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Interferon-delta: The first member of a novel type I interferon family
Bi~chimie (1~)8~ 80, 779-788 © Soci6t~ fnmqaise dc bi~whimie e~ bioh~ic m~k~cak4~c / Elsevic~~, P ~ s
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F r a n c o i s Lef~v~e "~*, M i c h e l G u i l l e m o t ~, Sabine D ' A n d r 6 a % S a b i n e B a t t e g a y ~, C l a u d e L a Bonnardi~re" '"Ut~itd de Viov!ogie et lmntu~w&~gie Moldcut¢ffrex, h~ra, 78352 Jouv-~dos-~s cede'x, France e~Umtd de Biologic Celhda~re e¢ Moldctdaire. L~tra, 78352 Jouy-E~-Josas (~dex~ Fram'e
~Received 2 February i998: accepted 9 July 1998) Abstract .......We have rccen|!y described a novel type I imerferon ~!FN) co-expressed with !FN-y by ~he trophcctod~nm of Hie pig ctmccplu~ bciv,+cC+~d~y 12 and da) 18 ~ff ge~t+~tion+ ~ development stage t!mt corresponds ~rOimplantation in the uterus~ This [FN, ~o,~,. oli~ciaUy ~.amcd |F'N b~ is recognized as ~he !irs~ member of a novel type I iFN tamilyo This paj~r reviews the main published dat~ on IFN4% toged~er wi|h some new data, showing that IFN~6, while being a true !ype I IFN, has some very specific structural and biological pro~nies. Sequences relaled 1o IFN=b coding sequence were fi~und in the gcnome of man and olher ungulates but the or,,~) other potentially functional gone was |~mnd. so far. in lilt: ht~tse. The pig IFN~b mature protein, wi!h 149 am~no acids, is the smailest
of all known type li IFNs. |t is unusually rich m cysteine~ (seven residues), and has a very basic is~leclric poinL Recombinant IFN-6 expressed in in~cc~ cells is glycosyla~ed and has a high anliviral actmty cm porcine cells, but not on human cells. |~ has high anfipmiiferative activity, which is significantly enhanced in lhe presence of IFNq.. Th~s new IFN was shown ~ bind or~ pig cells ~ ~he same type ! ~vceptor as IFN-,. !FN-~ and !FN.y genes are co-regulated in lhe pig trophectoderm, whose cells ~n day 14-16 of development simultaneously .~ecrete both IFN proteins. The bioh~gical r~le of pt~rci~e IFN-~ in early prcgmmcy has been fi~und unrelated to i!~e known an|d~|eotytic efl~'ct of tmphob!a~tic IFN~" in ruminants. @ Soci6t~ franqaise de biochimie et biologic moldculaire I Elsevier, Pan.~
ty~ 1 interferon t trophob!ast / pig / intefferon-~ / geslation 1. |nlrodt!ciion ~!3~ I imerferons {IFN~ t'epre~ent a tmique group ~t° t'elated antiviral cytokine~ with many original properties j l I. Tiley are matn!y virus-inducible and arc encoded by introntess genes located in tl~e ~amc region of a single chromostmlel2. 31. Although some members of |his iamily al~ ~ometinies distamty r~lated flor example, IFNoa and IFNq:-| polypeptide sequence.~ share only 20% identity 141), they all interact with the same membrane receptor [5=71. According to a well established theory, all t y ~ I IFN genes originated fi'om a common ancestor that expanded by successive duplications 121. Until recently, four distinct type I IFN families have been recognized in mammals according to their structural and antigenic pro~rttes: IFN-ct, -[k -to and -t. The three former families top,sent classical virus4nducible IFNs, existing in almost all mammalian species and mainly involved in nora s~ciflc protection against a wide variety of pathogens. mainly viruses 181. !nterferon-t or trophoblastins, discow ered about I0 years ago, represent the first example of a type ! IFN family specialized in a physiological role (see accompanying paper by Martal et al.). These proteins are secreted into the uterus during early pregnancy by the trophoblast of the embryo in some ruminant species. Their
ptoima13~biological role at the materno-fetal interface is not anti-infectious nor antiviral: they are antilutcolysins that represent the lir,~t embryonic signal preventing ~'cgression of tile corpus luteum and permitting omgoing gestation (Igl; see I10, Ill for reviews), Moreover JFNt rcpre~eat~ the first type I IFN fiuni!y that seems to be re~;lrictcd to a ,~;ingle taxonomic group among mammal~ [! 21, Presently, there i~ no spme~ m whici! the comptctc sequence of the type I IFN locus i~,;exhaustively known. Thus, the possibility of existence of yet unknown familie~ of type I IFNs havin$ orlgtna biological propez~ies and roles remains open. We have previously described that in the porcine species, the trophoblast expresses a type i iFN gene around imphmtation 113. 141. According to its ofi$io hal properties, the product of this gone is now considered as the first member of a new type 1 IFN gone tamily designated by a special Greek letter: iFN-5. In this paper. we will review data available on porcine IFN-b concerto ing the original properties of its gone and protein, its possible biological function and preJiminary data indicat° ing that IFN°8 could be present in other mammalian species.
2. The discovery of porcine IFN-b Altllough expression of IFN biological activily m the matento°l~tal unit has ~ e n known for a long time in man
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mouse t15o!71, it is lhe discove~ of ruminant IFNo¢ l~.t ~ the ~ a ~ h for tFN expression by the ~ast of ungula'.~ s~cies. Ungulates are mammalian spcc~ kaving ~ epithcliochorial placentati~ and late i m p t ~ t i o n II8, 191. At the end of the 1980s, several ~rs, the ~nce of ~-dertv~ antiviral activity in the uterine hums of ~ t ~ w s ~ n g early ges~ion [13, 14, 20-221. ~ t e r i z a t i o n of ~is ~ t i v i ~ activity reveMcd a ~ k i n g feature: most of it was IFN-y espres~d MI ~ cells ~ abundantly as IFNot in ~mi~nt~[13l, It ~ l d ~ rimed that strong IFN~y e~pcessh~ by the pig peaoimplan~aon trophoblast ran mainsa uniq~ feature of thi~ s~ies. It rai~ in.resting
questions abo~l tissue~speciflc ~ r i p t i o n a l
regulation
~ ~g l ~ o y ~ene as expression of this gent was thought ~ b¢ stfic!ly limiled to ~tivated T lymph~ytes ~turM killer eelh 1231. Interestingly, we found that a~r conceptusocleriv~ antiviral ~tivity, not neutraliz~ able by anti--l~-¥ ~mm, was also detectable. Surprisingly, i~ ~as n e u ~ ! i ~ by ~ f i ~ r a raised against crude p ~ a t i o n s of hum~m vims~induced leuk~y~--derived IFN (IFNo~, C~nlell ~y~). thus su~esting that itcould a t y ~ 1 IFN 113, 14]. As Su~Jea¢ ~r¢ phylogen¢tically c!o~ to mminm~ts, we first speculated thal this pulative ty~ I 1 ~ was the po~in¢ homologu¢ of IFN--~,However, IFNo~ probes did not deiect significant sign_al in pig ~ e p l u s l~nsc~tpls 12'0, 24] al~l ex~nsive ~reening of pig generate libraries with t y ~ ! [FN pro~s {even IFN--oh the t'~ily h~ving lhe highest homology with IFNor) did
not lead to isolation of porcine l ~ , t
alike genes 1251,
However~ using very low s~ringency h),bridi~alion, we w,e~ able to detect IFN--to~lated !r~n~¢ripls in_day 14 In d ~ 20 pig C'~cepIus mRNAs!251, This gave us the o~aun:ily 1o done ~he con~sponding cDNA~ from a day eDNA library 126t, Most of d~em ~a 1 ~ ~ r ~ o ~td (~a) p~p~otein rel~t~J to all other ty~ l IFNs. As ~ ~ , I 5~rm of this protein had a~ a~li-hum~n IFNo~ ~utr~lt~bl¢ anflvtral activity, Be.o cau~ ot~tl~ si~e ( 149 aa for the mature protein, ~he shortest of known ~ I IFNg Ji/~r~ IA), the provisional name *slrl IFN' (for ~ ~t~ t y ~ ! IFN) was coined for this ~ein !26!,According to sever~.l cdleria (see ~low), i~ b~am¢ evtd~ot th~ |his novel ~y~ 1 I ~ could not be lr~l~-d in any of the previously defined t y ~ 1 IFN t~ilies. It has ~ n officially recognized as the first ~ m b e r of a fifth lhmily of mammalian tyt~ ! IFNs: IPN.b 1271,
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family mem~rs. Fiw~,~t,it has 11o preferential homology with one of the tour other families (an identities ranging between 27% for routine IFN-[~ and 42% for human I F N - ~ [26]). This fact is best exemplified by phylogenefic analysis of the mature protein sequences. As shown in figure IB, phylogenetic trees generated from such analysis clearly demonstrated that [FN-~ is distinct and divergent from IFN-~, -~ a~ and -t families, although it appears more closely rcia~ed to the IFN-ct. -to, -r cluster than to the !FN-~. The cysteine content of porcine IFN°b has no ~uivalent among other type I IFNs as the p~p~tein and matu~ protein contains nine and seven Cys residues, res~ctively. Intere~ting!y, five of the seven cysteine residues of the matu~ p~tein (~silions 9, 56. 58, 107 and 145) are ~|ypical among |ypc I tFN~ (~gure IA), Acco~ing to the k~wn 3D strut|arcs of human IFNoa2a, o~2b and o1~and marine IFN ~ii~[2g~31L all t y ~ 1 IFN~ display the same characteristic stnlcmrc of helical cytokines with a bundle of four 'up-up-downo down* [~ helice~ and an additional at helix. Consequently, it is ~ason~b!e to ~lieve that IFNo6 has a structure ve~, similar Io human !FNoa2, lhe t y ~ I IFN with which it sha~s highest simih~rity0 !f ~o, it ran be deduced from the alignment of IFN-a2 and l ~ ° b sequences ej~ul~ IA~ that !bur of the atypical Cys residues toys56, Cys58, Cysl07 and Cys145) appeared gathe~d in the same region of tl~e molecule thus suggesting that additional disulfide linkage could• e~ist ~.wecn-t -~- these . . residues. . . . .According . . . . to. multiple . ~lignmem~ with o!her t y ~ 1 !FN proteins frigate tA~. the sho~ size of the molecule con ~ attributed to a eenmd ~a deletion ~twecn ~sidues !03~!~ and to ~t |rimmed Cqem~inal end, I| is inte~!ing to no!e !l~at the central deletion occur~ in the ~egion ~howing the highest level of v~r~iahility ~n~on~ ~yt~ 1 IFN~ ~nd corrc~p~nding to a loop sepa~!ing lwo ~ h¢tiee~. Ttlis s|tatctural pecuiiarity, to-. gethe~ with the wimmed C~enni~al end pr~hably t'esult~ in a mot¢cule h~ving t~ m o t compact structure ihan olher tyf~ I IFNs, The porcine genon,~ was shown to contain only two int~onless nonoallelic loci with strong homology 1o IFNo~ eDNA I261. One of them was most likely a pseudogene as no corresponding eDNA was found in pig concepas library and its c ~ i ~ s~uence complained a p~vmatu~ termination codon resulting in a protein devoid of antivital acavity 1261, ~ e other It~us a ~ d to ~ the one transcribed in t~ph.oblas11261, Organization of the IPN~b gent (/~gu~ 2A~ ~veals tl~e dassica! s~ructu~ of a RNA polytrcera~ II lransc~t~.~ gent with a single c~p silt and a TATA ~ x k~ttted in position -33 to ~o27. Hob.raver. the seque~vce of this tran~fiption unit displays several uniq~e l~tlu~s by coml~nson to olher t y ~ I IFNs. First, the tran~ribed mRNA has a 5' non-coding region of 218 nucleotidcs (nt~ which is unusually ~ong among type I IFNs ~no more than 80 nt in other t y ~ ! IFN genes) and Ihat c~id have important implications on transla6onal regulation. It is noticeable that except fi~r the c,~ng
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regiom IFNob gene has no signilicant homology with virusJnducible tyl~ I IFN genes nor with IFN-x genes 1261. Moreover, sequences located upstream from the °rATA box are almost devoid of potential IRF-I or IRF-2 binding sites and GAAANN and GAAATG sequences known to be the target of transcription factors t~egulating virus inducibility 13, 331, This fact is in good ag~ement with the apparent ~bsence of virus inducibility of the gene (Lef'evre, unpublished data). Instead, 5' proxi° real putative regulate D, regions contain two adjacent, almost I~rfect 40-41 bp direct repeats and a third imper= feet and 5'-trimmed copy of this repeat located further upstream (ligure 2A, B). Seven~l GC-rich sequences bearing elements resembling consensus binding sites lot transcription iactor APe2 are present in these repeat~ as
well as in ~equences located up~tre~mi arid dow~stt~e~m
(tigure 2B). Such ~ites are known to medi~ite tn~n~crip~ tional activation in re~ponse to phorbol e~ter~ ~ d cAMP 1341. Thi~ suggests that ~igmd m~n~duction p~tho ways involving protein kinase C and/or protein kinetic A could regulate !FN-~ gene expression t34!, Fin~dly~ just upstream from the mo~t distal repe~t, a retroposon belo~lg. ing to the pig PRE. 1 flintily of short interspersed repetilive elements (SINE) is present (jigure 2At, 111e hnporl~,c¢ of these elements in the transcriplional regulation of IFN~b is unknown. Itowever, it is interesting to note thu~ the 40 bp direct repeat proximal to the TATA box i:, the only one to be absent in the unexpressed highly homologous pseudo~ eerie, thus suggesting functional importance tbr lhi~ repeat 1261.
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~|~l~ ~. A, Nucleo~id~ ~qu¢~.ce of th~ po~in+++tFN+b gen.e. N~qlemide~ are+nmnt~red r¢la|i~.dy In ll~ecap ~it¢ tvc~!~¢~im+~o • | k ~ e T~TA ¢o.n+,en~u~~++qt_~en.ce,lhe ++~, at~d ~lop ct~+hm~~ bo~vd. T h e f o u r G A A A N N h e x a | t t e l ~ f o t l R d H~ tl~t+ u p M r e a m | , : g i o | t ~|!d lh? poy~d~nylalio|! ~ign~l a~ undertil~ed, Po~i0oo of the !~dy{A) tail i~ im]iea|¢d hy an on;o~, The large I~ox i|!t|ieate~ tilt* hw,a0mt ot t ~ PR~ i e!em~nl~ Hofl~on!al, hl~ck h+atf+-~ff~w~indicate ~+h~~ilit++i~ of th~ 40-41 hp direct ~l~i~|,~(RI! and Rll!~ and of th~ 5L+tfimnK+, ~! (RI) lhat l+++tCk~[h~ Ot~l 1t bp of R!t ~+adRill Sequence e!ement~ h~+'alt,d up~tremn li~m~ tiw cap ~itc and th~ ~t~qut~ I~.~. Tho~ lt+t~t~hilt~gtmo~t !~tq~¢0~' wiih AP2 ¢or+~,~u~ hilldil~g ~ite~ ~,,~ iodic~lled by haWhed ha~"~,B+ AliSnm++n:!of the ~ettuenqe~ of lh¢ !hree rel~ml.~R!. RI! ++~ndRill. th,.~itim~sof the end of ca+:h ~,t~,a| my mdic;~ed ~~lali~dy !o the c++p~ile, a ++++pw~,~ii~t++~! It+ m~iml~e h~m~oiog+vmad idcm~¢~l lra++c~are mda~>m+;+~lby a~ensk~. 4, IFN-~ pco'~tm ph~slco
Nqem~inal ~ q u e ~ of the puritied rcc~m~hinant Im~lein establis~-tl that the cleavage ~~f the signal ~ptid~
<~¢mtcd. ~ p~dict~d, !,~lw~n r~sidu~ 2l ~nd 22 of tll~ preprotein, thu~ c(miim~ing tha~ nm~u~ IFNob, wi0~ 149 ~sidues, possesses the shortest sequence of all kt~own [ y ~ I IFNs. Also atypical is the ve~y t~sic isoek~|ric ~int of IFN-6 ~¢~lcu!aled pl 9,07L a prol~zny Omt was exploiied fi~r its purification on a cationic exchanger [35}, The ~¢~-~mhinant IFNo~ eluted in gel filtration with a M~of 19 (~0, suggesting dmt the active p~x~lcin is essentially monomeri¢. The recombinant protein wus found to Nogly¢osylated. most probably on Ash 79 ffiga,~+ lab and Nogly¢osidase F t~catmem resulted in a protein of M,. lT i~0 which fully ~t~in~.~l its antiviml activity 1351, Antigenicity of fitis no~++elIFN is st~-cific, since a neutral+ i~ing mt +mti~rum ~o recombinant IFNob did not neutmli ~ any ot" several i y ~ I IFNs. whether po~ine (a, fi_,m)+ or ovine ('t) +~r human {otl. ~2) 135L Recip~ally. IFN+~ was not ~utPali~.cd by any ami~ra rated against ~veral rtx'ombinant porcine or human t y ~ 1 IFNs 1261. However.
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IFN.~. ~hat ~,as exp~',.'ted +f both +FN spcct+es brad ~o ~ °++,,~:
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4,2, Comm+:m pm++}er++esw++hhe+N++{++ IFN+6 shales seve~a~ physico+chemicat p~x>pemes with oll~er ~y~ i IFNs. namely s~aN~i~y of its active|) at pH 2. but sensitivity 1o reduction (t% 2-mercapt~'thanol} and heating ( i(R} ~:C), Also, like IFN-~, IFNo6 has the property of 'del)nsive reversible denaturation" [36~. namely resis+ lance to healing {IiRTI~+C) in the presence of urea and 2+mercapwethana! i36i The specific a|~t|iYiratl activity of a on M D B K CO+ischallenged w+ith ~esicutar pm+ified |I"N+~++ slomalit{s vires ga~c an_ esti+na~e +~15 :-< tO" to I(P lU/mg pmt¢im i+e+, in |he rm~gc of previously known iFN.{:~ activities t351+ !n c{}mn]~.oilwith IFN+{:~+IFN+6 was shown to e x e r t a synergistic antiprolifemtive effect with fFN+y on pig kidney ceils {iigure3~ This synergy with IFN-y could make sense from a functional point of ~icw, in connection with the known co-expression of both ty|Rs of !FNs b) :he tmphcct~em~ (see below}. In comrast, no synergistic ambgmwth effect ~,as observed between !FN-6 and
IFN-6 labeled with ~est was found to brad sb'~cif~ca~y to+ pig kidney cells with tk~st kme~ics ([~v, re4A,. This binding could be completed with eearly, egua} efficiency by porcine [FN-{,~ and by humm~ [FN<~ (tigu+e 4B,~ Upon cross+linking of radiolabeIed [FN+~ to pordne cel~s. a singte 120 kDa complex was formed, which ¢ott~d be prevented by an excess of unlabeled p{:.cine tFN+(:~, bm not of porcine IFN+y [37i, These data show that IFN+('~ binds to the same receptor bindmg chain as IFN ~ on pig cells+ On human celts, whereas p{m:inc !FN~e is capab.te of both antiviral and an~i+growth effects, IFN~6 exerts no nignificant activity [ t 7 i Accordingly+ bindmg and cross~ linking experiments peri~rmed with radio+inhered IFN+(~ on human WIStl cc~ls did +~ot detect any specific bind.* ing [371+ tt appem's therefore thai iFN-{'~ is devoid of significant affinity for the human type I |FN ieceptor,
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Absorbance at 6~0 nm Figure& Synergistic antiproliferative elTec! of IFN+h and IFN+y on potviue cp th h21 cells. Swine tcsti,,, cells 1~51 were s¢,ed.ed a| 50(RR)cells/well in DMEM medium + I1)++ FCS in 22 ram. 6+well multi+trays. One thousand antiviral tlllits of cacti IFN-{~, IFN-y or IFN+6 were added, either alone or in combined mixtttrcs (!12- !12), +u¢l+ that all monolayer~ received the ,,ame total amoum of IFN. All treatments were made in triplicate in randomized wells. Atier a lurlher 4B+h incubation, c¢II mono~ layers were lixcd with 2,5% glutaraldehyde and relative cell anmunls were estimated by methylene Idue uptake, exlraction, and measurement of ahsorbance at 65()rim, Bars represent the standard co'ors of the means.
FiRure 4, Kin~+lic~ of binding ~md ~m~pcmmn ~i~v~+~b~ di~t~, re.t IFN~ with s~"i 1t4N ~ ~m pi~ kidney c~|h {Ri:q~{-~~7~iJjm¢} Purified l~¢omlmmnt IFN+~ wa~ iodin~4ab¢l~d ~ i ~ |~h~m tluntcr reagent 1~1 kit (ICN, Radio~ghcmical~L Al| ~ p ~ r i i n ~ were p~rlk~rmed at 37 "C in 6+w,ell clu~ler plat¢~ (3~ mm m diameter) in the pre~nce of DMEM nt~dmm + 2{} mM ii~p+~,, ptl 7.4. I(Y~; t~tai calf serum (FCS) and 0,()5~;~ T~¢¢it 20, A, Kinetic study: abemt 800 antiviral tmit~ (2()0(~)0 ~'pm~ +~
~sI+IFN+~ were incubated ¢m RPTG cell monolay~r~ w~th { ~ or without (I~) an ¢x¢¢~,~ (100 ×) of tllllab~led iFN+& ~|nd at indicated tim¢,s h~¢uhathm medium from two ~elb+ ~,.as rcmo~ ved. cells washed 3 tim¢~ at 0 ~'(' with iY¢~t~m~dmm ¢~mh~mmg I~)~ FCS. and c¢ll4~smciatcd radtoa~|ivity wa~ dct~rmi,¢d, Sp¢ci|i¢ binding ( ~ ) wa.~ cah Elated a~ the dif't~ivnce bctw,¢¢t~ total a~ld |lOlVSp¢c+i|i¢ values for earth iitcuhalio|~ tittle, B, Conlpctition curves, hlctJbatioi~ ¢ol~dilioi~, ~¢r+¢ the ~am¢o ¢~+ cept Iha! °~al+lFN+,~ was il!¢ubalcd tiw ! h ill the Presritc'e cd' k!!oWll Coilc~l|tratio|ls of the ['t)ll+>~viiff+ ~Fut+~+mbii+ai+l !lN~: pt)IFN~(~.I ( I ) ; poil+N+h (O); p.olFN Y (/+ i; hu!FN ~tl ti'~i: am! ImlFN+(~2a (()+
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t~ 1t~ ,~ iA!, a~.ki~ !~t!hhii ~tlli~tautil tit !t:N ? ll!}, ~¢¢ond ~iep 17ft~ T~!tt~ t'i~t|--t~ilt~l!!~t~| 7otti ~llti~!'ttl t~{i l+{} itittl
Ft!'C .~'o!tttJ~l~d !~t~i t!tl!Vftlh!~it 17(I {!tl> Nott, ~ thit! i|l l~lth C i ~ 1t~ i~ lJnh!li¢il, Co|~l.tt~till!llttJilO~!tt!ltilt 7 ~qlh
with WN-y l t t a i i ~ , I~issthli
One of t~.~ m~l striking
i~atu~s
of IFNo~ is ils
det~t~-b~ in p~gnant uterus around d~y 13, l ~ k s on ihy 1~ot6 lind tl~n ~ ' ~ I t ~ s until ¢hy 201141, Analysis o{ dtff i4 to dily 20 Col~'¢ptus trim~ripts indicated tht~t evolimorl of both miNAs is .p~.all¢l durin I Ibis ~ ~nd Ihtit mil~imum m~IivirM ~ctivily is sligklly ¢1¢h~c~ ~s c~o~ to t-oa~h~l trtmscripl tev~ls lfifu.t~ .1i, At-o though l / f l i p , us quantitative an~lysi~ of the e,~olutkm of l~4h p!~Icins in the uied~ !un~n has nol l~'cn~d~ nnc~l, ii c1¢ dy ~ l t ~ Ihltl IFN-~ is expensed ill Inuch lower
hlasl i:+Cli4ct~ " "' , ' ~ith ~ntihocht, ~ c i i i c
ihr each IFN
derm ¢¢11s on d~y 16 of dcv¢!opmcm are t~siliv¢ tor IFNoy ~md IFN-6, ~,~ revealed by immui~ofluo~,~t,cace {fi{lu~ 6}, Second. Ihe subc¢llular l.t~alizai.ion (l~ue
Inlerf+-nm+{'~
?g5
7+ P~,+slMe ~+rlgia ami n+lv+ ++f |fN-i'+
7• J'. l.~ +'+Pc'++'+P+Pq~.+++~+,++,++g~ +. . . . .
+' '+ ++++p,+g++g++"
Goat
Cow
++'~++~,t++~++~+>?+&~ d +PC ++,+g++¢-'+"++.
iZ
+',~+'O+P+,~+++,+'+,~" ?
Horse
+++
ea As well as 1FNoT. il aNx>ars clear that ti:N+~ can be considered as a ~rue tmphoblas,.ic IFN. It seems very sinking lhal ia t,,m gr{mps of species which am dose mlati,+,es. ,'~amcty Suideae and ruminants, ~ype I IFNs cxpNssed lV one tissue of the same ontogenic origin ape+~r so distantly ~:;}afed. This raises the interesm-~g. question of lhe eompm+d evoh.mona U origin of boda type I !FN fi~milics. Sevend lines of evidence indicate that !FNT. which +tt-c ct~+sely w!ated to !FN-.m. have evolved nM'~er i.?ccnt~y from ~ m,is--ia{h~cih~et>p,.: t Ii::Ngenes that ii~'t_!~fir~dir~,+phob!as!~p¢+ii:}c ex.p~-cssi~m and lent almost ¢ompleleiy lheh" virus+inducibility m non+trophobhtstic tissues ]I 2, 26i, According to phyh:,gcne|ic data. it seems very !ikely that this event was limited to ruminants so thai IFN+T am no| pwscnt in other mammalian l*amilien i 12]+ By contra.~L |FN-~3 in much mo+e di++.e~gcm irom othct+ i y N ! IFN i)mi!ie~+ Pt!ylogenetic data O,~p+++++++..+'/B) . suggest that the duplication event leading to IFN-i5 was before the divergence of ItaN+¢,+ +m and -T famttie,,, but latter lhan the divegence of IFN-iL At'cm+ding to the divegence el+ e,t~ting sequences at replacement mites, it was suggested tha| IFN+6 aneestra! scqttenee diverged ahot!t I blO My agm !ong betbre mammalian radiation [26]. The presence uf homotogtm~ o1' l|:N-b in other nmmlnaliail species t~ lhcrclbre very coiisislcltl. Ti) lest this hylmthesi.,,, we tried
tO deicer i!:N+a-relaicd seqii¢llCeS ill the gt;llOilliC DNA ~1 ~e~,ei~;ii iiiai!tilllil~. Prdh/iitlai°) • data i[idiealc thai the hu l!ia!l g¢llon!¢ !:Olliaill,~ a +,lllgl¢ hicu~ clmel)-, rchlicd to pen:hie II:N-5 hilt thai it is a p~Ottditth+liP ilefbvre, untlut~i!i,,,hcd l'l:~ltlt~l, A~ ~htlt~,'ll iii fiRt+li" 7, gOllOli/iC ~ql|¢llC'7,~ hot!!olo~!it|~ tti !!':N ~ c'tlti he easily deter'ted ill +++tt~mi+ DNA of l+t)viiie+ t+aprin+ aiid horse mpc¢ies w!licti ilr,0ui!g!lhll+ iliitilinlMs phylogenetiea!ly ¢lmc to the pig, We have cloned one of the equine Io¢i which revealed a D~tetllially litnetional get,e+ i,e+, cnco.ding il prolehl with amiviM activity° ~hafi~lg highest homok)gy with porcine It,:N+~ (55¢;~ idcmity al the aa level i, thus .~trongly suggesting thai the I! ~N +~ thlnily e~itends to other illalllnlalian stle~ies (Lel}vre, unpublished rcsults), li is interesting to note the presence oi" homologous sequences in ruminants. If functional IFN:bdike genes are present in lhe~+ s~cies, they arc probably not !mphobhlsl+spc¢ilic, as no conceptus+derived antiviral activity distinct I'roni IFN-I has s~,! far ever been li)und in runliniinl species. Further work is required to determine whether these novel equine and bovine genes arc virusoinduciblc. For the time ~ing,
a quite plausible hyg~thcsis is thai porcine IFNoh
derived thml a t'amily (if virus+inducible type I !FNs nlore
or less widely distributed iilliOll ! illalllmiils. I+ikc II"N+i, they evolved towards lrophoblasi speci!ic expression in a narrow group of species.
kb
21,7 --
5+0--
+
....
4+ ',,3
+
+
~}i ¸¸
1.9
+
,
+
1.3"-
0.94
FiRure 7, Prc~c.¢e oI'IFN +6-wlated ~¢qu~a¢¢~ m m~.0g ~ m m ~ el
,~¢m aI uiID+h~w~llmmmal~. |}NA ffmn ~,~ch~pe~0i~,{ IB #~ii~+~ +lig~,~ttzdto comphmon vvilh the ~p¢~:ified ~])tr~t+¢ a!~d ~het~ loa,J~don a (l,~I+/,agmm{' ~cl,The Not wa,~hy!mdh'{:du~idc'~k~w, ~lringem:y with a probt., b~aling lhe IFN+6 coding ~¢ql+~nc¢~,The po,dihm,,, tff ~,i#e ~landa+d~+(in kbl m~e ~llo',*n oct lhe ~ ft,
7, 2, Wlrn mh' IHays II"N.+) i. pr¢,~mm~Lv:' The preci~,crole played by ll:N+b !a~ well ~, by IFNy} during early development is m far unknown, llowcver, we have precise indiealion~ on what functions arc' no! fulfil!cd
by II:N~. An experimcn! shnilar m dm~c done in rumi+ nants, which dcmonslralcd lh~I !FN-4 per .~c i,~ amitutcolylic in ovinc and bovine spc¢:i¢~ 139, 401. wa~ pt:r+
7~ f¢,em~l i~ ~ pi$, I~ th~ s!~ies, tL~ ~sult was ~~iy dl : i ~ t k m o¢ massive do~s of ~ o m . ~ IFNs (IFNoy ~ IFN-8 in mixing) in the uterus of m m o p ~ ~ gitt~ betw~n days i i ~ 14 of ihe estrus cyc~ ~ ~ ~ v e n t the coepus lu~um regression (ludid ~ signifi~tly delay the m~s !451, This provided a d~monstration of favo~ ~1 whar¢in estrogens ~reted y ~ pig i, fact tl'~ main ~tilu~olytic I ~ . ~refore, a ~4ike effect of Imwine l ~ s , ~ would '~.~ue" d~.. c o ~ s luteum (~ ~ ~ ~ what is callM ' ~ t e m a l recognition of ~Y'L ts v ~ unli~ly. It remains however m ~ g ~ d if IFNs ~ est~ens could ~ t in synergy on corpus luteum nmintenance 1451, s~ts oi' data strongly suggest that the most ~ ~ tm3get tiSS~ of #g ~ b l a s t IF)Is is the erdometri~ epitl~lial cell. which fao~ the trophectoo ~rm. First. ~ t r i M e # ~ l i a l ceils we~ tbund suso ~ i M ¢ to IFN ~tivity by ~veral cri~da !461. Second. we ~ M demons!~te wi~ a mnsitiv¢ and s~iti¢ ~L!SA 1Mr ~ l~--y ( ~ d probably no IFNob) crosses ~¢ ma:~rnal ¢pitha!ia! b ~ e r . what makes it ver~ unlikely that u~rl~ mucost,, which includes stmmal and lymphoid cells, would consfit~e a target lbr IFNs (D'Andr~a ot a!., UBlplJb|iS~ results). Third. the wophectoderm i t , ll was fo~M un¢~spo~slve m both !FNs, which piles out a ~ssihl¢ ~ u t ~ d ~ ¢lT~.'ton !rt~phoblast ceils I~t, As ~ immune !bmtion of !FNob is vee/unlikely and ~ a / ~ the ~tri¢t eplth¢ltt~ho~ta! pla¢¢ntatton tn Suideae ~v.ents ~.ny contact t~tween em~onk" o¢ l~t~l ¢¢!!s with tM maternal bl~M ! lgl), the ~)~sihility t~tnain~ that the ¢~tokine '¢ms.~ot~tk' ~two~n the tix~phec|~tent-t ~md the uterine epit~lium c ouM ¢0~t either: t) ~la¢ non. s ~ t 0 ¢ ~ s t s t a ~ of efldometrlum to infections; from thi~ tmint oi' view, IFN~b wa-s found ¢ill¢ient as ..n ~ativi~| t.~ttl~ is ¢l~rly )I¢ csp0flment~l a~h to veri~ this hyr~h¢.ts~ 2) o/ae or several ~(s) ~!n~d to p~pa~ tM to t t n ~ a t t o n , Another eytokine, nan~ly |¢ukcmi~ inhibitory fa¢tor (LIF), was shown to ~ ¢s~no tim for i m ~ a t i ~ in mice I471, This factor, as ~ll as oI~r cytoki~s and growth f ~ t ~ s such as interleukin 6, ~ W~ns,fomfing growth f.~to~ ~ insulinolike gix)wth f ~ 2 ~ arc ¢xpr~s~ at tl~: ~.~.~oofetal interi~ce during ~ ~ ~sto~yst att~hm~nt I~-50t, It s~:~ms likel-~ that ~ s e f ~ o r s m~y wo~ in ~ n ¢ ~ with ¢'o.¢xp~sgxl' t r o p ~ ~ i c IFNs to aff~t ~ri-implantation events, C~-et~i,~g IF.Ns, ©x viva studies with luminal ~Am~ctri~l co!Is in,eultu~ are umterw~Lvto exami~ their effects ~ th~s epiti~lium, ~ m t w ~ IFN-t_x,w~.~~ ~ t ~ atv~mg other t y ~ I ! ~ s f ~ , ~ ~ , n ~ bkmciivity (as ikatas we could ~-~: in ~,~11cultue, It~y are similar to tl~.~ of IFN-~L ~
i,efb~vre et ai, favor the hy~thesis that iFN-tb was selected on the basis of physicoochemical and phammcological pn~perties. leading to bi~is~nibility, stability and confinement Ihat ~st fitted to its sec~tion and function within the uterine environment. It is to be noted, in this ~sp~t. that the two l ~ s produced by the pig trophoblast are both highly basic proteins (pl over 9). Neve~eless. the syn~ orgy found ~ t w ~ n IFN-y and IFN-8 for the ~lipmlit~rative effects Oqgure3). probably applies to any other biological effect. Co-secretion of both t y t ~ might. therefor. ~ p ~ n t an efficient means of am#ification of each ~ingle biooactivity, and hence a po~ib!c ~¢lecd~e advantage over sec~tion of a single !FN si~.cies, In conclusion, 1FN-6 is another mcm~r of an h-tc~easo ing family of tY!~ 1 IFN~. It i~ a .at! tha~ iha~last lwo n~vei s~cie~ discove~d llt:No~ and iFN-a) are bofli ttopho~ bla~tic IFNs. each one s~cific to a d i ! ~ n t mammalian family. It is probable that other s~cies will ~, ibui~d, each gifted wid~ ~cu!iar physicoochemical, ph~aeological and biological pin,hies. Like IFN-t, IFN~b d~s not ~ , m to have a functional humaa homo!ague, in spite of this. it will b¢ of interest to study in tl~e pig or in other animal models, s ~ i f i c pham~acolog~cal and theraF,utic perthnnanee~ of this novel mol~ule as an antioinfe¢|ious and/or anti--tumor agent, in view of the known t~cufrenc:e of !FNob gen.e(s) in other animal s~cies. Acknow!~gmems
cmd A. Lo¢i~!~lli(Uai|e d~.~ Physioh~gie de la
I~t,p~u¢ii~.d ~
PRI)M ~ioIl'f~r p¢i1~ntlii!g ltltil~iill ¢~l~,O|iienlI~ii|td ~Utg¢iy
Referees
tal¢~ieg~m $ ~
et~ter tic!cra~it~tl f~oln a YAC ekta~: coaiig,
14~ 1Pgnigt~ch~T,, M~t~tei N~, ~.hwart~l¢in M,, N~g~a S., M~r,m~atsu I3~ U#~ G,, t.~i'|~th G., Gwx~:~ I., C~vcii~: Iransfcr of a functional h,z~ean t~l~e~'tm a ~,~'e~w int~ n ~ cdk: ckming a~td etp~es.
~?1 CoIK~ |t. No~h.'k D., B~rak S,, Rubtn~ein Me, Li~anddndut;t~tl ~ ¢ i ~ t ~ m ~II"0~c type ! in~cdenm r~,2ept~tr t'ompo~:ents, Me!, Call, Biul t3 iI~5~ agm~.~21a
ln~e~emm~
7
Ig] Sen G . C , R a n x ~ ! ~ R.M, Lmc~le~m ~.~a{~v¢~ ~ m ~ m q ~ B . ~ ,
!~,~i
irva~ iBdu,ib0ip, of ~ " human ~FN <~ . ~ (!990) 767--77~
{l l} RiR~It~, R.M., In~erf~-,m,a~ and pv'eb,~m~:y..f~ lmcrh.'nm Cymk1121 R¢~-~Is R.M., i,,m L . Atcxe'&k~ A.., New and atyp:cai lim~i~ies of lyp~ i inl~rt~m.,.1s m eaamm:a{x: C~mpan~ivc fun¢~ioax, ,w~c:un:,,. ]i3t
t l,l]
Se, { i~92} 287-.~25 1.¢f~{'¢ |=-',, M~lrlinat.-Bot~6 |{. t]~ilhsn~ot M . Ztmari K . {'h(~rtcx' |TL L,~ Bom~m~lien: (7 tatc~i¢-'lon-gamma gcn~c ,rod p~owm e~e •q~.~tl~tlieOtisI~ CXD~?~s~'~I b) fh.¢ pod's'me: lml~hcctodcrm c a r k m ~eMalitill, E~tt it. lmm~mol ).t} ~ tg~}l 2485-24~}O IAl t_ii.~l~i}al-t.|i{'t'~ {? Matitii*;it.B~-~tto i",, "[i;'i~ltit N| l.et)tfe t:
I151 17~ttc-G~ii'am !}. Ro!trt-il;dliol tt+, l,i~pct J, {'trait) { ' . {:nu,~iml ~ll)t~-liI'O~ll%, c'OllSltlttltvi~ i~lerlt, trt)tl~ ~.tlld ~tlllgtTottl~l~ tll lit,q~tol~t placenlal bioc~i, t:~i,/~. Nail At'~td. Rot 1L~S:t~7 l tt)S3~ 5tltlb-SOt4 1 ltq Chai-d T. Ctiig Ptt., Mituah~ii~,ey M . l.c~: {'.. S,lpha mlefb.'mn m t171 b~wle~ A.K.. Reed C D , Oim~ DA, tdel~l~ti~alioll t~l ~li tllt~'rlt'lott In ~itlarl!le pla~;'¢t!lii~, Nalitr¢ 2N~ 1 t~)SII} 2B6-2BT. 1181 Leis~ R., Kaulirtat~t~ P0 glaceaiM slrttemre in a ctmtparaitvc a~f~ei:l, tTi~p~flirt, tirtdt~wmt~ 102 ~1994} 122 i ~4 i 1~}t Ottiihml,at M.o l;ldeh~)t~ ItC, l,¢m): I- l,ll,i~l~)<~l de~ehipinc, m ,rod ~lt-itlkiillalmll lit l'hiblilth I[', l.,ev~t~,~eiir M.C., ||li~tler R It.F, tl.id-~.}. R~tl~u¢lton m Malllll-t~ll~ ~tlitt ttl~itL t{llipm t~itfi~, l lJlJJ,
~i~t p 12111 { " m ~ ]if",, fli~B~ii~ R,M.. ik~rcine conccplu~¢~ ~ecrc~c an mlc~ lelt~lt dulrtltg ih¢ |~lt'~liiit¢lititi?~ll t~tt~il ol eal-ty p~¢g~tan¢), i|ml R,¢p~i~i. 4{1 1 l~t~iJt i lil ~} 11 i~. 1211 ltt'lt~'~iitdo M,A0 tialiic'v JP, t'lecr~ N, thtnltc.~' {'.11, hlrr¢~ B.A. JtRm~mt tt,Mo f t a m t.]{%', ¢}l~nt,I [}i ~l?l/fC'llllll OI p~'t~lt'Hi~ %%Hh ~litt~iflii iii.'U%ll%7 b~ pig ¢i~lt~°tepltt,w~. ] Rc,pt~M l'c'rlil ~N I I~JtJlti |!}? 2t}.t 1!21 lte~i~ ~ 0 Mlriilidtt M.A.- lkml~+l.'i- tLIl. t-t~rnc'} J t': ]hllft.~ ii A ,Ihtllttt~ttti ti.M,, iia~¢t I:.W. ltll]il¢ll~:c, t~t thi.' t'tldtlttl~lllt|tii, pmw,l~¢ iil|li|till/t~
iiild lit~etttlg
iltg ¢oilceptti~'~
~iti i,itilit if~ll ~icltvli}'
ol [lltttt'|tl~,
] R~'t]md Fc¢iH ~I~4 i I~l~Jtli 71}% )11
~tpt-~ i~lleffem~l, (leite 77 I ItJ~l~l) ;t41 l.ilt. Mege i)., lxef~vf-e It, I.ii I]oililflldi~i'l~ C,, "File llt,ll°~>lill~ i'illliil~ ~}t tt!t~'l-ll~fl)tl<-~ °, eh~llilig, Ml~itl~lllllil littlilys~s and fuB¢lion~tl Mlidi~ of Iit'e I~l~ied gene.% ], hiierl"elOil !t¢,~. !1 I I9~)1t 341.--350, 1261 Lel~vi~a Fo, itoulay V., A novel lUld alypieal ly|l¢ lillt~ inlerfel'illi gene expressed by impllobla~l during early i~regnliney, J. ilii~l, Chem. 268 {1993} 197~}-.li}768. 1271 Lundgten F.., 1,anger J,A.. N¢~nlenehllure of inltarl¢ron reeeplop, and inlerfemn-~. 1, Inierlemn Res0 17 i ItJt)7} .t10, t2t!1 Kl~u~ W., ¢i~11 It,, Labhtifdl A., Wipf tt,,. S~nn II.. l'h¢ thr~¢di il~n~,tOltal llitlll ~¢solulion ,~iru¢iuri;~ ~1 litainlui inie~l'emn !~-2a dt~lenlfiu~d by hciemnu¢lear N M R Sl~¢irm~opy in ~ohiiion, !. Mot. Biol. 274 (1~{}7} 661-675. 1291 Radh,akri~hnan R,, Walll~r L,J,, Itfu#a A , Rell:h¢ii P., l'itlliii I:P. IN!lgabhu~han T,I,., Willler 1%1,171,,Ziu¢ niedililcd dhn~r of lltllilil!l hilerferon i!~, re~¢aled by X-ray cryslalhtglaph 5, Slru~'lur~? 4 I I{J96} 145]-,146,t. I;Oi Senda 'IL, Sailoh S,, Mir~ui Y., Relined erys!a! ,~lru¢iure ol lta¢ombintml muriue inlcrfi:rlm-171 al 2,1 $ A rtL~llhltlilll, ]. Micti, Biol. 2~), (!!}{}5) 187,.207.
and G A A A ( ; T generates sequences that me.~r~ptien f2semr AP 2 mediates mdt~ctmn by ~ dKfemm s~ignat ~ ~s~d~tc~m p~ah~,a}~< protein kiaase C and cAMP, C0~[ 5t (t987~ 251-260.
[/{5{ ~mrcmc ~ypc I intcrlkw~m. Bi~hcm~c~[ and B~tugic.q c{tamc~cr iAamn of ~he ~yc~mhinam ~ro~cm exprcx~ed ,s~ m,~cc~¢¢-}1~ E~r 1 giochcm 23t~ { i~)ggt 2{R~,-206
f17{
[b~}r,
Nm P|).. [.cl),,~e |: | a B~m:lard~¢,~c tL, \%p~ca~ ,,p[ ,qacr~cr~rt brad,, ~m porcine ¢e~b+ t~, a re
RS .RS. 1401 |~|}{[t{'he[ ~/%~;.. }[/.[[~?',C|} P ) . (;ri}7,"~ [ S . ~[g[~ }¢t' S [). ~}[,t.[}{~" ( ' .
1411
J. R c p m d FertH, {Su~pl.} ~
1421
{It}~){} [71 lg¢~
~,~c%i,flied 1-1%
A¢it! IRes, M~I, 1711o1,5~ I ic~{}7} iil~}--127, 1241 Charlier 1%t,, Ilue D,, Ma!lttl J.> Gta~e 1~, Chiiiiiig iiiid l~'t!lr~',~iOll !~t ~25[
t34
'i:X-!q ~c~c,, CctI c~O
14il J Repmd, I,¢i!il, 7~J {t{JR?} ~e3~ [72, 1441 Van ller Meuie~l 1, Helm~md F,A~ {.}ud~aa~td¢~ ¢ ' P J , [:,ff~¢t ,~ I ei~.'vre ~',, Mfmma!.lt{me 1'., l,~va~elti A0 N ~ PB,, h?r{l~,t M [,~ Bonnardi~N C , Inlrauteriee i~l~,i~n o¢ high do~e~ ~ gi 8 m~ph~:~ blast inlcrfemn~ ha~, rio aniilulet~I}ti¢ ¢He~:t m ~3,~,'h~ gitt~= fl0o~ R e p m d 5~ ~tq~lS} t026.1031, 14¢~1 D'Andrea S., Ch~u~t¢~-m~m S,, |'lOvh, m J [ ~ ia~ B~m~lafdiere ¢', Paramn¢ a¢livilie~ ~tf porcine wophoblami+ mlerlcmt+++, ] R~+pt~+d Feriil+ I02 { [~)04) Ig5 ~+4 14/I Sle~t~:l iLL,,: Ea,~p,~r P0 Btu~¢t | J , B~tbtt I I , {[;~di I0 iomge~ l , Ahb~fld~til~o S,J,, lihl~t~u~y~ mll}latllalio~i {j~p~d~ ~m malef~d
I.t51
7/~ 79,
14~1 t:t}ll¢:gptus illl;lgh|ll¢llt, Cyloli[|i¢ O { [t)93} 49f~, 499, 1491 Gilpl;l h,, l};Iz¢l ~ l;,~,',, Jileg¢|: I+,A,, [}iffer¢I;li;tl cxpw~:~m el bctt~ irallMornlillg growth la~'t~.}|?, I'i'GI bct~ [, I { } F b~-t,} 2 trod | t } | h¢la 3} and [heir rcccptor~ {type I and ly!w i ! } m l}.eri impt~!matuln l}or~'itl¢ ¢on~'eplu~es, BIol, Reprod, 55 t l{J{Rt} 7*}t~ a02,
788 1501 Sinamen R,C,M,, Green M,L., Simmen EA., IGF system in periimplantation uterus and embryonic development, in: Molecular and Cellular As~cts of Periimplantation Processes, S.K. Deyed Serono Symposia USA, Springer-Verlag, New York, 1995. 1511 Devereux J,, Haeberli P,, Smithies O,, A comprehensive set of ,sequence analysis programs for the VAX, Nucleic Acids Res. 12 (1984) 387-395. [521 Fitch W.M., Margoliash E., Construction of phylogenetic trees, Science 155 (1967) 279-284.
Lef~vre et al. 1531 Charlier M., Hue D., Boisnard M,, Martal J., Gaye P., Cloning and structural analysis of two distinct families of ovine interferonalpha genes encoding functional class 11 and trophoblast (oTP) alpha-interferons, Mol. Cell, Endocrinol. 76 ( 1991 ) 16 I- 17 !. [541 Ghosh D., A relational database of transcription factors, Nucleic Acids Res. 18 (1990) 1749-1756. 1551 McCiurkin A.W., Norman J.O., Studies on transmissible gastroenteritis of swine. II. Selected characteristics of a cytopathogenic virus commot~, to five isolates from transmissible gastroenteritis, Can. J. Comp, Med. Vet. Sci. 30 (1966) 190-198.
Inlerfen}n-del
a The fi ..t m e m b e r ot" a novel type [
aterferoa fanuly
F r a n c o i s Lef~v~e "~*, M i c h e l G u i l l e m o t ~, Sabine D ' A n d r 6 a % S a b i n e B a t t e g a y ~, C l a u d e L a Bonnardi~re" '"Ut~itd de Viov!ogie et lmntu~w&~gie Moldcut¢ffrex, h~ra, 78352 Jouv-~dos-~s cede'x, France e~Umtd de Biologic Celhda~re e¢ Moldctdaire. L~tra, 78352 Jouy-E~-Josas (~dex~ Fram'e
~Received 2 February i998: accepted 9 July 1998) Abstract .......We have rccen|!y described a novel type I imerferon ~!FN) co-expressed with !FN-y by ~he trophcctod~nm of Hie pig ctmccplu~ bciv,+cC+~d~y 12 and da) 18 ~ff ge~t+~tion+ ~ development stage t!mt corresponds ~rOimplantation in the uterus~ This [FN, ~o,~,. oli~ciaUy ~.amcd |F'N b~ is recognized as ~he !irs~ member of a novel type I iFN tamilyo This paj~r reviews the main published dat~ on IFN4% toged~er wi|h some new data, showing that IFN~6, while being a true !ype I IFN, has some very specific structural and biological pro~nies. Sequences relaled 1o IFN=b coding sequence were fi~und in the gcnome of man and olher ungulates but the or,,~) other potentially functional gone was |~mnd. so far. in lilt: ht~tse. The pig IFN~b mature protein, wi!h 149 am~no acids, is the smailest
of all known type li IFNs. |t is unusually rich m cysteine~ (seven residues), and has a very basic is~leclric poinL Recombinant IFN-6 expressed in in~cc~ cells is glycosyla~ed and has a high anliviral actmty cm porcine cells, but not on human cells. |~ has high anfipmiiferative activity, which is significantly enhanced in lhe presence of IFNq.. Th~s new IFN was shown ~ bind or~ pig cells ~ ~he same type ! ~vceptor as IFN-,. !FN-~ and !FN.y genes are co-regulated in lhe pig trophectoderm, whose cells ~n day 14-16 of development simultaneously .~ecrete both IFN proteins. The bioh~gical r~le of pt~rci~e IFN-~ in early prcgmmcy has been fi~und unrelated to i!~e known an|d~|eotytic efl~'ct of tmphob!a~tic IFN~" in ruminants. @ Soci6t~ franqaise de biochimie et biologic moldculaire I Elsevier, Pan.~
ty~ 1 interferon t trophob!ast / pig / intefferon-~ / geslation 1. |nlrodt!ciion ~!3~ I imerferons {IFN~ t'epre~ent a tmique group ~t° t'elated antiviral cytokine~ with many original properties j l I. Tiley are matn!y virus-inducible and arc encoded by introntess genes located in tl~e ~amc region of a single chromostmlel2. 31. Although some members of |his iamily al~ ~ometinies distamty r~lated flor example, IFNoa and IFNq:-| polypeptide sequence.~ share only 20% identity 141), they all interact with the same membrane receptor [5=71. According to a well established theory, all t y ~ I IFN genes originated fi'om a common ancestor that expanded by successive duplications 121. Until recently, four distinct type I IFN families have been recognized in mammals according to their structural and antigenic pro~rttes: IFN-ct, -[k -to and -t. The three former families top,sent classical virus4nducible IFNs, existing in almost all mammalian species and mainly involved in nora s~ciflc protection against a wide variety of pathogens. mainly viruses 181. !nterferon-t or trophoblastins, discow ered about I0 years ago, represent the first example of a type ! IFN family specialized in a physiological role (see accompanying paper by Martal et al.). These proteins are secreted into the uterus during early pregnancy by the trophoblast of the embryo in some ruminant species. Their
ptoima13~biological role at the materno-fetal interface is not anti-infectious nor antiviral: they are antilutcolysins that represent the lir,~t embryonic signal preventing ~'cgression of tile corpus luteum and permitting omgoing gestation (Igl; see I10, Ill for reviews), Moreover JFNt rcpre~eat~ the first type I IFN fiuni!y that seems to be re~;lrictcd to a ,~;ingle taxonomic group among mammal~ [! 21, Presently, there i~ no spme~ m whici! the comptctc sequence of the type I IFN locus i~,;exhaustively known. Thus, the possibility of existence of yet unknown familie~ of type I IFNs havin$ orlgtna biological propez~ies and roles remains open. We have previously described that in the porcine species, the trophoblast expresses a type i iFN gene around imphmtation 113. 141. According to its ofi$io hal properties, the product of this gone is now considered as the first member of a new type 1 IFN gone tamily designated by a special Greek letter: iFN-5. In this paper. we will review data available on porcine IFN-b concerto ing the original properties of its gone and protein, its possible biological function and preJiminary data indicat° ing that IFN°8 could be present in other mammalian species.
2. The discovery of porcine IFN-b Altllough expression of IFN biological activily m the matento°l~tal unit has ~ e n known for a long time in man
T~
Lei~vrc et al.
mouse t15o!71, it is lhe discove~ of ruminant IFNo¢ l~.t ~ the ~ a ~ h for tFN expression by the ~ast of ungula'.~ s~cies. Ungulates are mammalian spcc~ kaving ~ epithcliochorial placentati~ and late i m p t ~ t i o n II8, 191. At the end of the 1980s, several ~rs, the ~nce of ~-dertv~ antiviral activity in the uterine hums of ~ t ~ w s ~ n g early ges~ion [13, 14, 20-221. ~ t e r i z a t i o n of ~is ~ t i v i ~ activity reveMcd a ~ k i n g feature: most of it was IFN-y espres~d MI ~ cells ~ abundantly as IFNot in ~mi~nt~[13l, It ~ l d ~ rimed that strong IFN~y e~pcessh~ by the pig peaoimplan~aon trophoblast ran mainsa uniq~ feature of thi~ s~ies. It rai~ in.resting
questions abo~l tissue~speciflc ~ r i p t i o n a l
regulation
~ ~g l ~ o y ~ene as expression of this gent was thought ~ b¢ stfic!ly limiled to ~tivated T lymph~ytes ~turM killer eelh 1231. Interestingly, we found that a~r conceptusocleriv~ antiviral ~tivity, not neutraliz~ able by anti--l~-¥ ~mm, was also detectable. Surprisingly, i~ ~as n e u ~ ! i ~ by ~ f i ~ r a raised against crude p ~ a t i o n s of hum~m vims~induced leuk~y~--derived IFN (IFNo~, C~nlell ~y~). thus su~esting that itcould a t y ~ 1 IFN 113, 14]. As Su~Jea¢ ~r¢ phylogen¢tically c!o~ to mminm~ts, we first speculated thal this pulative ty~ I 1 ~ was the po~in¢ homologu¢ of IFN--~,However, IFNo~ probes did not deiect significant sign_al in pig ~ e p l u s l~nsc~tpls 12'0, 24] al~l ex~nsive ~reening of pig generate libraries with t y ~ ! [FN pro~s {even IFN--oh the t'~ily h~ving lhe highest homology with IFNor) did
not lead to isolation of porcine l ~ , t
alike genes 1251,
However~ using very low s~ringency h),bridi~alion, we w,e~ able to detect IFN--to~lated !r~n~¢ripls in_day 14 In d ~ 20 pig C'~cepIus mRNAs!251, This gave us the o~aun:ily 1o done ~he con~sponding cDNA~ from a day eDNA library 126t, Most of d~em ~a 1 ~ ~ r ~ o ~td (~a) p~p~otein rel~t~J to all other ty~ l IFNs. As ~ ~ , I 5~rm of this protein had a~ a~li-hum~n IFNo~ ~utr~lt~bl¢ anflvtral activity, Be.o cau~ ot~tl~ si~e ( 149 aa for the mature protein, ~he shortest of known ~ I IFNg Ji/~r~ IA), the provisional name *slrl IFN' (for ~ ~t~ t y ~ ! IFN) was coined for this ~ein !26!,According to sever~.l cdleria (see ~low), i~ b~am¢ evtd~ot th~ |his novel ~y~ 1 I ~ could not be lr~l~-d in any of the previously defined t y ~ 1 IFN t~ilies. It has ~ n officially recognized as the first ~ m b e r of a fifth lhmily of mammalian tyt~ ! IFNs: IPN.b 1271,
3, ~ U e l l l
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IFN-6 protein and
~veral inleresting t~atures distingui~ the p~rci~ IFN~ tg~li~ ~ q ~ ; ~ f~m tabor mammalian t y ~ I IFN
family mem~rs. Fiw~,~t,it has 11o preferential homology with one of the tour other families (an identities ranging between 27% for routine IFN-[~ and 42% for human I F N - ~ [26]). This fact is best exemplified by phylogenefic analysis of the mature protein sequences. As shown in figure IB, phylogenetic trees generated from such analysis clearly demonstrated that [FN-~ is distinct and divergent from IFN-~, -~ a~ and -t families, although it appears more closely rcia~ed to the IFN-ct. -to, -r cluster than to the !FN-~. The cysteine content of porcine IFN°b has no ~uivalent among other type I IFNs as the p~p~tein and matu~ protein contains nine and seven Cys residues, res~ctively. Intere~ting!y, five of the seven cysteine residues of the matu~ p~tein (~silions 9, 56. 58, 107 and 145) are ~|ypical among |ypc I tFN~ (~gure IA), Acco~ing to the k~wn 3D strut|arcs of human IFNoa2a, o~2b and o1~and marine IFN ~ii~[2g~31L all t y ~ 1 IFN~ display the same characteristic stnlcmrc of helical cytokines with a bundle of four 'up-up-downo down* [~ helice~ and an additional at helix. Consequently, it is ~ason~b!e to ~lieve that IFNo6 has a structure ve~, similar Io human !FNoa2, lhe t y ~ I IFN with which it sha~s highest simih~rity0 !f ~o, it ran be deduced from the alignment of IFN-a2 and l ~ ° b sequences ej~ul~ IA~ that !bur of the atypical Cys residues toys56, Cys58, Cysl07 and Cys145) appeared gathe~d in the same region of tl~e molecule thus suggesting that additional disulfide linkage could• e~ist ~.wecn-t -~- these . . residues. . . . .According . . . . to. multiple . ~lignmem~ with o!her t y ~ 1 !FN proteins frigate tA~. the sho~ size of the molecule con ~ attributed to a eenmd ~a deletion ~twecn ~sidues !03~!~ and to ~t |rimmed Cqem~inal end, I| is inte~!ing to no!e !l~at the central deletion occur~ in the ~egion ~howing the highest level of v~r~iahility ~n~on~ ~yt~ 1 IFN~ ~nd corrc~p~nding to a loop sepa~!ing lwo ~ h¢tiee~. Ttlis s|tatctural pecuiiarity, to-. gethe~ with the wimmed C~enni~al end pr~hably t'esult~ in a mot¢cule h~ving t~ m o t compact structure ihan olher tyf~ I IFNs, The porcine genon,~ was shown to contain only two int~onless nonoallelic loci with strong homology 1o IFNo~ eDNA I261. One of them was most likely a pseudogene as no corresponding eDNA was found in pig concepas library and its c ~ i ~ s~uence complained a p~vmatu~ termination codon resulting in a protein devoid of antivital acavity 1261, ~ e other It~us a ~ d to ~ the one transcribed in t~ph.oblas11261, Organization of the IPN~b gent (/~gu~ 2A~ ~veals tl~e dassica! s~ructu~ of a RNA polytrcera~ II lransc~t~.~ gent with a single c~p silt and a TATA ~ x k~ttted in position -33 to ~o27. Hob.raver. the seque~vce of this tran~fiption unit displays several uniq~e l~tlu~s by coml~nson to olher t y ~ I IFNs. First, the tran~ribed mRNA has a 5' non-coding region of 218 nucleotidcs (nt~ which is unusually ~ong among type I IFNs ~no more than 80 nt in other t y ~ ! IFN genes) and Ihat c~id have important implications on transla6onal regulation. It is noticeable that except fi~r the c,~ng
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regiom IFNob gene has no signilicant homology with virusJnducible tyl~ I IFN genes nor with IFN-x genes 1261. Moreover, sequences located upstream from the °rATA box are almost devoid of potential IRF-I or IRF-2 binding sites and GAAANN and GAAATG sequences known to be the target of transcription factors t~egulating virus inducibility 13, 331, This fact is in good ag~ement with the apparent ~bsence of virus inducibility of the gene (Lef'evre, unpublished data). Instead, 5' proxi° real putative regulate D, regions contain two adjacent, almost I~rfect 40-41 bp direct repeats and a third imper= feet and 5'-trimmed copy of this repeat located further upstream (ligure 2A, B). Seven~l GC-rich sequences bearing elements resembling consensus binding sites lot transcription iactor APe2 are present in these repeat~ as
well as in ~equences located up~tre~mi arid dow~stt~e~m
(tigure 2B). Such ~ites are known to medi~ite tn~n~crip~ tional activation in re~ponse to phorbol e~ter~ ~ d cAMP 1341. Thi~ suggests that ~igmd m~n~duction p~tho ways involving protein kinase C and/or protein kinetic A could regulate !FN-~ gene expression t34!, Fin~dly~ just upstream from the mo~t distal repe~t, a retroposon belo~lg. ing to the pig PRE. 1 flintily of short interspersed repetilive elements (SINE) is present (jigure 2At, 111e hnporl~,c¢ of these elements in the transcriplional regulation of IFN~b is unknown. Itowever, it is interesting to note thu~ the 40 bp direct repeat proximal to the TATA box i:, the only one to be absent in the unexpressed highly homologous pseudo~ eerie, thus suggesting functional importance tbr lhi~ repeat 1261.
782
Lef+vrc et al+
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~|~l~ ~. A, Nucleo~id~ ~qu¢~.ce of th~ po~in+++tFN+b gen.e. N~qlemide~ are+nmnt~red r¢la|i~.dy In ll~ecap ~it¢ tvc~!~¢~im+~o • | k ~ e T~TA ¢o.n+,en~u~~++qt_~en.ce,lhe ++~, at~d ~lop ct~+hm~~ bo~vd. T h e f o u r G A A A N N h e x a | t t e l ~ f o t l R d H~ tl~t+ u p M r e a m | , : g i o | t ~|!d lh? poy~d~nylalio|! ~ign~l a~ undertil~ed, Po~i0oo of the !~dy{A) tail i~ im]iea|¢d hy an on;o~, The large I~ox i|!t|ieate~ tilt* hw,a0mt ot t ~ PR~ i e!em~nl~ Hofl~on!al, hl~ck h+atf+-~ff~w~indicate ~+h~~ilit++i~ of th~ 40-41 hp direct ~l~i~|,~(RI! and Rll!~ and of th~ 5L+tfimnK+, ~! (RI) lhat l+++tCk~[h~ Ot~l 1t bp of R!t ~+adRill Sequence e!ement~ h~+'alt,d up~tremn li~m~ tiw cap ~itc and th~ ~t~qut~ I~.~. Tho~ lt+t~t~hilt~gtmo~t !~tq~¢0~' wiih AP2 ¢or+~,~u~ hilldil~g ~ite~ ~,,~ iodic~lled by haWhed ha~"~,B+ AliSnm++n:!of the ~ettuenqe~ of lh¢ !hree rel~ml.~R!. RI! ++~ndRill. th,.~itim~sof the end of ca+:h ~,t~,a| my mdic;~ed ~~lali~dy !o the c++p~ile, a ++++pw~,~ii~t++~! It+ m~iml~e h~m~oiog+vmad idcm~¢~l lra++c~are mda~>m+;+~lby a~ensk~. 4, IFN-~ pco'~tm ph~slco
Nqem~inal ~ q u e ~ of the puritied rcc~m~hinant Im~lein establis~-tl that the cleavage ~~f the signal ~ptid~
<~¢mtcd. ~ p~dict~d, !,~lw~n r~sidu~ 2l ~nd 22 of tll~ preprotein, thu~ c(miim~ing tha~ nm~u~ IFNob, wi0~ 149 ~sidues, possesses the shortest sequence of all kt~own [ y ~ I IFNs. Also atypical is the ve~y t~sic isoek~|ric ~int of IFN-6 ~¢~lcu!aled pl 9,07L a prol~zny Omt was exploiied fi~r its purification on a cationic exchanger [35}, The ~¢~-~mhinant IFNo~ eluted in gel filtration with a M~of 19 (~0, suggesting dmt the active p~x~lcin is essentially monomeri¢. The recombinant protein wus found to Nogly¢osylated. most probably on Ash 79 ffiga,~+ lab and Nogly¢osidase F t~catmem resulted in a protein of M,. lT i~0 which fully ~t~in~.~l its antiviml activity 1351, Antigenicity of fitis no~++elIFN is st~-cific, since a neutral+ i~ing mt +mti~rum ~o recombinant IFNob did not neutmli ~ any ot" several i y ~ I IFNs. whether po~ine (a, fi_,m)+ or ovine ('t) +~r human {otl. ~2) 135L Recip~ally. IFN+~ was not ~utPali~.cd by any ami~ra rated against ~veral rtx'ombinant porcine or human t y ~ 1 IFNs 1261. However.
Imer~:mn~6
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IFN.~. ~hat ~,as exp~',.'ted +f both +FN spcct+es brad ~o ~ °++,,~:
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t I FN reeept~.~r
4,2, Comm+:m pm++}er++esw++hhe+N++{++ IFN+6 shales seve~a~ physico+chemicat p~x>pemes with oll~er ~y~ i IFNs. namely s~aN~i~y of its active|) at pH 2. but sensitivity 1o reduction (t% 2-mercapt~'thanol} and heating ( i(R} ~:C), Also, like IFN-~, IFNo6 has the property of 'del)nsive reversible denaturation" [36~. namely resis+ lance to healing {IiRTI~+C) in the presence of urea and 2+mercapwethana! i36i The specific a|~t|iYiratl activity of a on M D B K CO+ischallenged w+ith ~esicutar pm+ified |I"N+~++ slomalit{s vires ga~c an_ esti+na~e +~15 :-< tO" to I(P lU/mg pmt¢im i+e+, in |he rm~gc of previously known iFN.{:~ activities t351+ !n c{}mn]~.oilwith IFN+{:~+IFN+6 was shown to e x e r t a synergistic antiprolifemtive effect with fFN+y on pig kidney ceils {iigure3~ This synergy with IFN-y could make sense from a functional point of ~icw, in connection with the known co-expression of both ty|Rs of !FNs b) :he tmphcct~em~ (see below}. In comrast, no synergistic ambgmwth effect ~,as observed between !FN-6 and
IFN-6 labeled with ~est was found to brad sb'~cif~ca~y to+ pig kidney cells with tk~st kme~ics ([~v, re4A,. This binding could be completed with eearly, egua} efficiency by porcine [FN-{,~ and by humm~ [FN<~ (tigu+e 4B,~ Upon cross+linking of radiolabeIed [FN+~ to pordne cel~s. a singte 120 kDa complex was formed, which ¢ott~d be prevented by an excess of unlabeled p{:.cine tFN+(:~, bm not of porcine IFN+y [37i, These data show that IFN+('~ binds to the same receptor bindmg chain as IFN ~ on pig cells+ On human celts, whereas p{m:inc !FN~e is capab.te of both antiviral and an~i+growth effects, IFN~6 exerts no nignificant activity [ t 7 i Accordingly+ bindmg and cross~ linking experiments peri~rmed with radio+inhered IFN+(~ on human WIStl cc~ls did +~ot detect any specific bind.* ing [371+ tt appem's therefore thai iFN-{'~ is devoid of significant affinity for the human type I |FN ieceptor,
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Absorbance at 6~0 nm Figure& Synergistic antiproliferative elTec! of IFN+h and IFN+y on potviue cp th h21 cells. Swine tcsti,,, cells 1~51 were s¢,ed.ed a| 50(RR)cells/well in DMEM medium + I1)++ FCS in 22 ram. 6+well multi+trays. One thousand antiviral tlllits of cacti IFN-{~, IFN-y or IFN+6 were added, either alone or in combined mixtttrcs (!12- !12), +u¢l+ that all monolayer~ received the ,,ame total amoum of IFN. All treatments were made in triplicate in randomized wells. Atier a lurlher 4B+h incubation, c¢II mono~ layers were lixcd with 2,5% glutaraldehyde and relative cell anmunls were estimated by methylene Idue uptake, exlraction, and measurement of ahsorbance at 65()rim, Bars represent the standard co'ors of the means.
FiRure 4, Kin~+lic~ of binding ~md ~m~pcmmn ~i~v~+~b~ di~t~, re.t IFN~ with s~"i 1t4N ~ ~m pi~ kidney c~|h {Ri:q~{-~~7~iJjm¢} Purified l~¢omlmmnt IFN+~ wa~ iodin~4ab¢l~d ~ i ~ |~h~m tluntcr reagent 1~1 kit (ICN, Radio~ghcmical~L Al| ~ p ~ r i i n ~ were p~rlk~rmed at 37 "C in 6+w,ell clu~ler plat¢~ (3~ mm m diameter) in the pre~nce of DMEM nt~dmm + 2{} mM ii~p+~,, ptl 7.4. I(Y~; t~tai calf serum (FCS) and 0,()5~;~ T~¢¢it 20, A, Kinetic study: abemt 800 antiviral tmit~ (2()0(~)0 ~'pm~ +~
~sI+IFN+~ were incubated ¢m RPTG cell monolay~r~ w~th { ~ or without (I~) an ¢x¢¢~,~ (100 ×) of tllllab~led iFN+& ~|nd at indicated tim¢,s h~¢uhathm medium from two ~elb+ ~,.as rcmo~ ved. cells washed 3 tim¢~ at 0 ~'(' with iY¢~t~m~dmm ¢~mh~mmg I~)~ FCS. and c¢ll4~smciatcd radtoa~|ivity wa~ dct~rmi,¢d, Sp¢ci|i¢ binding ( ~ ) wa.~ cah Elated a~ the dif't~ivnce bctw,¢¢t~ total a~ld |lOlVSp¢c+i|i¢ values for earth iitcuhalio|~ tittle, B, Conlpctition curves, hlctJbatioi~ ¢ol~dilioi~, ~¢r+¢ the ~am¢o ¢~+ cept Iha! °~al+lFN+,~ was il!¢ubalcd tiw ! h ill the Presritc'e cd' k!!oWll Coilc~l|tratio|ls of the ['t)ll+>~viiff+ ~Fut+~+mbii+ai+l !lN~: pt)IFN~(~.I ( I ) ; poil+N+h (O); p.olFN Y (/+ i; hu!FN ~tl ti'~i: am! ImlFN+(~2a (()+
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t~ 1t~ ,~ iA!, a~.ki~ !~t!hhii ~tlli~tautil tit !t:N ? ll!}, ~¢¢ond ~iep 17ft~ T~!tt~ t'i~t|--t~ilt~l!!~t~| 7otti ~llti~!'ttl t~{i l+{} itittl
Ft!'C .~'o!tttJ~l~d !~t~i t!tl!Vftlh!~it 17(I {!tl> Nott, ~ thit! i|l l~lth C i ~ 1t~ i~ lJnh!li¢il, Co|~l.tt~till!llttJilO~!tt!ltilt 7 ~qlh
with WN-y l t t a i i ~ , I~issthli
One of t~.~ m~l striking
i~atu~s
of IFNo~ is ils
det~t~-b~ in p~gnant uterus around d~y 13, l ~ k s on ihy 1~ot6 lind tl~n ~ ' ~ I t ~ s until ¢hy 201141, Analysis o{ dtff i4 to dily 20 Col~'¢ptus trim~ripts indicated tht~t evolimorl of both miNAs is .p~.all¢l durin I Ibis ~ ~nd Ihtit mil~imum m~IivirM ~ctivily is sligklly ¢1¢h~c~ ~s c~o~ to t-oa~h~l trtmscripl tev~ls lfifu.t~ .1i, At-o though l / f l i p , us quantitative an~lysi~ of the e,~olutkm of l~4h p!~Icins in the uied~ !un~n has nol l~'cn~d~ nnc~l, ii c1¢ dy ~ l t ~ Ihltl IFN-~ is expensed ill Inuch lower
hlasl i:+Cli4ct~ " "' , ' ~ith ~ntihocht, ~ c i i i c
ihr each IFN
derm ¢¢11s on d~y 16 of dcv¢!opmcm are t~siliv¢ tor IFNoy ~md IFN-6, ~,~ revealed by immui~ofluo~,~t,cace {fi{lu~ 6}, Second. Ihe subc¢llular l.t~alizai.ion (l~ue
Inlerf+-nm+{'~
?g5
7+ P~,+slMe ~+rlgia ami n+lv+ ++f |fN-i'+
7• J'. l.~ +'+Pc'++'+P+Pq~.+++~+,++,++g~ +. . . . .
+' '+ ++++p,+g++g++"
Goat
Cow
++'~++~,t++~++~+>?+&~ d +PC ++,+g++¢-'+"++.
iZ
+',~+'O+P+,~+++,+'+,~" ?
Horse
+++
ea As well as 1FNoT. il aNx>ars clear that ti:N+~ can be considered as a ~rue tmphoblas,.ic IFN. It seems very sinking lhal ia t,,m gr{mps of species which am dose mlati,+,es. ,'~amcty Suideae and ruminants, ~ype I IFNs cxpNssed lV one tissue of the same ontogenic origin ape+~r so distantly ~:;}afed. This raises the interesm-~g. question of lhe eompm+d evoh.mona U origin of boda type I !FN fi~milics. Sevend lines of evidence indicate that !FNT. which +tt-c ct~+sely w!ated to !FN-.m. have evolved nM'~er i.?ccnt~y from ~ m,is--ia{h~cih~et>p,.: t Ii::Ngenes that ii~'t_!~fir~dir~,+phob!as!~p¢+ii:}c ex.p~-cssi~m and lent almost ¢ompleleiy lheh" virus+inducibility m non+trophobhtstic tissues ]I 2, 26i, According to phyh:,gcne|ic data. it seems very !ikely that this event was limited to ruminants so thai IFN+T am no| pwscnt in other mammalian l*amilien i 12]+ By contra.~L |FN-~3 in much mo+e di++.e~gcm irom othct+ i y N ! IFN i)mi!ie~+ Pt!ylogenetic data O,~p+++++++..+'/B) . suggest that the duplication event leading to IFN-i5 was before the divergence of ItaN+¢,+ +m and -T famttie,,, but latter lhan the divegence of IFN-iL At'cm+ding to the divegence el+ e,t~ting sequences at replacement mites, it was suggested tha| IFN+6 aneestra! scqttenee diverged ahot!t I blO My agm !ong betbre mammalian radiation [26]. The presence uf homotogtm~ o1' l|:N-b in other nmmlnaliail species t~ lhcrclbre very coiisislcltl. Ti) lest this hylmthesi.,,, we tried
tO deicer i!:N+a-relaicd seqii¢llCeS ill the gt;llOilliC DNA ~1 ~e~,ei~;ii iiiai!tilllil~. Prdh/iitlai°) • data i[idiealc thai the hu l!ia!l g¢llon!¢ !:Olliaill,~ a +,lllgl¢ hicu~ clmel)-, rchlicd to pen:hie II:N-5 hilt thai it is a p~Ottditth+liP ilefbvre, untlut~i!i,,,hcd l'l:~ltlt~l, A~ ~htlt~,'ll iii fiRt+li" 7, gOllOli/iC ~ql|¢llC'7,~ hot!!olo~!it|~ tti !!':N ~ c'tlti he easily deter'ted ill +++tt~mi+ DNA of l+t)viiie+ t+aprin+ aiid horse mpc¢ies w!licti ilr,0ui!g!lhll+ iliitilinlMs phylogenetiea!ly ¢lmc to the pig, We have cloned one of the equine Io¢i which revealed a D~tetllially litnetional get,e+ i,e+, cnco.ding il prolehl with amiviM activity° ~hafi~lg highest homok)gy with porcine It,:N+~ (55¢;~ idcmity al the aa level i, thus .~trongly suggesting thai the I! ~N +~ thlnily e~itends to other illalllnlalian stle~ies (Lel}vre, unpublished rcsults), li is interesting to note the presence oi" homologous sequences in ruminants. If functional IFN:bdike genes are present in lhe~+ s~cies, they arc probably not !mphobhlsl+spc¢ilic, as no conceptus+derived antiviral activity distinct I'roni IFN-I has s~,! far ever been li)und in runliniinl species. Further work is required to determine whether these novel equine and bovine genes arc virusoinduciblc. For the time ~ing,
a quite plausible hyg~thcsis is thai porcine IFNoh
derived thml a t'amily (if virus+inducible type I !FNs nlore
or less widely distributed iilliOll ! illalllmiils. I+ikc II"N+i, they evolved towards lrophoblasi speci!ic expression in a narrow group of species.
kb
21,7 --
5+0--
+
....
4+ ',,3
+
+
~}i ¸¸
1.9
+
,
+
1.3"-
0.94
FiRure 7, Prc~c.¢e oI'IFN +6-wlated ~¢qu~a¢¢~ m m~.0g ~ m m ~ el
,~¢m aI uiID+h~w~llmmmal~. |}NA ffmn ~,~ch~pe~0i~,{ IB #~ii~+~ +lig~,~ttzdto comphmon vvilh the ~p¢~:ified ~])tr~t+¢ a!~d ~het~ loa,J~don a (l,~I+/,agmm{' ~cl,The Not wa,~hy!mdh'{:du~idc'~k~w, ~lringem:y with a probt., b~aling lhe IFN+6 coding ~¢ql+~nc¢~,The po,dihm,,, tff ~,i#e ~landa+d~+(in kbl m~e ~llo',*n oct lhe ~ ft,
7, 2, Wlrn mh' IHays II"N.+) i. pr¢,~mm~Lv:' The preci~,crole played by ll:N+b !a~ well ~, by IFNy} during early development is m far unknown, llowcver, we have precise indiealion~ on what functions arc' no! fulfil!cd
by II:N~. An experimcn! shnilar m dm~c done in rumi+ nants, which dcmonslralcd lh~I !FN-4 per .~c i,~ amitutcolylic in ovinc and bovine spc¢:i¢~ 139, 401. wa~ pt:r+
7~ f¢,em~l i~ ~ pi$, I~ th~ s!~ies, tL~ ~sult was ~~iy dl : i ~ t k m o¢ massive do~s of ~ o m . ~ IFNs (IFNoy ~ IFN-8 in mixing) in the uterus of m m o p ~ ~ gitt~ betw~n days i i ~ 14 of ihe estrus cyc~ ~ ~ ~ v e n t the coepus lu~um regression (ludid ~ signifi~tly delay the m~s !451, This provided a d~monstration of favo~ ~1 whar¢in estrogens ~reted y ~ pig i, fact tl'~ main ~tilu~olytic I ~ . ~refore, a ~4ike effect of Imwine l ~ s , ~ would '~.~ue" d~.. c o ~ s luteum (~ ~ ~ ~ what is callM ' ~ t e m a l recognition of ~Y'L ts v ~ unli~ly. It remains however m ~ g ~ d if IFNs ~ est~ens could ~ t in synergy on corpus luteum nmintenance 1451, s~ts oi' data strongly suggest that the most ~ ~ tm3get tiSS~ of #g ~ b l a s t IF)Is is the erdometri~ epitl~lial cell. which fao~ the trophectoo ~rm. First. ~ t r i M e # ~ l i a l ceils we~ tbund suso ~ i M ¢ to IFN ~tivity by ~veral cri~da !461. Second. we ~ M demons!~te wi~ a mnsitiv¢ and s~iti¢ ~L!SA 1Mr ~ l~--y ( ~ d probably no IFNob) crosses ~¢ ma:~rnal ¢pitha!ia! b ~ e r . what makes it ver~ unlikely that u~rl~ mucost,, which includes stmmal and lymphoid cells, would consfit~e a target lbr IFNs (D'Andr~a ot a!., UBlplJb|iS~ results). Third. the wophectoderm i t , ll was fo~M un¢~spo~slve m both !FNs, which piles out a ~ssihl¢ ~ u t ~ d ~ ¢lT~.'ton !rt~phoblast ceils I~t, As ~ immune !bmtion of !FNob is vee/unlikely and ~ a / ~ the ~tri¢t eplth¢ltt~ho~ta! pla¢¢ntatton tn Suideae ~v.ents ~.ny contact t~tween em~onk" o¢ l~t~l ¢¢!!s with tM maternal bl~M ! lgl), the ~)~sihility t~tnain~ that the ¢~tokine '¢ms.~ot~tk' ~two~n the tix~phec|~tent-t ~md the uterine epit~lium c ouM ¢0~t either: t) ~la¢ non. s ~ t 0 ¢ ~ s t s t a ~ of efldometrlum to infections; from thi~ tmint oi' view, IFN~b wa-s found ¢ill¢ient as ..n ~ativi~| t.~ttl~ is ¢l~rly )I¢ csp0flment~l a~h to veri~ this hyr~h¢.ts~ 2) o/ae or several ~(s) ~!n~d to p~pa~ tM to t t n ~ a t t o n , Another eytokine, nan~ly |¢ukcmi~ inhibitory fa¢tor (LIF), was shown to ~ ¢s~no tim for i m ~ a t i ~ in mice I471, This factor, as ~ll as oI~r cytoki~s and growth f ~ t ~ s such as interleukin 6, ~ W~ns,fomfing growth f.~to~ ~ insulinolike gix)wth f ~ 2 ~ arc ¢xpr~s~ at tl~: ~.~.~oofetal interi~ce during ~ ~ ~sto~yst att~hm~nt I~-50t, It s~:~ms likel-~ that ~ s e f ~ o r s m~y wo~ in ~ n ¢ ~ with ¢'o.¢xp~sgxl' t r o p ~ ~ i c IFNs to aff~t ~ri-implantation events, C~-et~i,~g IF.Ns, ©x viva studies with luminal ~Am~ctri~l co!Is in,eultu~ are umterw~Lvto exami~ their effects ~ th~s epiti~lium, ~ m t w ~ IFN-t_x,w~.~~ ~ t ~ atv~mg other t y ~ I ! ~ s f ~ , ~ ~ , n ~ bkmciivity (as ikatas we could ~-~: in ~,~11cultue, It~y are similar to tl~.~ of IFN-~L ~
i,efb~vre et ai, favor the hy~thesis that iFN-tb was selected on the basis of physicoochemical and phammcological pn~perties. leading to bi~is~nibility, stability and confinement Ihat ~st fitted to its sec~tion and function within the uterine environment. It is to be noted, in this ~sp~t. that the two l ~ s produced by the pig trophoblast are both highly basic proteins (pl over 9). Neve~eless. the syn~ orgy found ~ t w ~ n IFN-y and IFN-8 for the ~lipmlit~rative effects Oqgure3). probably applies to any other biological effect. Co-secretion of both t y t ~ might. therefor. ~ p ~ n t an efficient means of am#ification of each ~ingle biooactivity, and hence a po~ib!c ~¢lecd~e advantage over sec~tion of a single !FN si~.cies, In conclusion, 1FN-6 is another mcm~r of an h-tc~easo ing family of tY!~ 1 IFN~. It i~ a .at! tha~ iha~last lwo n~vei s~cie~ discove~d llt:No~ and iFN-a) are bofli ttopho~ bla~tic IFNs. each one s~cific to a d i ! ~ n t mammalian family. It is probable that other s~cies will ~, ibui~d, each gifted wid~ ~cu!iar physicoochemical, ph~aeological and biological pin,hies. Like IFN-t, IFN~b d~s not ~ , m to have a functional humaa homo!ague, in spite of this. it will b¢ of interest to study in tl~e pig or in other animal models, s ~ i f i c pham~acolog~cal and theraF,utic perthnnanee~ of this novel mol~ule as an antioinfe¢|ious and/or anti--tumor agent, in view of the known t~cufrenc:e of !FNob gen.e(s) in other animal s~cies. Acknow!~gmems
cmd A. Lo¢i~!~lli(Uai|e d~.~ Physioh~gie de la
I~t,p~u¢ii~.d ~
PRI)M ~ioIl'f~r p¢i1~ntlii!g ltltil~iill ¢~l~,O|iienlI~ii|td ~Utg¢iy
Referees
tal¢~ieg~m $ ~
et~ter tic!cra~it~tl f~oln a YAC ekta~: coaiig,
14~ 1Pgnigt~ch~T,, M~t~tei N~, ~.hwart~l¢in M,, N~g~a S., M~r,m~atsu I3~ U#~ G,, t.~i'|~th G., Gwx~:~ I., C~vcii~: Iransfcr of a functional h,z~ean t~l~e~'tm a ~,~'e~w int~ n ~ cdk: ckming a~td etp~es.
~?1 CoIK~ |t. No~h.'k D., B~rak S,, Rubtn~ein Me, Li~anddndut;t~tl ~ ¢ i ~ t ~ m ~II"0~c type ! in~cdenm r~,2ept~tr t'ompo~:ents, Me!, Call, Biul t3 iI~5~ agm~.~21a
ln~e~emm~
7
Ig] Sen G . C , R a n x ~ ! ~ R.M, Lmc~le~m ~.~a{~v¢~ ~ m ~ m q ~ B . ~ ,
!~,~i
irva~ iBdu,ib0ip, of ~ " human ~FN <~ . ~ (!990) 767--77~
{l l} RiR~It~, R.M., In~erf~-,m,a~ and pv'eb,~m~:y..f~ lmcrh.'nm Cymk1121 R¢~-~Is R.M., i,,m L . Atcxe'&k~ A.., New and atyp:cai lim~i~ies of lyp~ i inl~rt~m.,.1s m eaamm:a{x: C~mpan~ivc fun¢~ioax, ,w~c:un:,,. ]i3t
t l,l]
Se, { i~92} 287-.~25 1.¢f~{'¢ |=-',, M~lrlinat.-Bot~6 |{. t]~ilhsn~ot M . Ztmari K . {'h(~rtcx' |TL L,~ Bom~m~lien: (7 tatc~i¢-'lon-gamma gcn~c ,rod p~owm e~e •q~.~tl~tlieOtisI~ CXD~?~s~'~I b) fh.¢ pod's'me: lml~hcctodcrm c a r k m ~eMalitill, E~tt it. lmm~mol ).t} ~ tg~}l 2485-24~}O IAl t_ii.~l~i}al-t.|i{'t'~ {? Matitii*;it.B~-~tto i",, "[i;'i~ltit N| l.et)tfe t:
I151 17~ttc-G~ii'am !}. Ro!trt-il;dliol tt+, l,i~pct J, {'trait) { ' . {:nu,~iml ~ll)t~-liI'O~ll%, c'OllSltlttltvi~ i~lerlt, trt)tl~ ~.tlld ~tlllgtTottl~l~ tll lit,q~tol~t placenlal bioc~i, t:~i,/~. Nail At'~td. Rot 1L~S:t~7 l tt)S3~ 5tltlb-SOt4 1 ltq Chai-d T. Ctiig Ptt., Mituah~ii~,ey M . l.c~: {'.. S,lpha mlefb.'mn m t171 b~wle~ A.K.. Reed C D , Oim~ DA, tdel~l~ti~alioll t~l ~li tllt~'rlt'lott In ~itlarl!le pla~;'¢t!lii~, Nalitr¢ 2N~ 1 t~)SII} 2B6-2BT. 1181 Leis~ R., Kaulirtat~t~ P0 glaceaiM slrttemre in a ctmtparaitvc a~f~ei:l, tTi~p~flirt, tirtdt~wmt~ 102 ~1994} 122 i ~4 i 1~}t Ottiihml,at M.o l;ldeh~)t~ ItC, l,¢m): I- l,ll,i~l~)<~l de~ehipinc, m ,rod ~lt-itlkiillalmll lit l'hiblilth I[', l.,ev~t~,~eiir M.C., ||li~tler R It.F, tl.id-~.}. R~tl~u¢lton m Malllll-t~ll~ ~tlitt ttl~itL t{llipm t~itfi~, l lJlJJ,
~i~t p 12111 { " m ~ ]if",, fli~B~ii~ R,M.. ik~rcine conccplu~¢~ ~ecrc~c an mlc~ lelt~lt dulrtltg ih¢ |~lt'~liiit¢lititi?~ll t~tt~il ol eal-ty p~¢g~tan¢), i|ml R,¢p~i~i. 4{1 1 l~t~iJt i lil ~} 11 i~. 1211 ltt'lt~'~iitdo M,A0 tialiic'v JP, t'lecr~ N, thtnltc.~' {'.11, hlrr¢~ B.A. JtRm~mt tt,Mo f t a m t.]{%', ¢}l~nt,I [}i ~l?l/fC'llllll OI p~'t~lt'Hi~ %%Hh ~litt~iflii iii.'U%ll%7 b~ pig ¢i~lt~°tepltt,w~. ] Rc,pt~M l'c'rlil ~N I I~JtJlti |!}? 2t}.t 1!21 lte~i~ ~ 0 Mlriilidtt M.A.- lkml~+l.'i- tLIl. t-t~rnc'} J t': ]hllft.~ ii A ,Ihtllttt~ttti ti.M,, iia~¢t I:.W. ltll]il¢ll~:c, t~t thi.' t'tldtlttl~lllt|tii, pmw,l~¢ iil|li|till/t~
iiild lit~etttlg
iltg ¢oilceptti~'~
~iti i,itilit if~ll ~icltvli}'
ol [lltttt'|tl~,
] R~'t]md Fc¢iH ~I~4 i I~l~Jtli 71}% )11
~tpt-~ i~lleffem~l, (leite 77 I ItJ~l~l) ;t41 l.ilt. Mege i)., lxef~vf-e It, I.ii I]oililflldi~i'l~ C,, "File llt,ll°~>lill~ i'illliil~ ~}t tt!t~'l-ll~fl)tl<-~ °, eh~llilig, Ml~itl~lllllil littlilys~s and fuB¢lion~tl Mlidi~ of Iit'e I~l~ied gene.% ], hiierl"elOil !t¢,~. !1 I I9~)1t 341.--350, 1261 Lel~vi~a Fo, itoulay V., A novel lUld alypieal ly|l¢ lillt~ inlerfel'illi gene expressed by impllobla~l during early i~regnliney, J. ilii~l, Chem. 268 {1993} 197~}-.li}768. 1271 Lundgten F.., 1,anger J,A.. N¢~nlenehllure of inltarl¢ron reeeplop, and inlerfemn-~. 1, Inierlemn Res0 17 i ItJt)7} .t10, t2t!1 Kl~u~ W., ¢i~11 It,, Labhtifdl A., Wipf tt,,. S~nn II.. l'h¢ thr~¢di il~n~,tOltal llitlll ~¢solulion ,~iru¢iuri;~ ~1 litainlui inie~l'emn !~-2a dt~lenlfiu~d by hciemnu¢lear N M R Sl~¢irm~opy in ~ohiiion, !. Mot. Biol. 274 (1~{}7} 661-675. 1291 Radh,akri~hnan R,, Walll~r L,J,, Itfu#a A , Rell:h¢ii P., l'itlliii I:P. IN!lgabhu~han T,I,., Willler 1%1,171,,Ziu¢ niedililcd dhn~r of lltllilil!l hilerferon i!~, re~¢aled by X-ray cryslalhtglaph 5, Slru~'lur~? 4 I I{J96} 145]-,146,t. I;Oi Senda 'IL, Sailoh S,, Mir~ui Y., Relined erys!a! ,~lru¢iure ol lta¢ombintml muriue inlcrfi:rlm-171 al 2,1 $ A rtL~llhltlilll, ]. Micti, Biol. 2~), (!!}{}5) 187,.207.
and G A A A ( ; T generates sequences that me
[/{5{ ~mrcmc ~ypc I intcrlkw~m. Bi~hcm~c~[ and B~tugic.q c{tamc~cr iAamn of ~he ~yc~mhinam ~ro~cm exprcx~ed ,s~ m,~cc~¢¢-}1~ E~r 1 giochcm 23t~ { i~)ggt 2{R~,-206
f17{
[b~}r,
Nm P|).. [.cl),,~e |: | a B~m:lard~¢,~c tL, \%p~ca~ ,,p[ ,qacr~cr~rt brad,, ~m porcine ¢e~b+ t~, a re
RS .RS. 1401 |~|}{[t{'he[ ~/%~;.. }[/.[[~?',C|} P ) . (;ri}7,"~ [ S . ~[g[~ }¢t' S [). ~}[,t.[}{~" ( ' .
1411
J. R c p m d FertH, {Su~pl.} ~
1421
{It}~){} [71 lg¢~
~,~c%i,flied 1-1%
A¢it! IRes, M~I, 1711o1,5~ I ic~{}7} iil~}--127, 1241 Charlier 1%t,, Ilue D,, Ma!lttl J.> Gta~e 1~, Chiiiiiig iiiid l~'t!lr~',~iOll !~t ~25[
t34
'i:X-!q ~c~c,, CctI c~O
14il J Repmd, I,¢i!il, 7~J {t{JR?} ~e3~ [72, 1441 Van ller Meuie~l 1, Helm~md F,A~ {.}ud~aa~td¢~ ¢ ' P J , [:,ff~¢t ,~ I ei~.'vre ~',, Mfmma!.lt{me 1'., l,~va~elti A0 N ~ PB,, h?r{l~,t M [,~ Bonnardi~N C , Inlrauteriee i~l~,i~n o¢ high do~e~ ~ gi 8 m~ph~:~ blast inlcrfemn~ ha~, rio aniilulet~I}ti¢ ¢He~:t m ~3,~,'h~ gitt~= fl0o~ R e p m d 5~ ~tq~lS} t026.1031, 14¢~1 D'Andrea S., Ch~u~t¢~-m~m S,, |'lOvh, m J [ ~ ia~ B~m~lafdiere ¢', Paramn¢ a¢livilie~ ~tf porcine wophoblami+ mlerlcmt+++, ] R~+pt~+d Feriil+ I02 { [~)04) Ig5 ~+4 14/I Sle~t~:l iLL,,: Ea,~p,~r P0 Btu~¢t | J , B~tbtt I I , {[;~di I0 iomge~ l , Ahb~fld~til~o S,J,, lihl~t~u~y~ mll}latllalio~i {j~p~d~ ~m malef~d
I.t51
7/~ 79,
14~1 t:t}ll¢:gptus illl;lgh|ll¢llt, Cyloli[|i¢ O { [t)93} 49f~, 499, 1491 Gilpl;l h,, l};Iz¢l ~ l;,~,',, Jileg¢|: I+,A,, [}iffer¢I;li;tl cxpw~:~m el bctt~ irallMornlillg growth la~'t~.}|?, I'i'GI bct~ [, I { } F b~-t,} 2 trod | t } | h¢la 3} and [heir rcccptor~ {type I and ly!w i ! } m l}.eri impt~!matuln l}or~'itl¢ ¢on~'eplu~es, BIol, Reprod, 55 t l{J{Rt} 7*}t~ a02,
788 1501 Sinamen R,C,M,, Green M,L., Simmen EA., IGF system in periimplantation uterus and embryonic development, in: Molecular and Cellular As~cts of Periimplantation Processes, S.K. Deyed Serono Symposia USA, Springer-Verlag, New York, 1995. 1511 Devereux J,, Haeberli P,, Smithies O,, A comprehensive set of ,sequence analysis programs for the VAX, Nucleic Acids Res. 12 (1984) 387-395. [521 Fitch W.M., Margoliash E., Construction of phylogenetic trees, Science 155 (1967) 279-284.
Lef~vre et al. 1531 Charlier M., Hue D., Boisnard M,, Martal J., Gaye P., Cloning and structural analysis of two distinct families of ovine interferonalpha genes encoding functional class 11 and trophoblast (oTP) alpha-interferons, Mol. Cell, Endocrinol. 76 ( 1991 ) 16 I- 17 !. [541 Ghosh D., A relational database of transcription factors, Nucleic Acids Res. 18 (1990) 1749-1756. 1551 McCiurkin A.W., Norman J.O., Studies on transmissible gastroenteritis of swine. II. Selected characteristics of a cytopathogenic virus commot~, to five isolates from transmissible gastroenteritis, Can. J. Comp, Med. Vet. Sci. 30 (1966) 190-198.