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JOURNAL OF VIROLOGY, Mar. 1990, p. 1171-11810022-538X/90/031171-11$02.00/0Copyright © 1990, American Society for Microbiology
Vol. 64, No. 3
Cloning and Sequence Analyses of cDNAs for Interferon- and Virus-Induced Human Mx Proteins Reveal that They Contain Putative
Guanine Nucleotide-Binding Sites: Functional Study of theCorresponding Gene Promoter
MICHEL A. HORISBERGER,l* GARY K. McMASTER,1 HELMUT ZELLER,lt MARC G. WATHELET,2JOELLE DELLIS,3 AND JEAN CONTENT3
Pharmaceuticals Research, Ciba-Geigy Ltd., CH4002 Basel, Switzerland,' and Laboratoire de Chimie Biologique,Universite Libre de Bruxelles, B-1640 Rhode St-Genese,2 and Institut Pasteur du Brabant, B-1180 Brussels,3 Belgium
Received 14 August 1989/Accepted 6 November 1989
The human protein p78 is induced and accumulated in cells treated with type I interferon or with someviruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-lengthcDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frameof 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in goodagreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein.The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acidprotein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysateswhere it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs containedwithin the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alphainterferon (IFN-a) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cellstreated with IFN-a. In contrast, the p78-related protein was not expressed at detectable levels. The rate ofdecay of p78 levels in diploid cells after a 24-h treatment with IFN-a was much slower than the rate of decayof the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein isprobably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as thehomologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic ofGTP-binding proteins.
The p78 protein is encoded by the MX1 gene located onthe distal part (21q22.3) of the long arm of human chromo-some 21 (23; K. Gardiner, M. A. Horisberger, and D.Patterson, manuscript in preparation) in the region patho-genic for Down's syndrome. The gene is induced by type Iinterferon (IFN-a/,B) and by some viruses. Furthermore, itsexpression is modulated by biological response modifiersinvolved in viral infection, inflammation, and immune re-sponse (12). However, the real function of p78 is stillunknown.The human p78 protein has been shown to be homologous
to the mouse Mxl protein by several criteria such as size, pI,amino acid composition, antigenic determinant(s), and IFNinducibility (21, 22). The intracellular localization of p78implies that the protein is involved in cytoplasmic functions(21). p78 may be inhibitory for influenza virus, as is theIFN-induced mouse protein Mx (33). In contrast to themouse Mx system, however, there might be no strict corre-lation between the induction of the p78 protein and theantiviral activity of IFN. Thus both IFN-a and gamma IFN(IFN-y) protect human cells against influenza virus infec-tion, whereas only IFN-a is a potent inducer of p78 proteinin vitro (12, 19). IFN-y-primed cells, however, are able toaccumulate p78 protein during viral infection, suggestingthat IFN--y programs cells to full antiviral activity upon virus
* Corresponding author.t Present address: Institute of Experimental Dermatology, Uni-
versity of Munster, Munster, Federal Republic of Germany.
infection (12). We have no evidence that p78 gene expressionplays a pathophysiological role in specific diseases. Allindividuals tested so far are positive for the protein p78 afterexposure to IFN (36). However, p78 is a sensitive marker forbiological activity of IFN, including acid-labile IFN indiseases such as systemic lupus erythematosus (36; D.Jakschies, H. K. Hochkeppel, M. A. Horisberger, H.Deicher, and P. von Wussow, J. Biol. Response Modif., inpress) or acquired immunodeficiency syndrome (P. vonWussow, D. Jakschies, B. Block, I. Schedel, M. A. Horis-berger, H. Hochkeppel, and H. Deicher, AIDS, in press).
Expression and analysis of cDNAs may help to define thefunction of the p78 protein. In this report, we describe thecloning of two types of full-length cDNAs which are homol-ogous to the mouse Mx cDNA. Structural analysis of pro-teins encoded by these clones revealed the presence of threeconsensus sequence elements with distinct spacing whichcould confer a guanine nucleotide-binding domain to humanp78 and p78-related proteins and mouse Mx protein.
MATERIALS AND METHODS
Abbreviations. The human proteins p78 and p78-relatedprotein (old nomenclature) could be named Hu-Mxl andHu-Mx2, respectively, and the corresponding genes MX1and MX2. To avoid confusion between the abbreviationsHu-Mx and Mu-Mx (murine protein Mx), we have kept theold nomenclature throughout the report.
Cells, viruses, infections, and IFN. Human embryonic lungcells (HEL, Flow Laboratories 2002) were grown in minimal
1171
1172 HORISBERGER ET AL.
Eagle medium-Earle salts supplemented with 10% fetal calfserum. They were infected with 1 PFU per cell for thestudies on virus replication. Working stocks of vesicularstomatitis virus and of influenza A virus, strain fowl plague(Rostock/34/H7N1), were prepared from the allantoic cavityof 10-day-old embryonated eggs. Titers of viruses weredetermined on primary cultures of calf kidney cells by theprocedure for plaque assay as previously described (19).Recombinant human IFN-cxB (rHuIFN-cx8 in the numericdesignation; >95% pure) was produced in yeasts and puri-fied by affinity chromatography with monoclonal antibodies.IFN-a2c was from Boehringer Ingelheim.
Escherichia coli strain, plasmids, and gene library. cDNAwas synthesized from cytoplasmic poly(A)+ RNA that wasisolated from human embryonic lung cells treated for 6 hwith 1,000 IU of rIFN-otB per ml (12, 23). The cDNA wascloned into EcoRI-cleaved arms of Xgtll as previouslydescribed (28). Positive plaques were detected with anoligo-labeled cDNA probe comprising upstream noncodingsequences and coding sequences for p78 mRNA (clone B1.1[23]). Positive cDNA inserts were subcloned into the EcoRIsite of Bluescript M13 vector (Stratagene, San Diego, Calif.)for sequencing. Both strands of positive clones were se-quenced by the dideoxy method (Sequenase; U.S. Biochem-ical Corp.).
In vitro synthesis of mRNAs. A typical in vitro reactionmixture contained 30 mM dithiothreitol, 0.4 mM ATP, CTP,and UTP, 0.2 mM GTP, 1 mM 7mGpppG (Pharmacia,Uppsala, Sweden), 25 U of RNasin (Promega Biotec, Mad-ison, Wis.), 1 p.g of template, and 10 U of T7 RNApolymerase (Stratagene) in a final concentration of 40 mMTris hydrochloride (pH 8), 8 mM MgCl2, 2 mM spermidine,and 50 mM NaCl. Incubation was performed at 37°C for 60min. DNase I (1 U) was then added, and the mixture wasincubated at 37°C for 10 min. The reaction mixture wasphenol extracted, and RNA transcripts were precipitatedwith 0.6 volume of isopropanol containing 0.3 volume of 3 Msodium acetate (pH 6).
In vitro synthesis of proteins. About 1/10th to 1/15th of anin vitro transcription reaction was used in a typical transla-tion reaction. The rabbit reticulocyte lysate (micrococcalnuclease digested; Amersham International, Amersham,United Kingdom) was used at an 80% concentration andsupplemented with 7 ViCi of [35S]methionine per 6 1J ofreaction. The reaction mixture was incubated at 30°C for 60min.
Immunoprecipitation. The in vitro-synthesized proteinswere treated with 1% sodium dodecyl sulfate and diluted in10 mM Tris hydrochloride (pH 7.5)-50 mM NaCl. They wereincubated for 3 h at 4°C with the different antibodies (seeResults) and then incubated for two more hours at 4°C withprotein A-Sepharose CL-4B (Pharmacia LKB Biotechnol-ogy) as a 50% suspension in buffered phosphate salinecontaining 0.5% bovine serum albumin. Immunoprecipitateswere washed as described previously (31) and analyzed bypolyacrylamide gel electrophoresis (26).
NH2-terminal amino acid sequence. Protein p78 was puri-fied as described elsewhere (21) and its amino acid sequencewas analyzed in a Beckman 8906 sequencer as described byChang et al. (6).
Isolation of genomic clone containing p78 (MX1) genepromoter and fusion of two derived fragments with the catreporter gene. A chromosome 21-Hindlll library (AmericanType Culture Collection-National Institutes of Health hu-man chromosome-specific libraries, 1988) was screened withthe partial cDNA clone B1.1 (23). A positive clone also
hybridizing with an oligodeoxynucleotide (5'-CAGTGCTGGAGTGCGGCCTCCGCTC-3') corresponding to the mostupstream region of the cDNA sequence (see Fig. 4) wassequenced and subcloned to fuse it, in toto or in part, withthe cat reporter gene derived from a pRSVCAT plasmid(American Type Culture Collection-National Institutes ofHealth repository of probes and cloned genes, 1988).
Transfection and chloramphenicol acetyltransferase assay.After two rounds of CsCl-ethidium bromide gradient purifi-cation, 6 pLg of the various plasmid-cat constructs weretransfected with 106 human L-132 cells (ATCC CCL 5) in thepresence of 12 Fig of herring sperm carrier DNA by thecalcium phosphate precipitation method (15). To eliminate a
major bias caused by variability in transfection efficiencyfrom one dish to another, we introduced, as an internalmarker, plasmid pCH110 (16), which expresses ,B-galactosi-dase under the control of the simian virus 40 early promoter.Each cat construct to be tested was cotransfected withpCH110 in a 3:1 molar ratio. A colorimetric assay based onthe use of O-nitrophenyl-3-D-galactopyranoside was used tomeasure the P-galactosidase activities in cytoplasmic ex-tracts from transfected cells (16). After 48 h, the cells wereeither treated or not treated with 3,000 IU of IFN-cx2c per mlfor 16 h. The chloramphenicol acetyltransferase assay was
performed as described previously (14).
RESULTS
Cloning of cDNAs encoding the entire p78 protein. Toisolate cDNAs encoding the human p78 protein, we con-structed a Agtll library of cDNAs prepared from mRNAsisolated from human embryonic lung cells which had beeninduced with IFN-ot. The library was screened with a partialcDNA clone (referred to as the B1.1 clone in reference 23)encoding 5' untranslated sequence and NH2-terminal se-
quence of the p78 protein. A total of 1.5 x 105 recombinantsgave 22 positive bacteriophage clones, of which 10 wererandomly selected and 9 contained an EcoRI insert of 2,600base pairs (bp) (an open reading frame of approximately2,100 nucleotides would suffice to encode the entire p78protein). These inserts were subcloned into the EcoRI site ofBluescript vector DNA, and plasmid DNA was amplified inbacteria. Restriction analysis of the purified DNA indicatedthat two types of cDNAs had been cloned. The partialrestriction map of a group of seven cDNA clones wasconsistent with that of the partial cDNA clones describedearlier (23) encoding part of the p78 protein. These cloneswere named p78 cDNA (Fig. 1). The partial restriction mapof the other group composed of two cDNA clones differedextensively from the p78 cDNA group (Fig. 1). These cloneswere designated p78-related cDNAs because they hybrid-ized very strongly with the other group of cDNAs.
Proteins encoded by p78 and p78-related cDNAs. Previ-ously, we have shown that the partial p78 cDNA B1.1 cloneused to screen the Xgtll cDNA library coded for the p78protein by using the hybrid selection procedure followed byimmunoprecipitation of the translation product (23). Sincethe cDNAs cloned in the present work were long enough toencode the p78 protein, we transcribed mRNA in vitro fortranslation in the reticulocyte lysate system. By this proce-dure, clone 2-8b cDNA (representative of the p78 cDNAgroup) directed the synthesis of a polypeptide with the sameapparent molecular weight as the p78 protein synthesized invivo (Fig. 2A). Clone 1-1 cDNA (representative of thep78-related cDNA group) directed the synthesis of a poly-peptide with a slightly faster migration rate, indicating that
J. VIROL.
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS 1173
0 1kilobases
2 3
p78 cDNA clone 2-8b
p78 rel. cDNA clone 1-la TTUTTT-DrallO SaclO Pstlu Bglllo SmalA Sal Il\
FIG. 1. Restriction endonuclease maps of p78 and p78-related cDNA clones 2-8b and 1-la, respectively. The maps are shown in theconventional 5' to 3' orientation. The solid lines indicate the coding sequences predicted from the nucleotide sequence data. Symbols: 0,DraII; 0, Sacl; *, PstI; i, BglII; A, SmaI; A, Sall.
the p78-related protein was smaller than the p78 protein byapproximately 2 kilodaltons (Fig. 2A). Two-dimensional gelanalysis of in vitro translation products showed that the p78protein was more acidic than the p78-related protein (Fig.2B). The p78 protein synthesized in vitro had the sameantigenic properties as the p78 protein isolated from IFN-induced cells since it was immunoprecipitated by specificmonoclonal antibodies (Fig. 2C). The p78-related proteinreacted with polyclonal antibodies (data not shown) or withmonoclonal antibodies directed against the p78 protein,demonstrating that the two proteins shared common anti-genic determinants (Fig. 2C).
In vivo expression of p78 and p78-related proteins. The in
A1 2 3 4 5
i_
BK
vivo expression of protein p78 is well documented (21, 23).The expression of p78-related protein in human cells has notyet been described. We addressed the question of in vivoexpression of p78-related protein since we knew that thecorresponding mRNA was present in IFN-induced cells. Invitro translation studies (see above) showed that mRNAsencoding p78-related protein could be translated into proteinwhich was recognized by antibodies directed against p78.We therefore analyzed total extracts of HEL cells inducedwith 5,000 IU of rIFN-aB per ml for the accumulation ofp78-related protein. Western blotting (immunoblotting) oftwo-dimensional gels with polyclonal antibodies revealedonly one strong spot corresponding to protein p78 (Fig. 3).
C1 2 3 4 5 6 7
NEPHGE
92.5 basic acidic-N 69 1 -
"~~1
46 C2
30 rn
3
W| 14.3 *X
FIG. 2. In vitro synthesis and characterization of proteins encoded by p78 and p78-related cDNAs. mRNAs were transcribed from cDNAclones 2-8b and 1-la and translated in the reticulocyte lysate with [35S]methionine as a radioactive marker. (A) One-dimensional gel analysisof radioactive polypeptides synthesized in vitro with mRNAs from clone 2-8b (p78 protein) (lane 1), or clone 1-la (p78-related protein) (lane3); lane 2, mixture of lanes 1 and 3; lane 4, control lysate (no mRNA added); lane 5, "4C-labeled molecular weight markers (K, x 103).Arrowheads indicate in vitro-synthesized p78 and p78-related proteins, respectively. (B) Two-dimensional analysis of "S-labeled polypep-tides synthesized in vitro with mRNAs from clone 2-8b (p78 protein) (panel 1) or 1-la (p78-related protein) (panel 2); panel 3, mixture ofsamples from panels 1 and 2. Only the region of the film showing p78 and/or p78-related proteins is shown. First dimension; NonequilibriumpH gradient electrophoresis (NEPHGE). Second dimension, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (C)Reticulocyte lysates were immunoprecipitated with monoclonal antibodies to the IFN-induced human p78 protein (21). The immunoprecip-itates were collected on protein A-Sepharose, and the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Control lysate (no mRNA added) before (lane 1) or after (lane 2) immunoprecipitation. Lysate with mRNA derived from clone 1-la before(lane 3) or after (lane 4) immunoprecipitation. Lysate with mRNA derived from clone 2-8b before (lane 5) or after (lane 6) immunoprecipi-tation. Lane 7, Same as lane 3, but with a shorter film exposure. Arrowheads point to the immunoprecipitated p78-related and p78 proteins.
VOL. 64, 1990
1174 HORISBERGER ET AL.
--- NEPHGEbasica b
rt
acidic
i:
c
Al
e
...ii...
f
(0cn
-U
.g
.....-X...
..w*i
.s:w:
*1mK '#:,. + - \ _ _:~~~~~.a
.|@w* r~~~~~~~~~~~~~~e:uaw
immunoblot autorad iographyFIG. 3. In vivo expression of p78 and p78-related proteins. Monolayers ofHEL cells were treated or not treated with 5,000 IU of rIFN-cXB
per ml for 20 h in the presence of [35S]methionine in the medium. Proteins from total cell extracts were separated by two-dimensional gelelectrophoresis, and they were then transferred onto nitrocellulose. (a, c, and e) Pieces of nitrocellulose were assayed by an enzyme-linkedimmunosorbent assay with mouse polyclonal antibodies raised against the pure p78 protein (21). (b, d, and f) The same pieces were thenexposed to an X-ray film to reveal the radioactive proteins. For proteins from IFN-induced cells, a 5-,ul sample was analyzed in panels a andb and a 50-,ul sample was analyzed in panels c and d. For proteins from uninduced cells, a 50-,lI sample was analyzed in panels e and f.NEPHGE, Nonequilibrium pH gradient electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Arrow-heads depict the position of the p78 protein.
p78-related protein was not detected, even when the gel wasoverloaded (Fig. 3c), indicating that it is not accumulated invivo in HEL cells.
Structure of cDNAs encoding p78 or p78-related proteins.The composite sequence of the cDNA for p78 mRNA isshown in Fig. 4. Two partial cDNA clones, B1.1 and C'l,which we have described earlier (23), comprise upstreamnoncoding sequences and coding sequences for p78 mRNA.The B1.1 clone spans from nucleotides 1 to 720, and cloneC'1 spans from nucleotides 615 to 1680. The cDNA clone2-8b extends from the EcoRI site in position 137 to the 3' end(2788). The three cDNA clones were completely sequencedin both directions, and no single-nucleotide difference was
found in the regions of overlap. The upstream noncodingsequence contains an alternative exon (249 to 324) whichwas present in clone B1.1 but absent in clones C'1 and 2-8b.The absence of this exon created a Hindlll site at thejunction AAG...CTT. The alternative exon was present in 5
of 10 cDNA clones analyzed. The mouse Mx cDNA alsocontains an optional exon resulting from an alternativesplicing in its upstream noncoding region (24), but thepresence of this exon seems to be less frequent than in thecDNA for p78. The cDNA clone 2-8b had a polyadenylationsite (AATAAA) at position 2768.
Nucleotide sequence analysis of cDNA clone 2-8b re-vealed an open reading frame of 662 residues with the
'I
J. VIROL.
.4w14
4p, jok "Ili*.
p
L_ Bl. 1
* 10 30 50 70 90AGAGCGGAGCCGCACTCCAGCACGCGCAGGGACCGCCACCGGACCC0GCCAGGCATCCCAGTGTCACGGTGGACACGCCrCCC cCC
110 130 _- 2-8b 150 170 190TTGCCGCCCACCTGCTCACC__TCGTCTTCTGTCCCC :A CCACT
210 230 tiO 270 290CCCTGAAC ACGCCGCCC
310 330 350 370 390TATTTGAAGGAACGTATA CACC
MetValValS.rGluValAspIleAlaLysAlaAspProAlaAlaAlaS.rHisP410 430 450 470 490
CTCTATACTGAATGGAGATGCTACTGTGGCCCTGTTGCAGCCAGTATGAGGAGAAGGTGCGCCCCTGroLouLeuLouAsnGlyAspAlaThfirVaLAlaGlnLysAsnProGlySerValAlaGluAsnAsnLeuCysS-rGlnTyrGluGluLysValArgProCy
510 530 550 570 590CACACTCATCTCr_CG_N_sIleAsprAuIleAspSerLouArgAlaLeuGlyValGluGInAspL*uAlaL*uProAlaIlvAlaVal1l GlyAspGlnSorS-r,GlyLysS-rSer
610 F-.- C'1 630 650 670 690GTTG GCACTGTAGGTCCTCC_CAGAGACGACTACG CVaLouGluAlaL.uSerGlyValAlaLeuProArgGlyS.rGlyIleValThrArgCysProLeuValL.uLyszauLysLysLeuValAsnGluAspL
710 B1.1 ..- 730 750 770 790AGTGGAGAGCCAAGGTCAACCAACAATAAAGCCATCGCCGGysTrpArgGlyLysValSerTyrGlnA,spTyrGlull Glull-SerAspAlaS-rGluValGluLysGlulleAsnLysAl&GlnAsnAl&Il-AlaG1
810 830 850 870 890_ AAT~~~~~~~CTCcc5%ArACAGCTcCCCGAGATGTC
yGluGlyM.tGlyIleSerHisGluLeuIleThrLuGluIleSorSerArgAspValProAspLeuThrL.uIl.ASpL.uProGlyIleThrArgvaI910 930 950 970 990
GCTGTGGGCATCAGCCTG CCCAAlaValGlyAsnGlnProAlaAspIleGlyTyrLysIleLysThrL.uIleLysLysTyrIl*GlnArgGlnGluThrIleSrL.uValValValProS
1010 1030 1050 1070 1090_ _ _ _ _ _CGAGTCCGC~GCCGGG~GCCCAGGGCAGCCATcCGAATCFrGACGAAGCCTGATCTGGT
*rAsnValAspIleMaThrThrGluAlaLouSerM?tAlaGlnGluValAspProGluGlyAspArgThrIllGlyIl.L.uThrLysProAspL.uVa1110 1130 1150 1170 1190
lAspLysGlyThrGluAspLysValValAspValValArgAsnL*uValPheHisL.uLysLysGlyTyrM.tIl.ValIaysCysArgGlyGlnGlnGlu1210 1230 1250 1270 1290
ATCCAGGACChGCCAGMCCTGTCGACCGAAAAAAC71YAACACAArCGGTYCGAGGAAGCAIleGlnAspGlnLuSerLeuSerGluAlaLeuGlnArgGluLysIlPhePh.GluAsnlHisProTyrPheArgAspLuL.uGluGluGlyLysAlaT
1310 1330 1350 1370 1390
hrValProCysLuAlaGluLysLeuThrS.rGluL uIllThrHisIleCysLysSorLuProL.uLuGluAsnGln1leLysGluThrHisG1nAr1410 1430 1450 1470 1490_ A = C A G G A ~~~~~~~~~~~~~~~~~~~CAGA
gIleThrGluGluLIuGlnLysTyrGlyValAspIllProGluAspGluAsnGluyqsM.tPh.PheLeuIllAspLysIllMnAlaPhAsnGlnAsp1510 1530 1550 1570 1590
A _ ~ TPGGG ACTcG C cA CLAJG ACAATAAT;IloThrAlaLuMstGlnGlyGluGluThrValGlyGluGluAspIl.ArgL.uPh.ThrArgL.uArgHisGluPheHisLIsTrpSerThrIleIleG
1610 1630 1650 1670 C'1 -f 1690
luAsnAsnPh GlnGluGlyHisLysIl-LouS-rArgLysIl GlnLysPh GluAsnGlnTyrArgGlyArgGluLsuProGlyPh ValAsnTyrAr1710 1730 1750 1770 1790
gThrPheGluThrIleValLysGlnGlnlleLysAlaLeuGluGluProAlaValAspMstL uHisThrValThrAspM.tValArgLzuAlaPhbThr1810 1830 1850 1870 1890
AspValSorIleLSysAsnPh GluGluPh-PheAsnLouHisArgThrAlaLysS*rLysIl GluAspll*ArgAlaGluGInGluArgGluGlyGlulL1910 1930 1950 1970 1990
ysduIrlArgLauHisPheGlnM.tGluGlnlleValTyrrysGlnAspGlnValTyrArgGlyAlaL.uGlnLysVa1ArgGluLysGluL.uGluG12010 2030 2050 2070 2090
uGluLydsLysLsLysSerTrpAspPhoGlyAlaPh*GlnS-rS-rSerAlaThirAspS rS-rMotGluGlull-Ph GlnHisL*uMetAlaTyrHis2110 2130 2150 2170 2190
GlnGluAlaS*rlysArgleSorS-rHislleProLeulalellGlnPh PhotLouGlnThrTyrGlyGllnGlLoeuGlnLysAlaMetLouGlnL2210 2230 2250 2270 2290
*uLauGlnAspLysAspThrTyrS.rTrpL.uLauLysGluArgS.rAspThrSerAspLysArgLysPheL.uLysGluArgLauAlaArgL.uThrGl2310 2330 2350 2370 2390
nAlaArgArgkrgLeuAlaGlnPh.ProGlyEnd2410 2430 2450 2470 2490
2510 2530 2550 2570 2590
2610 2630 2650 2670 2690_ _ _ TACc~mCTA:TrrAT TGCCcrCACAAA
2710 2730 2750 2770 2-Sb -l__-CrCACrAGCCaG
FIG. 4. The nucleotide sequence of p78 cDNA and the predicted amino acid sequence of p78 protein. The composite cDNA sequenceshown was obtained from three clones, B1.1, C'l, and 2-8b. The presumed cap site nucleotide corresponds to the A* (residue 1). The overlineindicates the position of the oligodeoxynucleotide used to screen a genomic library for isolation of the promoter sequence. Vertical arrowsshow the boundaries of an alternative exon. A putative polyadenylation signal is underlined at approximately position 2770.
1175
1176 HORISBERGER ET AL.
potential to encode a protein of Mr 75,500 (Fig. 4). This is inclose agreement with the apparent molecular weight of p78(Mr 78,000) determined by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis for the purified natural p78 protein(21). Moreover, the NH2-terminal sequence of the naturalpurified p78 protein, namely, VVXDIAKA (X was an aminoacid not determined), corresponded exactly to the aminoacid sequence predicted from cloned cDNAs.The cDNA clone 1-la which encodes the p78-related
protein was 2,740 bp long (Fig. 5). It contained a long openreading frame extending from nucleotides 2 to 2062. Theprecise nature of the NH2 terminus of p78-related proteinremains uncertain. A tandem of AUG codons in position 44is not likely to serve as initiation site since these codons arenot in an ideal context for translational initiation (25).Furthermore, the corresponding 77-kDa protein of 673amino acids (larger than the p78 protein) was not synthesizedin the reticulocyte lysate (Fig. 2A). The AUG at position 164could serve as an initiation codon. Since there is an openreading frame upstream of this codon, it is possible thattranslation is initiated further upstream, beyond the bound-ary of the clone. Nevertheless, because a G and a C residueoccur at positions -3 and -2, respectively (with respect tothe first base of the triplet) while G occurs at the +4 position,it is reasonable to assume that this AUG acts as the initiationcodon, giving rise to a 72,500-kDa protein of 633 aminoacids. This would be in close agreement with the Mr of therecombinant polypeptide synthesized in vitro and is slightlysmaller than that of protein p78 (Fig. 2A). Moreover, thehomology with the mouse Mx protein starts precisely at themethionine coded by this AUG in position 164.
It has been proposed that AU-rich regions, particularlythose with the motif AUUUA, play a role in controllingmRNA stability (5, 32). It is interesting that the sequenceATTTA is present once within the alternative exon (5'noncoding region) and once within the 3' nontranslatedregion of the p78 cDNA (positions 278 and 2469 on Fig. 4).The same motif is present in four positions in the p78-relatedcDNA, namely, three times in the coding region and once inthe 3' nontranslated region (positions 1667, 1855, 2003, and2556 in Fig. 5). These sequences may regulate gene expres-sion at a posttranscriptional level by making the mRNAunstable.
Protein sequence analysis. The p78 protein has a 34-amino-acid segment at its NH2 terminus which was notfound in p78-related protein or in Mu-Mxl protein (Fig. 6).p78 and p78-related protein had 63% homology in theircommon sequence, whereas they had 67 and 58% homology,respectively, with the Mu-Mxl protein. The calculatedamino acid composition of p78 was in very good agreementwith that experimentally determined previously with thenatural p78 protein (21). p78 had a slight excess of acidicresidues (94 Asp plus Glu versus 88 Arg plus Lys), whereasp78-related protein had almost equimolar amounts of acidic(80) and basic (81) residues, confirming that p78 was moreacidic than p78-related protein (Fig. 2B).The Mx-related proteins had the highest homologies in
their NH2-terminal halves (Fig. 6). p78 contained sevencysteines (positions 42, 52, 105, 280, 322, 336, and 533)which are highly conserved in the mouse Mxl protein. TheMu-Mxl protein contained four extra cysteines which wereall located in the second half of the protein. p78-relatedprotein had eight cysteines, but only four occupied positionscommon with the p78 protein (positions 52, 105, 280, and533). Five differences in cysteine position occurred in theNH2-terminal half of p78-related protein (positions 42, 115,
218, 322, and 336), indicating that the folding of p78 may bequite different from that of p78-related protein.Near the NH2 terminus we found three domains with the
proper spacing thought to serve collectively as a GTP-binding site (10). The three consensus sequence elementsand corresponding sequences in p78 and p78-related proteinsare shown in Fig. 7. The general features of these domainsare discussed in detail below.
Structural and functional analysis of p78 gene promoter.Primer extension analysis indicated that the p78 cDNAsequence (clone B1.1) is complete at its 5' end and that thepresumed cap site nucleotide corresponds to the A* (residueat position 1) in Fig. 4 (data not shown).
Transient transfection with the 1,800-bp human genomicfragment containing the putative p78 (MX1) gene promoter(Fig. 8A) fused to the cat reporter gene (pHHcat) showedthat this fragment suffices to confer the IFN response to thecat reporter gene (Fig. 8B).To further localize the functional element responsible for
this activity, we fused a limited region containing only 83nucleotides upstream from the presumed cap site and the 5'-27 first nucleotides of the first exon (pNFcat) to the catreporter gene. This short fragment was apparently as activeas the entire genomic fragment described above (Fig. 8B).
In vivo levels of p78 protein do not correlate with antiviralstate. We examined whether p78 protein had an antiviralaction by comparing its intracellular levels with the antiviralstate at various times after IFN treatment. Human embry-onic lung cells were treated with 100 IU of rIFN-aoB per ml.The medium containing IFN was removed from the culturesafter 24 h of incubation, and the cultures were furtherincubated in medium lacking IFN. At the times indicated inFig. 9, cell cultures were either extracted for measurementof p78 content or infected with virus. Virus yields weremeasured in medium harvested at 20 h after infection, andthey were taken as a measure of the antiviral state at the timeof infection.The p78 protein was rapidly induced and reached maxi-
mum levels 16 to 24 h after IFN treatment. The protein levelswere very stable thereafter, and they decreased to one-thirdof the maximum level at day 7 after induction by IFN (Fig.9). These late p78 levels represented full-size protein asdetected on a Western blot (data not shown). The stablelevels of p78 protein were in contrast with the antiviral statesagainst influenza virus and vesicular stomatitis virus, whichhad reverted to preinduction levels within 4 to 7 days. Wetherefore did not observe a strict correlation between theantiviral state and p78 protein levels in human fibroblaststreated with IFN.
DISCUSSION
In the course of cloning cDNA encoding the p78 proteinwhich we had purified and characterized earlier (21), weisolated a cDNA encoding a protein closely related to p78,sharing extensive amino acid sequence homology and anti-genic determinants. Sequence analysis revealed that the twocorresponding mRNAs cannot originate from the same genebut are derived from two separate but closely related genes.Evidence for the existence of two Mx-related genes inhuman cells has already been described by others (1). Thepresence of more than one Mx-related gene has been iden-tified in mice (34) and rats (27). Moreover, two distinctMx-related proteins exist in cattle (17) and in several othermammals (18).The unambiguous assignation of cDNA clone p78 to the
J. VIROL.
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS 1177
10 30 50 70 90CTTcCAGCAAcAGCcAcCAcCATTcGGCACAGTGccAcCACAAACGATGTTTcCTccAAAcTGGCAGGGGGCAGAGAAoGACGCTGCITTcCTcGccAAGPheGlnGlnGlnProProProPheGlyThrValProProGlnM.tHtPhProProAsnTrpGlnGlyAlaGluLysAspAlaAlaPhOLuAlaLys
110 130 150 170 190GACTTCAACTTTCTCACTTTGAACAATCAGCCACCACCAGGAAACAGGAGCCAACCAAGGGCAA7GGGGCCCGAGAACAACCTGTACAGCCAGTACGAGCAspPheAsnPheLouThrLeuAsnAsnGlnProProProGlyAsnArgS-rGlnProArgAlaMbtGlyProGluAsnAsnLouTyrS-rGlnTyrGluG
210 230 250 270 290AGANGGTGCGCCCCTGCATTGACCTCATCGACTCCCTGCGGGCTCTGGGTGTGGAGCAGGACCTGGCCCTGCCAGCCATCGCCGTCATCGGGGACCAGAGlnLysValArgProCysIleAspLeuIleAspSerLuArgAlaLeuGlyValGGluGlnAspLIuAlaLuProAlaIleAlaValIleGlyAspGlnSe
310 330 350 370 390CTCGGGCAGAGCTGCTGT T CCCAGAGGCAGCGGAATCGTAACCAGGTGTCCGCTGGTGCTGAAACT&AAAAAGrSerGlyLysSerSerValL.uGluAlaLeuSerGlyValAlaLeuProArgGlyS@rGlyIleValThrArgCysProLeuValLuLysLuLysLys
410 430 450 470 490CAGCCCTGTGG=GCATGGGCCGGAAGGATCAGCTACCGGAACACCGAGCTAGAGCTTCAGGACCCTGGCCAGGTGGAGAAAGAGGDATAAAAAGCCCAGAGlnProCysGluAlaTrpAlaGlyArgIleSerTyrArgAsnThrGluL uGluIAuGlnAspProGlyGlnValGluLysGluIleHisLysAlaGlnA
510 530 550 570 590ACGTCATGGCCGGGAATGGCCGGGGCATCAGCCAGAGCCATCAGCCTGGAGATCACCTCCCCTGAGGTTCCAGACCTGACCATCATTGACCTTCCCGGsnValM.tAlaGlyAsnGlyArgGlyIleSerHisGluLouIleSerLouGluIleThrSerProGluValProAspLeuThrIleIleAspLeuProGl
610 630 650 670 690CATCACCAGGGTGGCTGTGGACAACCAGCCCCGAGACATCIGGACTC GATCAAGGCCTC TCAAGAAGTACATCCAAGACGCGATCAACTTGyIleThrArgValAlaVaLAspAsnGlnProArgAspIllGlyleuGlnIlLysAlaLeuIleLysLysTyrIleGlnArgGlnGlnThrIleAsnLeu
710 730 750 770 790G CTTGTTCDIAACCGTGGACATTCCACCACGGAGGCGCTGAGCATGGCCCATGAGGTGGAcCCCGAAGGGGACAGGACCATCGGTATCCTGACCAValValValProCysAsnValAspIleAlaThrThrGluAlaLeuSerMetAlaHisGluValAspProGluGlyAspArgThrIleGlyIleLeuThrL
810 830 850 870 890AACCAGATCTAATGGACAGGGGCACDCAGAAAAGCGTC.ATGAATGTGGTGCGGAACCTCACGTACCCCCTCAAGAAGGGCTACATGATTGTGAAGTGCCGysProAspLeuMetAspArgGlyThrGluLysSerValM.tA,snValValArgAsnLouThrTyrProLouLysLysGlyTyrMOtIleValLysCysAr
910 930 950 970 990GGGCCAGCAGGAGATCACAAACACTGAGCTTGGCAGAGWCAACCAAGAAAGAAA ACATTCTTTCAACACATCCATATTCAGAGTTCTCCTGGAGgGlyGlnGlnGluIleThrAsnArgLeuSerLeuAlaGluAlaThrsLsysGluIleThrPhePheGlnThrHisProTyrPh*ArgValLuLuGlu
1010 1030 1050 1070 1090GAGGTCAGCCACGGTTCCCCGAcCTGCAGAAAGACTTACCACTGAACTCATCATGCATATCCAAAAATCGCTCCCTGAAGAAAAGGGluGlySerAlaThrValProArgLeuAlaGluArgLeuThrThrGluLeuIleMbtHisIllGlnLysSerLeuProLeuLeuGluGlyGlnIl-ArgG
1110 1130 1150 1170 1190AGAGCCACCAGAAGGCGACCGAGGAGCTGCGGCGTTGCGGGGCTGACATCCCCAGCCAGGAGGCCGA=AsAGAGTTCTTTCAATGAGAAACAAGATluS-rHisGlnLysAlaThrGluGluLeuArgArgCysGlyAlaAspIleProS-rGlnGluAlaAspLysMbtPh*Ph-LouIllGluLysIleLysMe
1210 1230 1250 1270 1290GTMrAATCAGGACATMAAAAGAT=GtPheAsnGlnAspIleGluLysL.uValGluGlyGluGluValValArgGluAsnGluThrArgLOuTyrAsnLysIleArgGluAspPhOLysAsnTrp
1310 1330 1350 1370 1390GTAGGCATACTTGCAACTAATACCCAAAAAGTTAAAAATATTATCCAGAAGTAAAAG ACAGTATCGAGGCAAGGAGCTTCTGGGATValGlyIleL.uAlaThrAsnThrGlnLysValLysAsnI1lOIlHisGluGluValGluLysTyrGluLysGlnTyrArgGlyLysGluLOuLuGlyP
1410 1430 1450 1470 1490TGTCAACTACAAGACATTAGATCATCGTGCATCAGTACATCCAGCAGcGGTGGAGCCCGCCC'TTAGCATGCTCCAGAAAGCCATGGAAATTATCCAh.ValAsnTyrLysThrPh.GluIllIleValHisGlnTyrIl@GlnGlnLOuValGluProAlaL.uSerMetLuGlnLysAlaMetGluIleIleGl
1510 1530 1550 1570 1590GCAAGCTTTCATTAACGTGGCCAAAAAACATGGCGAATTCAACCTAAC GTC A GAGCAnGlnAlaPheIleAsnValAlaLysLysHisPheGlyGluPhePh AsnL uAsnGlnThrValGlnSerThrIl GluAspIleLysValLysHisThr
1610 1630 1650 1670 1690GCAAAGGCAG AAAACATGATCCAACTTCAGTTCGA GACGTGTTTGCAAGATCAGATTTACAGTGTTGTTCTGAAGAAAGTCCGAGAAGAlaLysAlaGluAsnMetIleGlnLeuGlnPheArgMetGluGlnM4etValPheCysGlnAspGlnI leTyrSerValValL uLysLysValArgGluG
1710 1730 1750 1770 1790AGATrTTTAACCCrTGGGGACGCCTrCACAGAATATGAAGTTGAACTCTCATTTTCCCAGTAATGAGTCTTCGGTTCCTCC rACTGAAATAGGCATluIloPheAsnProLeuGlyThrProSerGlnAsnMetLysLeuAsnSerHisPhOProSorAsnGluSerSerValSerSerPheThrGluIleGlyIl
1810 1830 1850 1870 1890CCACCrGAATGCCTACTTCTGGAAACCAGCAAACGTCTCGCCAACCAGATCCCATTATAATTCAGTATTTATGCrCCGAGAGAATGGTGACTCCTTGeHisLeuAsnAlaTyrPheLeuGluThrSerLysArgLeuAlaAsnGlnIleProPheIleIleGlnTyrPheMetLeuArgGluAsnGlyAspSerLou
1910 1930 1950 1970 1990CAGAAAGCCATGATGCAGATACTACAGGAAAAAAATCGCTATCCTGGCTGC1CTCAAGAGCAGAGTGAGACCGCTACCAAGAGAAAATCCTTAAGGAGAGlnLysAlaMetMetGlnIleLeuGlnGluLysAsnArgTyrSerTrpLeuLuGlnGluGlnSerGluThrAlaThrLysArgArgIleLeuLysGluA
2010 2030 2050 2070 2090GAATTTACCGGCTCACTCAGGCGCGACACGCACTCTGTCAATTCTCCAGCAAAGAGATCCACTGAAGGGCGGCGATGCCTGTrGTI TCITGTGCGTrgIleTyrArgLeuThrGlnAlaArgHisAlaLuCysGlnPheSerSerLysGluIleHisEnd
2110 2130 2150 2170 2190ACTCCArTTCTAAAGGGGAGTCGGTGCAGGATGCCGCTTCTG GGGGCCAAACTCTTCTGTCACTATCAGTGTCCATCTCTACTGTACTCCCTCAG
2210 2230 2250 2270 2290~~~~CATCGGAGATAGGCACACAGCTCAGCTCTCTCCACCACCCAGCTCTTCCCTGACCTACA7GAWCTCCATCTGGGTCC2310 2330 2350 2370 2390
CGTAGCACACAGTTACAGTGTCCTAAGATACTGCTATCATTCTTCGCTAATTTGTATTTGTATTCCCTTCCCCCTACAAGATTATGAGACCCCAGAGGGG2410 2430 2450 2470 2490
GAAGGTCTGGGTCAAATrCCTCACTGTGTAGTCCAGCACCrGCAGCAC _ACTCACTGAAOG AACGAATGAG2510 2530 2550 2570 --- 2590
TGCTGTGTAAGTGATGGAGATACCTGAGGCTATTGCTCAAGCCCAGGCCTTGGACATTTAGTGACTGTTAGCCGGTCCCTTTCAGATCCAGTGGCCATGC2610 2630 2650 2670 2690
CCCTGCTTCCCATGGTTCACTGTCATTGTGTTTCCCAGCCTCTCCACTCCCCCGCCAGAAAGGAGCcTGAGTGATTCTCTTrCTTCrGTTTCCCTGA2710 2730
TTATGATGAGCrTCCArTGTCrCTGTrAAGTCTrTGAAGAGGFIG. 5. The nucleotide sequence of p78-related cDNA (clone 1-la) and the predicted amino acid sequence. Putative initiation codons at
approximately positions 50 and 170 and a polyadenylation signal at approximately position 2470 are underlined.
VOL. 64, 1990
1178 HORISBERGER ET AL.
p78p78-relMu-Mx1
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551 EEEKKKKSWD. .FGAFQSSSATDSSME ...EIFQHUM4YHQEASKRISSHIPLIIQFFMRQYGQQLQKAMLQLLQDKDT 625516 NPLGTPSQNMKNSH-P-NESSV-FT.FT-GI-N-FL-T-LAQ-F-Y-REN-DS !4-I-E-NR 592517 -K-T-ALIN. PAT-NN-QFPQKG Lr- -K-Y--CRN-GRQ--KY-I-K-F-EEIE-M ----TSK 694
626 YSWLLKESDTSDKRKFLKERLARLTQARRRLAQFPG 662593 -QQ-E-AT-RI-IY HA-C-SSKEIH 633595 C-F-E-Q-RE-K-R-L-DE--QK-K-SD 631
FIG. 6. Comparison of the deduced amino acid sequences of the p78 protein with those of p78-related and Mu-Mxl proteins. Deletions(.) were introduced to optimize the alignment. Dashes represent amino acid identity. Putative GTP-binding domains are indicated by solidoverlines. Sequence of Mu-Mxl was taken from reference 33.
cellular gene product p78 relies on several parameters suchas molecular weight, pl, amino acid composition, and iden-tity of the NH2-terminal amino acid sequence determinedchemically on the purified protein with that predicted fromthe cDNA clone. cDNA clones encoding the p78-relatedprotein were estimated to be fivefold less abundant than
those encoding protein p78, reflecting most likely a corre-sponding lower steady state of transcripts. These genes areregulated at the transcriptional level, and a preliminaryfunctional analysis of the p78 (MX1) gene promoter showedthat a very short sequence suffices to confer inducibility byIFN-ox. Other constructions are already available that will
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FIG. 7. Amino acid sequences at the presumptive GTP-binding regions of p78, p78-related, mouse Mxl, and fish Mx (35) proteins incomparison with that of H-ras protein, E. coli elongation factor (EF-Tu), G protein ot subunit (bovine Gsa) (sequences from reference 11),SRP54, and SRP receptor (sequences from references 4 and 30). Boxes surround residues that are identical to the consensus sequence.Number in front of the sequence motifs refers to amino acid positions in the respective proteins.
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J. VIROL.
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS 1179
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FIG. 9. Induction and decay of p78 protein and of the antiviral state in cell treated with IFN. HEL cell cultures were treated with 100 IUof IFN-a-B per ml. One day later, the culture medium was removed, cells were washed, and fresh medium lacking IFN was added for furtherincubation of cultures. At the time points indicated, cell in cultures were infected with either influenza virus or vesicular stomatitis virus(VSV). Supernatant fluids were harvested 18 h later. The mean virus yields from two cultures in each of two independent experiments aregiven. Virus yields are expressed as the percentage of yields in control cultures not treated with IFN. The levels of p78 in parallel cultureswere determined on Western blots as described elsewhere (21).
permit a more detailed dissection of the functional domainsof this promoter (J. Dellis and J. Content, manuscript inpreparation). The p78-related gene may also be regulated ata posttranscriptional level in vivo since we were unable todetect p78-related protein in diploid fibroblasts treated withIFN, using a very sensitive immunoassay (21). Therefore,we assume that it is the p78 which is the functional protein inthis small gene family, at least in diploid fibroblasts.The mouse Mx system is characterized by a slow rate of
decay of the antiviral state against influenza virus (2) corre-lating most likely to the rate of decay of Mx protein, whichis a very stable protein with intrinsic anti-influenza virusactivity (20, 33). This has not been found in human diploidcells. Moreover, there was no correlation between proteinp78 levels and the antiviral state induced by IFN-a. Thus, 30to 50% of maximum levels of p78 were still present at a timewhen cells had regained full susceptibility to both virusestested. These observations suggest that the p78 protein is notinvolved in a specific antiviral mechanism against influenzavirus and that its function is more general than just partici-pating in the antiviral activity of IFN.p78/Mu-Mx proteins have their cysteines conserved at
their NH2 termini, which may indicate that a functionaldomain is in this region. We discovered three consensussequence elements defining a putative GTP-binding domainprecisely in this region of strongest sequence homology. Theexact sequence of the three elements is conserved in thefamily of p78/Mu-Mxl (Fig. 7) as well as in a fish gene withhomology to MX (35). There is a mismatch in the thirdelement with the strict consensus sequence, which is TKxDinstead ofNKxD (10) and which is found, for instance, in theelongation factor, in the ras protein or in G-binding proteins(Fig. 7). However, the N->T deviation has been very re-cently found in two proteins involved in the vectorial trans-port of proteins, namely, the 54-kDa protein of the signalrecognition particle (SRP54) and the a-subunit of the SRPreceptor in the endoplasmic membrane which has been
shown to bind GTP (4, 8, 30). We therefore infer that p78 andits homologs belong to a distinct subgroup of guanosinenucleotide-binding protein (4).The N--T deviation in the third consensus element, which
is the guanine-binding domain (10), may alter the affinityand/or the strict specificity of this domain for GTP and mayindicate a low affinity of Mx homologs for nucleotide bindingwhich might be compensated by the abundance of theprotein in IFN-induced cells (21). Alternatively, it mayindicate a specificity for bases related to guanosine such asxanthosine or inosine. Base aberrations in nucleic acids maybe more common than previously thought. For instance, adouble-stranded-RNA-unwinding activity is capable of con-verting adenosine residues to inosine residues, which havechemical properties similar to those of guanosine residues(3).
Guanylate-binding proteins of Mrs 67,000 and 56,000 areinduced by IFN in human cells, and they have been isolatedby GMP-agarose affinity chromatography. These proteins,however, do not seem to correspond to p78 or to Mxhomologs because they differ in Mr and because they are alsoinduced in mouse cells defective in MX gene expression (7).Several IFN-induced proteins and IFN-regulated enzymesreact with either nucleotides or nucleic acids (9). Otherproteins with the GxxxxGS/T domain react with nucleicacids, namely, unwinding proteins or viral replication pro-teins (13). We are currently testing the hypothesis thatprotein p78 binds a nucleotide or a nucleic acid. We plan toexpress p78 or a truncated form of it in E. coli to use in invitro studies. A major task may be the expression of anactive form of protein p78 since this protein seems to have avery low solubility.
ACKNOWLEDGMENTSWe thank M. C. Gunst and K. de Staritzky for skillful technical
assistance.Part of this work was supported by grants 3.4542.88 and 3.4518.89
J. VIROL.
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS 1181
from the Fund for Medical Scientific Research (Belgium) and by theCancer Research Foundation of the Belgian General Savings andRetirement Fund. M.G.W. is research assistant of the NationalFund for Scientific Research (Belgium). J.D. is supported by a grantfrom the Institute for encouragement of Scientific Research inIndustry and Agriculture (Belgium).
LITERATURE CITED1. Aebi, M., C. E. Samuel, H. Arnheiter, 0. Haller, and C.
Weissmann. 1987. Isolation and expression of cDNA clonesencoding human Mx and Mx-related protein. J. Interferon Res.7:719.
2. Arnheiter, H., and 0. Haller. 1983. Mx gene control of inter-feron action: different kinetics of the antiviral state againstinfluenza virus and vesicular stomatitis virus. J. Virol. 47:626-630.
3. Bass, B. L., and H. Weintraub. 1988. An unwinding activity thatcovalently modifies its double-stranded RNA substrate. Cell55:1089-1098.
4. Bernstein, H. D., M. A. Poritz, K. Strub, P. J. Hoben, S.Brenner, and P. Walter. 1989. Model for signal sequence recog-nition from amino-acid sequence of 54K subunit of signalrecognition particle. Nature (London) 340:482-486.
5. Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer,and A. Cerami. 1986. Identification of a common nucleotidesequence in the 3'-untranslated region of mRNA moleculesspecifying inflammatory mediators. Proc. Natl. Acad. Sci. USA83:1670-1674.
6. Chang, J. Y., H. Herbst, R. Aebersold, and D. G. Braun. 1983.A new isotype sequence (VK27) of the variable region of K-lightchains from a mouse hybridoma-derived anti-(streptococcalgroup A polysaccharide) antibody containing an additionalcysteine residue. Biochem. J. 211:173-180.
7. Cheng, Y.-S. E., R. J. Colonno, and F. H. Yin. 1983. Interferoninduction of fibroblast proteins with guanylate binding activity.J. Biol. Chem. 258:7746-7750.
8. Connolly, T., and R. Gilmore. 1989. The signal recognitionparticle receptor mediates the GTP-dependent displacement ofSRP from the signal sequence of the nascent polypeptide. Cell57:599-610.
9. Content, J. 1986. Biochemical aspects of interferon action, p.163-189. In R. Perez Bercoff (ed.), The molecular basis of viralreplication. Plenum Publishing Corp., New York.
10. Dever, T. E., M. J. Glynias, and W. C. Merrick. 1987. GTP-binding domain: three consensus sequence elements with dis-tinct spacing. Proc. Natl. Acad. Sci. USA 84:1814-1818.
11. Gilman, A. G. 1987. G proteins: transducers of receptor-gener-ated signals. Annu. Rev. Biochem. 56:615-649.
12. Goetschy, J. F., H. Zeller, J. Content, and M. A. Horisberger.1989. Regulation of the interferon-inducible IFI-78K gene, thehuman equivalent of the murine Mx gene, by interferons,double-stranded RNA, certain cytokines, and viruses. J. Virol.63:2616-2622.
13. Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko, and V. M.Blinov. 1988. A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involvedin duplex unwinding in DNA and RNA replication and recom-bination. FEBS Lett. 235:16-24.
14. Gorman, C. 1985. High efficiency gene transfer into mammaliancells, p. 143-190. In D. M. Glover (ed.), DNA cloning, apractical approach, vol. 2. IRL Press Limited, Oxford, England.
15. Graham, F. L., and A. J. Van der Eb. 1973. A new technique forthe assay of infectivity of human adenovirus S DNA. Virology52:456-467.
16. Herbomel, P., B. Bourachot, and M. Yaniv. 1984. Two distinctenhancers with different cell species specificities coexist in theregulatory region of polyoma. Cell 39:653-662.
17. Horisberger, M. A. 1988. The action of recombinant bovineinterferons on influenza virus replication correlates with theinduction of two Mx-related proteins in bovine cells. Virology162:181-186.
18. Horisberger, M. A. 1988. Homologs of the IFN-induced mouseMx protein in 14 animal species. J. Interferon Res. 8:S102.
19. Horisberger, M. A., and K. de Staritzky. 1985. Sensitivity ofinfluenza A viruses to human interferons in human diploid cells.FEMS Microbiol. Lett. 29:207-210.
20. Horisberger, M. A., and K. de Staritzky. 1989. Expression andstability of the Mx protein in different tissues of mice, inresponse to interferon inducers or to influenza virus infection. J.Interferon Res. 9:583-590.
21. Horisberger, M. A., and H. K. Hochkeppel. 1987. IFN-a inducedhuman 78 kD protein: purification and homologies with themouse Mx protein, production of monoclonal antibodies, andpotentiation effect of IFN-y. J. Interferon Res. 7:331-343.
22. Horisberger, M. A., P. Staeheli, and 0. Hailer. 1983. Interferoninduces a unique protein in mouse cells bearing a gene forresistance to influenza virus. Proc. Natl. Acad. Sci. USA80:1910-1914.
23. Horisberger, M. A., M. Wathelet, J. Szpirer, C. Szpirer, Q.Islam, G. Levan, G. Huez, and J. Content. 1988. cDNA cloningand assignment to chromosome 21 of IFI-78K gene, the humanequivalent of murine Mx gene. Somatic Cell Mol. Genet.14:123-131.
24. Hug, H., M. Costas, P. Staeheli, M. Aebi, and C. Weissmann.1988. Organization of the murine Mx gene and characterizationof its interferon- and virus-inducible promoter. Mol. Cell. Biol.8:3065-3079.
25. Kozak, M. 1987. At least six nucleotides preceding the AUGinitiator codon enhance translation in mammalian cells. J. Mol.Biol. 196:947-950.
26. Laemmli, U. K. 1970. Cleavage and structural proteins duringthe assembly of the head of bacteriophage T4. Nature (London)227:680-685.
27. Meier, E., J. Fah, S. Grob, R. End, P. Staeheli, and 0. Haller.1988. A family on interferon-induced Mx-related mRNAs en-codes cytoplasmic and nuclear proteins in rat cells. J. Virol.62:2386-2393.
28. Nielsen, P. J., G. K. McMaster, and H. Trachsel. 1985. Cloningof eukaryotic protein synthesis initiation factor genes: isolationand characterization of cDNA clones encoding factor eIF-4A.Nucleic Acids Res. 13:6867-6880.
29. Porter, A. C. G., Y. Chernajovsky, T. C. Dale, C. S. Gilbert,G. R. Stark, and I. M. Kerr. 1988. Interferon response elementof the human gene 6-16. EMBO J. 7:85-92.
30. Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron,and B. Dobberstein. 1989. Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteinswith putative GTP-binding domains. Nature (London) 340:478-482.
31. Rubin, B. Y., S. L. Anderson, R. M. Lunn, G. R. Hellermann,N. K. Richardson, and L. J. Smith. 1988. Production of amonoclonal antibody directed against an interferon-induced56,000-dalton protein and its use in the study of this protein. J.Virol. 62:1875-1880.
32. Shaw, G., and R. Kamen. 1986. A conserved AU sequence fromthe 3' untranslated region of GM-CSF mRNA mediates selec-tive mRNA degradation. Cell 46:659-667.
33. Staeheli, P., 0. Haller, W. Boll, J. Lindenmann, and C. Weiss-mann. 1986. Mx protein: constitutive expression in 3T3 cellstransformed with cloned Mx cDNA confers selective resistanceto influenza virus. Cell 44:147-158.
34. Staeheli, P., and J. G. Sutcliffe. 1988. Identification of a secondinterferon-regulated murine Mx gene. Mol. Cell. Biol. 8:4524-4528.
35. Staeheli, P., Y.-X. Yu, R. Grob, and 0. Haller. 1989. Adouble-stranded RNA-inducible fish gene homologous to themurine influenza virus resistance gene Mx. Mol. Cell. Biol.9:3117-3121.
36. Von Wussow, P., D. Jakschies, H. K. Hochkeppel, M. A.Horisberger, K. Hartung, and H. Deicher. 1989. Mx-homologousprotein in mononuclear cells from patients with systemic lupuserythematosus. Arthritis Rheum. 33:1-5.
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