), 50 vnM sodiumphosphate, pH 6.5, and 100 |xg/ml of denatured E. coli DNA.After hybridization, the filters were washed in 2 x SSC, 0.1%sodium dodecyl sulfate three times for 10 min at room temper-ature and twice for 15 min at 55°C in 0.1 x SSC, 0.1% SDS.For Northern blot analysis, 10 |Ag of each sample of poly(A) +

RNA was fractionated on a 1.5% agarose-methylmercury hy-droxyde gel and blotted onto nitrocellulose."8' Hybridizationwas performed at 37°C in a standard hybridization buffer con-

taining 30% formamide and blots were washed at 58°C in2 x SSC, 0.1% SDS prior to autoradiography with an intensi-fying screen.

Isolation and Mapping of Genomic DNA Clones and DNASequencing: A porcine genomic library was prepared as a par-tial Sau 3A digest of total liver genomic DNA cloned into theBam HI site of XEMBL4."9' A total of 200,000 recombinantphage XEMBL4 were plated on E. coli 803 and screened byusing high-stringency conditions for hybridization and subse-quent washings.'201 X clones were mapped according to stan-dard methods,"8'2" and the suitable hybridizing fragmentswere isolated and subcloned into pTZ vectors (Pharmacia) or

into M13 vectors. The nucleotide sequences of the inserts were

determined by the Sanger's dideoxy-chain terminationmethod.'22'23'

Computer Analysis: Pairwise comparisons and alignmentswere performed with the help of the Microgenie software byQueen et a/.,'24' installed on an IBM PC XT computer. Multi-ple alignments for maximum homology were made by using theMultalin program on a Data System AT computer with MS-DOS.'25'

In vitro Expression and Antiviral Bioassay: Enzymatic am-

plification of mature IFN-co coding sequences covalently linkedto T7 phage promoter was conducted as follows: 500 ng ofrecombinant X. phage DNAs were subjected to amplificationwith 5 units of Taq DNA polymerase from Thermus aquaticus,as described previously.'26' Gel-purified 5'-oligonucleotideprimer contained a 23-base T7 promoter sequence followed bya 14-base translation initiation signal'27' and a 19-base sequencecomplementary to the first six codons of the mature and con-

served coding sequence of porcine IFN-cüm genes, in frame withthe initiating ATG. The 3' primer hybridized with a 28- to33-base sequence lying between positions 48 and 80 of the 3'noncoding regions (numbering as in Fig. 3). The reaction mix-ture, including 250 pmoles of the 5' primer (5'-GG-TACCTAATACGACTCACTATAGGGAGACCACCATGT-GTGACCTGTCTCAGAACC-3') and 500 pmoles of the 3'primer (5'-CAGAAATAGGAGTCTTTTGAAGTGACCA-3')was subjected to a first amplification round of 5 cycles of 1 minat 90°C, 1 min at 37°C, and 3 min at 65°C, then to a second one

of 30 cycles of 1 min at 90°C, 1 min at 45°C, and 2 min at 75°Cby using a HYBED thermocycler. After the final cycle, tubeswere placed at 75°C for 15 min. Primer-free PCR-generatedfragments were recovered by precipitation with 1 volume of6.5% polyethylene glycol (PEG 8000, 0.8 M NaCl) for 1 h on

ice.'28' Transcription reactions were performed according to themanufacturer's specifications, by using 25 units of T7 RNApolymerase (Bethesda Research Laboratories) and 120 |o,g/mlDNA template. The RNA pellets were obtained as previouslydescribed'291 and then resuspended at a concentration of400 |xg/ml in DEPC (diethyl pyrocarbonate)-treated water. Inví'íTO-synthesized RNAs (1 |xg) were translated in nuclease-treated rabbit reticulocyte lysates (Promega-Biotech, MadisonWI) in the presence of [35S]methionine, according to the manu-

facturer's protocol. The translation products were assayed forantiviral activity by inhibition of the cytopathic effect of vesic-ular stomatitis virus on bovine MDBK cells as described previ-ously.30

THE PORCINE IFN-w MULTIGENIC FAMILY 343

RESULTS

The porcine genome contains a family of IFN-u> genesIn a previous study concerning the porcine IFN-a family, a

recombinant phage X clone referred to as X17 (see Fig. 2) was

isolated, containing two human IFN-a,-related loci separatedby 6.4 kb. DNA sequencing revealed that one of them belongedto the IFN-a family. The other locus was a pseudogene(i|jPoIFN-a>|) that might belong to a putative IFN-to family,since it was more homologous to IFN-to genes of other species(79%) with HuIFN-to, and 83% with BoIFN-to,) than to theporcine IFN-a gene (only 67%).'5) Genomic DNA was ana-

lyzed according to Southern and hybridized with the codingsequence of PoIFN-a,'5' and the 900-bp Bst EII-£eo RV frag-ment of iJ/PoIFN-io, (encompassing the region homologous tothe coding sequence of other known IFN-to genes) and a and to

probes, respectively. Under stringent conditions, chosen to pre-vent any cross-hybridizations between the sequences of a and to

loci, a distinct set of bands was obtained with each probe (Fig.1). Since the restriction enzymes chosen for this analysis are not

expected to cut within the sequences of both probes and, sincenone of the Type I IFN genes contains introns, it is likely thatmost of the observed hybridizing bands represent a completegene; this suggests the existence of a minimum of 10 IFN-a and6 IFN-co members in the porcine genome.

Isolation and sequence analysis offive nonallelicporcine IFN-u> loci

Using the porcine IFN-to probe, five distinct genomic clones,referred to as Xn10, X,,25, X„23, X,,9, and X,,17, bearing 5nonallelic IFN-to loci were isolated from a porcine genomiclibrary, as described. Five of the 6 fragments observed on

Southern blotting (Fig. 1) in Xba I-digested porcine genomicDNA corresponded in length to the hybridizing Xba I fragmentsfrom isolated porcine genomic X clones (2.1-kb, 2.6-kb, 2.8-kb, 4-kb, and 6.5-kb Xba I fragments from X„ clones 10, 25,23, 9, and 17, respectively). The presence of an additional1.9-kb Xba I fragment on Southern blotting suggests the exist-ence of at least one additional IFN-to locus which was notisolated in this screening. Figure 2 shows the physical maps offive distinct porcine genomic DNA fragments containing fivedifferent chromosomal IFN-to-related loci. The overlappingclones X17 and X,,10 define a genomic 24.6-kb fragment con-

taining a common 2.1-kb Xba I fragment bearing theujPolFN-to, probe, as shown by restriction-hybridization analy-sis (data not shown).

The nucleotide sequences of two pseudogenes (ujPoIFN-w,and iCPoIFN-íü2) as well as three potentially functional genes(PoIFN-to,, -to4, -to5) were determined and aligned for maxi-mum homology in Fig. 3. All five clones contain closely relatedsequences (which are at least 92% homologous), but only threeof them present an open reading frame (ORF) of sufficientlength to encode an intact IFN-to protein: to3 and to5 genescontain an uninterrupted ORF of 191 codons whereas to4 genehas an ORF of 180 codons. A TGA stop codon is also present at

position 191 in the o)4 sequence. Figure 3 shows the alterationof the 5' transcriptional control sequence as well as the presenceof multiple frameshift alterations within the coding sequencesof ujPoIFN-io, and -oo2; this probably caused toi and to2 genes

to be inactivated. Alignment with porcine IFN-a, gene showsthat all five IFN-to sequences display a common in-frame five-codon deletion between positions 405 and 406. This is a uniqueand general feature of porcine IFN-to sequences, occurring in a

highly variable region of Type I IFN both at the nucleotide andamino acid levels (see Discussion).

Sequence data obtained for all five genes extended —200 to-270 bp upstream from the translation initiation codon (Fig.3). The degree of homology between the 5' noncoding regionsof PoIFN-to,, -o)5, u)PoIFN-to, and -ü>2 was similar to that seen

among the transcription units (about 90% of homology whencomparing the 210 bp upstream from the initiating ATGcodon). However, to4 promoter region is somewhat divergentfrom that of to3 and oo5 genes (only 78% of homology). Atapproximately 100 nucleotides upstream from the ATG, a

TGTTTAA sequence (in the to3 and to5 genes) and a TATT-TAA sequence (in the to4 gene), representing a modified TATAbox,'1" were found. The promoter sequence of PoIFN-to4 genewas remarkable by other features: a putative CAT box conform-able to the consensus sequence GGPyCAAT/AC'32' could be

EcoRI BamHI Bgilla co a to a ©

Hindlll Xbala co a «

5.Q_

W4

m

FIG. 1. Demonstration of the existence of IFN-a and -to multi-genic families in the porcine genome. Total genomic DNA was

digested with various restriction endonucleases (Eco RI, BamHÍ. BglU. Hind III, Xba I), electrophoresed through a 0.8%agarose gel, and subsequently transferred to nitrocellulose fil-ters. Hybridizations were performed under stringent conditionswith porcine IFN-a and -to probes: IFN-a probe is a 800-bp EcoRl-Hpa I fragment containing the complete coding region ofPoIFN-al gene'5'; IFN-to probe is a 900-bp Bst EU-Eco RIfragment containing 4<PoIFN-to, pseudogene. Both probes were32P-labeled by random priming.149' Size markers (kilo-basepairs) are indicated on the left.

344 MEGE ET AL.

xn

XnlO

Xn23

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PoIFN-oJ

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v„25 \L

m r.

yPoIFN-col

ti^L^yPoIFN-co2

WTH

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I ^¿?î t ? T t

PoIFN-co3

,„9 ?,,! ?,?^t¿TL_:

PoIFN-cu4

T T

J

t t?.?Ti

^ Eco RI

| //índlll

| /'s/ i

kb 0 10 15 20—I

25

FIG. 2. Restriction maps of six genomic DNA fragments bearing five distinct PoIFN-to-related loci. Restriction map of clone X17,which contains the linked PoIFN-a3 (hatched box) and iIjPo-IFN-co, (black box) genes, is shown. Xn10 genomic insert overlapsX17 insert, each containing the same 2.1 -kb Xba I fragment bearing i|/PoIFN-co, pseudogene. For clones X17, X,,23, XN10, Xn17,and X||9, the position and the orientation of the gene or gene-like sequence have been determined, and are shown by arrows. Pst Isites were only localized on the 5.9-kb Bgl II-W/>idIII fragment of clone X„17.

identified 83 bp upstream from the TATA box; a 16-bp deletionat positions —34 to —51 (numbering as in Fig. 3) lying in thepresumed nontranslated leader sequence of the mRNA was

specific to this gene. By analogy with HuIFN-a, gene, wheresequences required for virally induced transcription have beenidentified,'33' a homologous purine-rich 46-bp region, likely to

represent virus responsive element (VRE) is found between

positions —179 and -136 from the ATG in the five IFN-cosequences. No polyadenylation site of the AATAAA or AT-TAAA type was found in the 210 to 270 bp extending down-stream from the stop codon in any of the sequences. The co4gene bears several unique features also in the 3' noncodingregion (Fig. 3). It contains three contiguous copies of theTTATTTAT consensus sequence followed by an additional one

separated by a fifth nearly perfect copy (TTATTTAC, shown initalics), between positions 192 and 233 downstream from thestop codon; a single copy is found between positions 197 and204 of cü3 gene and no copy is detectable in the available 3'sequence of to5 gene. Finally, the examination of the nucleotidesequence of co4 gene also revealed the presence of a single copyof the estrogen-responsive consensus element (ERE) consistingof a perfect palindromic 13-bp sequence GGTCANNNTGACC(where NNN is TCA) in the vicinity of the A-T rich sequence,between positions 162 and 175.

Three porcine IFN-u> genes (PoIFN-Wf, -u>4, and -u>^)encode proteins with antiviral activity

Analysis of Hanking and coding sequences suggested thatthree of the five isolated genes were likely to encode biologi-cally active IFN-co proteins. The porcine IFN-co polypeptidesare unusual in length since they all lack five amino acids in thenonconserved region between positions 113 and 117 of IFN-aand IFN-co mature proteins. The deduced encoded proteins are

aligned in Fig. 4: by analogy with other sequences of IFN-co,the three PoIFN-co sequences possess a 23-residue signal se-

quence with a conserved Ser-Leu-Gly preceding cysteine 1 ; theputative proteins are variable in length and always shorter thantheir counterparts in other animal species, where they have beencharacterized as 172-amino-acid-long mature proteins. co3 andco5 genes would encode 167-amino-acid mature proteinswhereas co4 protein would be 11 amino acids shorter at itscarboxy-terminal end. These proteins, as well as leader se-

quences, are very homologous since they share 88% of theirresidues (without consideration of the lacking 11 amino acids ofco4 protein).

The co3 and co5 genes could encode six additional carboxy-terminal amino acids found in all other known IFN-co se-

quences. Several amino acid residues found in most IFN-a and

-270 -260 -250yPoIFNOl CATTTGAATGGGCATACCTGGAA

yPoIFNö2PoIFNffl3PoIFNCM TTGGACTGACACATCCAAAGTTPoIFN05 CATTTGCATGGACATATCTGAAGPoIFNal AGGAAAAATAATCTGAAAAGG

(b) •_VRE__

*

-240 -230 -220 -210 -200 -190 -1Í5 ^TTO" -160 ^TSO" ^TÏO" -1301 TTATTGAGAAAATTAAAATTCTT**GTGAGATTAAATAGTGTAGATTGAA»TATCTTTTAGACCATATTGAGAAGACAAAGG2 GGAAAACTAAACCTCTATGACAAAATTAATGGTTTAGAGTAATGACTTTGTCTTATACCATATGGAAAATACATAAATGAAAACCA*AAAAAGAAGTG"AAAGTGTAGAGAAATGTTT**A3 GTTGTCTTGGACCATAGGCAAAATACATAAATGAAAACTA*AAAAAGAAGTG«AAAGAGTAGAGAAATGTTT**A4 AATGGGGAAAATTAAACTTTTATTGTAAAAATCAGGAGTTTAGAGTGATTACATATTCTCAG^TÇAAACAGAAAAT^^5 TTTCTGGGGAAATTAAACTTCTACGACAAAATTAATGGTTCACAGTTATGATGTTGTCTTGGACCATATCGAGAATACATAAATGAAAACCA*AAAAAGACGTG«AAAGTGTAGAGAAATGTTT**Aa CTCTGCAGGCAGAGGAAAGGAAGGCAATAATGAAAAAGAAATGGTTGCGAAATGTTGCCCTAACCCATTTGGAAAGTACAAAGTGAAAAGCAGAAACAGAAGTGGAAAGTAAGAGGGAACATTC**A

( )-120 -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -1

1 ATAAATfAAAATTACTGTGTTCACTATTTACTGGCCATGCTTAGAGAGAA2 r.AAAATr,AAAATTAC:TfiTfiTTCCCTGTTTAGTGGCCTTGCTTAGArJk^3 ftAAAATrJVAAATTACTnTr/rTCCCTGTTTAATGGCCTTGCTTAGAflAnAATr.GCATCA^^4 **AGAAAATGGAAACTGTGTTCCTlAlllAAAAGCCATGCTTAGGGAGAATGTCATGfcOfca*ACCTACCTGCAAGGT*************•••CTCAGCCAGGCCAGTTGCACCCTCATTTTCCCC"5 r.AftAKTftAAARTTACTftTGTTCCrTrvrTTAATGSCCTTfiCTTAGAt^a GAAAATGGAAATGAATTCATTCC*IAXtlAA^ACAMGGCCT*GAGGGAAGGTCTTC*QKaTCCTAGAGAGCAGGGTCACAGAGTCACCCACCTCAGCC^^

2 ^_^_*.T.A. ..C...

.A.G. TGA.T.C3 ATG GCC TTT GTG TTC TCT CTA CTG ACG GCC CTG GTG GTG TTC AGC TAT GGC CCT GGG GCA TCT CTG **GGC TGT GAC CTG TCT CAG AAC CAC GTG CAT4 _^_l..C

...

C.T...

**.T.

5.^^.T.**.C

a ^^

. . .

CCA ACC .CAG.CT.C ..C.C.AC.TGC AAT G.C ATC TGC. **.C.C....

AGC CTG

100 110 120 130 140 150 160 170 160 1901

...

A.. ..•.G .C.GTG.A.A.G.T.2

...

A.G.GTG. ..A.A.GG.3 GTC GGC AGG AAG AAC CTA GTG CTT CTA CAT CAG ATG AGG AGA CTC TCC CCT TCC TTC TGT CTG AAG GAC AGA AAA GAC TTC GGG TTC CCC CAG GAG4

...

A.G.G.G.T.5...A.G.G.C.a .CT CA. .CC .G. GC.

.

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.

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.T G.C A.GC. .GG

390 400 410 420 430 440 450 4601.G.. ..CT.. T.*** *** *** *** **•.A.C.T.A.T2.GT.«* •** «** **« *** *** *** *** **.CC.C

.

.C.

.T.A .A.G3 GAG CAG ACA TCT GCC CTG GGA •** •** «** *** **» ATG GCT GTG AAG AGG TAC TTT GAG GGA ATC CAT CTT TAC CTG AAA GAG AAG ATA TAC AGC4.G.A. •«• «•« **» *** ***.C.T.A.T5.A. *** •** *** **»***.C.A.a CT. G.A GGG A.C C.CTG GAG GAG GAC TCC ATC CG.G. .AA.CC.CA.. C. . ACC

.

.C

.

.T

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470 460 490 500 510 520 530 540 550 5601.T.T.TG .A.CCT.AA..A GTT

.

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.

.C.

. . .

A..

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ACT.

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CCC .A. ..GG.. .G.3 GAC TGT GCC TGG GAG ACT GTC AGA GTG GAA ATC ACA AGA CCC TTG TCT TTA «TCC ACC TAC TTA CAA GAA AGG CTG CAA ATT GCT AAT GTA GAC CTG4.G.T.TG

...

.G. *... -TGA.T ..T.JGA AG. . .. A.G G..

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...

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T.. ..C...

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570 573+1 10 20 30 40 50 60 70 B0 90 1001.CT J_^_ AATGACTGTCAGACT***AAGAATGCCATCTCACCCT*GTACA**«TTGTCTTTGGTCAC*TTCAAAAGACTC*CTAT*****CTTTGGCTACAGAACTTATTGA**AATAA2 .CA

...

CG -A. CATGATTCTCATTGA**TTAAGATGCCAAATCACC"TTGTACA**TTCGACTCTGGTCAT*TTCACAAAATTC*ATTTCT**GTTTTAGTCACAAAACT««TATTGAA*ATA3 GGG TCA TCC ISA CATGATTCTCATTCA-*CTAAGATGCCAACTCACT*TTGTACA**TTCAACGCTGGTCAC«TTGAAAAGACTC*CTTTTT**GCTTTAATCACAAAACT**TACTG*AATTA4 .T.

...

CA

...

AATAATTCTCTGTGA"*CTAAGATGCCATCTCACCCTTGCACAA*TTCATCTTTGGTCAT*TTCAAAAGACTC*TTATTTCTGCTTTAGTTACAAAACT«*TATTG*AATTA5.J_u. CÄTGATTCTCATTGA**CTAAGATGCCATCTCATCCTAGCACA**TTCATCTTTTGTCAT*TTTAAGAACCTC-TTATTTTGGATCTACCCACAAAATG**CATTG*AATTAa CCT GGT ..A .C CAGACACCTGGTTCATCATAGAAATGCTTCTTACAGACTAACAAGCCCATTCTTCCTCCTGTTCTGCCATGTCACGGCCTCTCATTTCTGTATCATCCTGCCCGTGCCCGTG

(d) (e>110 120 130 140 150 160 170 180 190 200 210 220

1 *TTTGAGTAATATTTTTTCAGTAGTAATAAGCATTCATATGTCAAAA«TTACTCAGCTCCAGGGGCA*TCA»GTCTTAGGAGATG»ACTGTCCTGAGGTGATTTATCTA*CTGTTTGTTTATGTTTAT2 ATTCGAGTAATACTTTTTCAGTAGTAAAACTTCCCTGGACATCAGGC*CCTAAGAGCTGTTTGCTCTGATGTGATTTATTTATTG»CTATTTACTAATTTCTATXTATTTAJ3 ATTCCATTAATACTTTTTCAGTAGTAGTAAGCAATTATGTGTTAAAA«TATTTCAGCTCCCGAGACA^4 ATTC*ACAAATATTTTGTCAGTAGTATTATGAAAAGTTATGTTAAAC'AGTT*CAGCTCCAGGGTCA*TCA^5 ATTCAGGTTAGTCCTTTTCAGTAGTAATAAGAATTAATGTTTCAAAA*TTTTTCAACTCCAGTGTTA*TCTGTCCCTTAGCAATG*ACTGTCCTGATGTTACTCATTTATATa AAGG*TGACTCAATTTGTCAAAGGTTTTCAGGAGTGTTAACAACATCTTGAT*CTACTCTATAGGCA*CTGGTTCTGTACAG^

230 240 250 260 270 280TCTTGTACCATATAAAATTTCATATAAAATATTTATTATTACATTGTAATTC IT/PoIFNCOl

yP0lFN(&2TTATTAATCTTGTATTTTATTTAAAATTTCATATAAAAGGAATTC PoIFN03AT*TTATTCATTCCTTCTTATACCTACCACATTTATAATATTTGGAA PoIFNiM

PoIFNODS

AX*TTATCTAAAGATTTATATGGAT*TCATCCTTATA PolFHOl

FIG. 3. Nucleotide sequences of the five PoIFN-w loci. Sequences are aligned with PolFN-a, gene to detect the best homology,which requires the insertion of gaps (*). Identical bases among the six coding or pseudo-coding sequences are shown with dots.Numbering is on PoIFN-ü>3 sequence. Initiation and termination codons are underlined. The TATA box is underlined (a), as well as

the putative CAT box (b) of the PoIFN-co4 gene. The putative mRNA cap site is indicated in bold letters. A region corresponding to

—

136 to —179 bp upstream from the translation initiation site is homologous to the 46-bp region responsible for viral induction inHuIFN-at; it is shown between two symbols (°). In the 3' noncoding regions, the TTATTTAT consensus sequences are underlined(c) (one imperfect repeat in co4 gene is in italics) as well as the ERE sequence of PoIFN-cd4 gene (d).

346 MEGE ET AL.

SI SIO S20 1 10 20 30 40 50 60 70 80 90PoIFNol ..PTSAF.LL.CNAIC.P.T.SLAHTRA.R..A_I. .FS..DH.R. ..S.H.AFG.N.V..A. .MALV.Q.FS. .G.A.

.

.NES. .HQFYT..D. ..R.PoIFNO>3 MAFVFSLLTALWFSYGPGASLGCDLSQNHVHVGRKNLVLLHQMRRLSPSFCLKDRKDFGFPQEMVEGSQLQKTQAISVLHEMLQQTFLLLHTERSSAAWDSTLLDKLCSGLHQQLEDPoIFN0>4 _L.F.S.R.H...PoIFNGtó .V.S.P.F.

100 110 120 130 140 150 160 167al .EA.VM..AGLEGTP.LEEDSIR..RK..HRLT...Q..S..P_I..A.VM.SF.S.RN..D..RKKEca3 LDPCLVQEMEEQTSALG*****MAVKRYFEGIHLYLKEKIYSDCAWETVRVEITRPLSLSTYLQERLQIANVDLGSS0>4 .ES_VRG. .A...E*****.K.I.M.R_MNF. . .UR.TnA. .V.P

a>5 .V.G.e*»***.K.M.A..ST.K..Q..R.MD.

FIG. 4. Amino acid sequences of three PoIFN-co proteins predicted from the DNA sequences shown in Fig. 3; alignment formaximum homology between the porcine IFN-a and -co primary sequences requires the introduction of gaps (*). The sequence ofthe signal peptide is designated as S1-S23, and the mature proteins as 1-167. Numbering is on PoIFN-co primary sequences.Residues identical in all sequences are changed into dots. The letter U stands for a termination translation codon. Underlinedresidues are not translated on PoIFN-co4 sequence.

IFN-co so far characterized are conserved in the porcine IFN-comolecules. These include cysteines at positions 1, 29, 99, and134 and the Cys-Ala-Trp-Glu sequence (residues 134-137); inaddition, a fifth cysteine residue was found at position 86 as inseveral IFN-a'34'35' and IFN-co sequences.'2'36' None of thenew sequences contains the Asn-X-Thr/Ser motif recognized as

a potential A'-glycosylation site.To know whether the naturally deleted PoIFN-co proteins

retained antiviral activity, we obtained corresponding mature

proteins using an in vitro expression system. The sequence ofT7 phage polymerase promoter was covalently linked to themature IFN-co coding regions by PCR amplification as de-scribed in Materials and Methods. After in vitro transcription,each RNA product was translated with [35Slmethionine intorabbit reticulocyte lysates. Electrophoresis of in vitro synthe-sized PoIFN-co products in reducing conditions showed theexpected bands with different apparent molecular weights (Fig.5): 17,000 for PoIFN-co4, 19,000 for PoIFN-co, and -co5 mature

proteins; no radioactive band could be detected in the controlwithout RNA.

IFN activity in samples was determined on bovine MDBKcells after half dilution in cell culture medium. The IFN titers(mean of two values) of the diluted lysates containing PoIFN-co,, -co5, and -co4 gene products were 170 IU/ml, 2,600 IU/ml,and 1,500 IU/ml, respectively (Table 1). We concluded thatneither the naturally occurring five amino acid deletion in thethree porcine IFN-co proteins nor the lack of 11 carboxy-termi-nal amino acids in IFN-co4 are detrimental to their antiviraleffect on bovine cells. The case of PoIFN-co4 is reminiscent ofprevious report which demonstrated that the 11 COOH-terminalamino acids of human IFN-a are not essential for antiviralactivity.'37'IFN-u) related sequences are expressed by the porcineconceptus in early gestation

Conceptus poly(A)+ RNA, sampled between days 14 and 20of gestation, was blotted according to Northern and probed withPoIFN-co, DNA. Under stringent hybridization conditions, no

RNA band was detected (not shown). Under nonstringent con-

ditions (see Materials and Methods), the probe detected at leastfive distinct transcripts during this period (Fig. 6): the threemajor ones were 1.4, 1.8, and 3 kb in size; among the three

FIG. 5. SDS-poIyacrylamide gel electrophoresis of in vitroporcine IFN-co translation products. In vitro transcripts weremade from synthetic primers carrying the T7 polymerase recog-nition site as described. Translation was performed in nuclease-treated rabbit reticulocyte lysates. [35S]Methionine-labeledsamples were run on 12% SDS-polyacrylamide gel and radioac-tivity was visualized using fluorography enhancer (Amplify,Amersham). Protein standards with the indicated sizes (kD) areshown on the right. Translation products correspond toPoIFN-co, (lane 2), PoIFN-co4 (lane 3), and PoIFN-co5 (lane 4)genes. Lane 1 is a control without RNA.

minor transcripts, one was larger than 3 kb. Maximal expres-sion of these transcripts appeared to occur on days 14-15 ofgestation. No signal was detected when the samples were

probed in the same conditions with a porcine IFN-a probe (notshown).

DISCUSSION

This study demonstrates the existence of a porcine IFN-cofamily, which comprises about six members. Five nonallelicPoIFN-co loci were isolated and sequenced, which might repre-sent all but one of the related genes of this family. Since genes

THE PORCINE IFN-w MULTIGENIC FAMILY 347

Table 1. Antiviral Activity of Three Mature Porcine IFN-coTranslation Products

No RNA P0IFN-W3 P0IFN-0J4 PoIFN-w5IFNtiter 0 170 1,520 2,600

IU/ml after 90 min30°C

Translation—

>500 51 108efficiencyIU/jug ARN/90 min

The translation products were assayed for antiviral activity by inhibition of the cytopathic effect ofvesicular stomatitis virus on bovine MDBK cells; they were obtained as described in the legend for Fig. 5.

from each family were both located on the same X insert (cloneX17), the porcine IFN-a and -co genes are likely to be clusteredon the same chromosome as shown for human genes'38'39'; as a

consequence, porcine IFN-a and -co genes might be localizedon the same region of chromosome 1, on which IFN-a geneshave been mapped.'401

We described the nucleotide and deduced amino acid se-

quences of three genes encoding biologically active PoIFN-coproteins, referred to as PoIFN-co,, -co4, and -cov The in-framefive-codon deletion of porcine IFN-co genes occurred in a re-

gion that showed low conservation among Type I IFN se-

quences at both the nucleotide and amino acid levels. Recentdata about the tertiary structure of recombinant MuIFN-ß ob-tained by the group of Senda'4" indicated that the poorly con-

served region, lying between residues 102-115 (Fig. 4) corre-

sponds to a remote loop, with the highest flexibility. Inaddition, these authors suggested that the spatially close assem-

bly of the other three polypeptide loops would be directly in-volved in the antiviral and antiproliferative activities of Type I

DAYS OF GESTATIONkb 14 15 16 17 20

7.5-4.4-

51«M M

0.24-

FIG. 6. Identification of mRNAs cross-hybridizing to the por-cine IFN-co, probe in porcine conceptuses during the implanta-tion period. Poly(A)+-enriched fractions (10 ixg per lane) fromconceptus tissues sampled at the appropriate day of gestationwere analyzed by Northern blot and hybridized with the 795-bpEco 01091 fragment of PoIFN-co, (containing the completecoding region) under low stringency. Positions of RNA sizemarkers are indicated.

IFN molecules. This is in agreement with our functional studiesdemonstrating that the 5-amino-acid deleted PoIFN-co proteins(between positions 113 and 117) retain an antiviral activity. Itwould be worth testing the PoIFN-co protein products for otherbiological activities such as immunomodulatory and antiprolif-erative properties. Interestingly, this five-codon deletion was

also found in both pseudogenes tliPoIFN-co, and -co,, whichsuggests that this mutational event would have occurred beforethe accumulation of other inactivating mutations all along theancestral transcription units.

From the comparison of PoIFN-co sequences with other al-ready known IFN-co genes, it appears that PoIFN-co coding andflanking regions are more related to IFN-co members of otherspecies than to the porcine IFN-a, both at the nucleotide andamino acid levels (Table 2). The most extensive similarity ofthe porcine co sequences is to bovine leukocyte IFN-co' ' ' and thelowest one is to equine IFN-co.'21 In addition to the conservedamino acid clusters between positions 29-41 and 123-142,which are common to IFN-a and -co proteins, IFN-co proteinsappear to share another conserved amino acid cluster betweenpositions 71-82. These conserved regions might be importantfor their function.

One of the purposes of this study was to compare the pro-moter sequences of porcine co genes with other mammalianIFN-a and -co genes. The VRE-like sequence of these genesmight be described like in human IFN-a, gene as an imperfectdirect repeat of two sequence elements RepA and RepB,'42'organized as: RepA = Rl-a-R2/RepB = R'l-b-R'2, where a

and b are spacer elements, and R represents conserved elementsamong functional genes. Considering only the three potentiallycoding genes PoIFN-co,, -co4, and -co5, the sequence elementsRl. R'l, R'2 would be ATGAAAC/A, GTGAAAG andAGAAA, respectively; R2 element is not conserved in the co4gene (Fig. 3). Further experiments would be required to assess

the regulatory role of such conserved upstream sequences invirus inducibility of PoIFN-co genes. It should be noticed that inthe porcine species homologies between putative VRE of bothIFN-a and -co genes are only 71%. This is comparable to ho-mologies found by Capon and co-workers' ' ' between the corre-

sponding VRE of a human or a bovine IFN gene of each family;these authors already pointed out the apparent paradox of a poorhomology in this region, although both IFN-a and -co genes are

coordinately induced in response to viral infection.The PoIFN-co4 gene is in part divergent from the other two

functional genes and possesses unique features in the flanking

348 MEGE ET AL.

sequences. The promoter of this gene has an apparently intactTATTTAA box, which is also present at a similar position inporcine IFN-a, gene as well as in most other IFN-a and -to

genes. In to3 and to5 sequences, an A G transition changes thebox into TGTTTAA, raising a doubt concerning the effectivetranscription of these otherwise potentially functional genes.The 3' downstream sequence of the to4 gene displays an exten-sive 41 bp AT-exclusive rich region, which was neither foundin other known porcine IFN genes nor in mammalian IFN-togenes. The repeat of the TTATTTAT motif has been found innumerous genes encoding inflammatory cytokines, as well as incertain proto-oncogenes.'43' The UA-exclusive composed se-

quence in the 3' untranslated regions has been demonstrated toconfer message instability in vivo, in a manner depending on thenumber of copies of inserted consensus octanucleotide'44'; fur-thermore, this sequence could act as a translation inhibitingelement.'45' In this respect, 3' untranslated regions of ruminanttrophoblast IFN-to mRNA seem characteristically deprived ofthe AU-rich sequence because of interspersed Gs and Cs. Inaddition, only the to4 gene among all IFN-to sequences reportedso far contains a perfect ERE consensus lying 27 nucleotidesupstream from the AT-rich region. Although unusual in itslocalization and nature, this ERE could yet represent a func-tional cíí-acting regulatory element; a few copies of degener-ated consensus ERE motif were originally found in the 5'-flanking region of liver estrogen-responsive Xenopusvitellogenin and chicken apo-VLDLII genes.'46' The Xenopusvitellogenin ERE was characterized as a transcriptional induc-ible enhancer, since it could confer estrogen inducibility to a

heterologous promoter, even when placed in an inverted orien-tation or far upstream from the transcription unit. Furthermore,a single perfect palindromic sequence was shown to be neces-

sary and sufficient to act as a fully active ERE, when placeddownstream from the transcription unit.'47' These data indicatethat PoIFN-to4 has the structural characteristics of a highly

regulated gene both at the transcriptional and post-transcrip-tional levels. The combined occurrence of such features, not

previously reported in any other IFN-a or -to genes, suggeststhat the expression of this gene may be controlled by both virusinfection and estrogens.

Several IFN-to-related mRNAs of various sizes were identi-fied by Northern blot analysis, using IFN-to, as a probe undernonstringent hybridization conditions (Fig. 6). However, thesetranscripts were not detected under stringent conditions, indi-cating that they are not expressed from the IFN-to genes de-scribed here. Moreover, previous reports showed that similartranscripts were not detected by an ovine trophoblastin (orOTP-1) probe.'48' These transcripts are probably expressedfrom an additional Type I IFN family, which is more related toIFN-to than to IFN-a. One or several of these mRNAs are likelyto encode the HuIFNLe-related antiviral activity that was re-

cently detected in conceptus culture supernatants and in uterineflushings from pregnant gilts at the time of implantation.'I416'The structure and sequence(s) of this family of IFN genes willhave to be identified.

These results indicate that expression of Type I IFN, andmore precisely of IFN-to-related genes by the implanting con-

ceptus, is not restricted to ruminants. In these species, tropho-blastic IFNs are known to mediate maintenance of the corpusluteum at the beginning of gestation. The fact that embryonicIFN is also present in the pig, in which this phenomenon ismediated through distinct mechanisms (probably by estrogens),could indicate that IFN molecules play other roles in the physi-ology and/or immunology of gestation. In this regard, the pig so

far represents a unique situation in which clearly the two Types(I and II) of IFNs are produced at the same time at the maternal-fetal interface."617'

The question of the expression of the IFN-to genes describedin this work remains therefore open. It is very likely that thesegenes are co-expressed with IFN-a in virus-induced leukocytes,

Table 2. Pairwise Comparison of Homology in Coding Regions of P0IFN-C03 Sequence, Five otherIFN-to Genes, and the PoIFN-ai Gene3

P0IFN-W3 BoIFN-o>ib HuIFN-wib EqIFN-w,b OTP-11 BTP-lb PoIFN-alb

PoIFN-<"3

BoIFN-toi

HuIFN-a>i

EqlFN-coi

OTP-1

BTP-1

83.3(71.6)

80.5(66.8)77.6

(62.6)

75.4(58.4)75.9

(62.6)74.4

(59.0)

78.8(65.3)84.7

(70.3)71.8

(56.4)70.4

(53.3)

78.4(66.8)84.8

(73.8)70.9

(57.9)71.4

(53.8)89.7

(81.5)

67.2(50.5)68.4

(55.5)65.6

(51.3)63.0

(49.7)60.1

(50.3)61.7

(51.9)

Only PoIFN-£U3 gene was compared here because of the high homology (both at the nucleotide and amino-acid levels) between members ofthe porcine IFN-to family.

a Shown are the percent nucleotide sequence homologies between pairs of coding regions and the percent amino acid homology between thecorresponding preproteins (in brackets). The six additional carboxy-terminal amino acid residues of IFN-oi proteins as well as the five missingamino acid residues of PoIFN-co proteins (Figs. 3 and 4) are not included in comparisons with PoIFN-aj.

bReferences of sequence data of BoIFN-toi, HuIFN-cot, EqlFN-coi, OTP-1, BTP-1, PoIFN-ai are (1), (1), (2), (48), (50), and (5),respectively.

THE PORCINE IFN-co MULTIGENIC FAMILY 349

as was shown for human IFN-co.' ' 3' Concerning their presencein the reproductive sphere, the possible participation of thematernal tissues to the secretion of antiviral activity cannot beruled out.

ACKNOWLEDGMENTS

We thank F. Martinat-Botte (Physiologie de la Reproduc-tion, INRA Tours, France) who provided us with the pig con-

ceptus samples. We also thank J. Martal (Endocrinologie del'Embryon, INRA Jouy-en-Josas, France) for help and advice.We also thank J. Buri for critical comments on the manuscript.This work was supported in part by a grant from the Institut deBiologie Animale, Montélimar, France.

The nucleotide sequences reported in this paper have beenassigned the following accession numbers in the EMBL DataBank: X57191 for PoIFN-a, gene and X57192-6 for the fivePoIFN-co sequences.

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Address reprint requests to:Dr. C. La Bonnardière

Laboratoire INRA de Virologie et d'Immunologie78350 Jouy-en-Josas, France

Received 13 March 1991/Accepted 31 July 1991

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