interferon (IFN-a) inducibility to the cat reporter gene. The p78 ...... G K S. G KT. G K S. G K S. G K S. 178. 143. 144. 57. 80. 223. 190. 520. D7. D. D. D. D. D. D. D.
JOURNAL OF VIROLOGY, Mar. 1990, p. 1171-1181
Vol. 64, No. 3
0022-538X/90/031171-11$02.00/0 Copyright © 1990, American Society for Microbiology
Cloning and Sequence Analyses of cDNAs for Interferon- and VirusInduced Human Mx Proteins Reveal that They Contain Putative Guanine Nucleotide-Binding Sites: Functional Study of the Corresponding Gene Promoter MICHEL A. HORISBERGER,l* GARY K. McMASTER,1 HELMUT ZELLER,lt MARC G. WATHELET,2 JOELLE 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 some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement 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 NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-a) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-a. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-a was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins.
The p78 protein is encoded by the MX1 gene located on the distal part (21q22.3) of the long arm of human chromosome 21 (23; K. Gardiner, M. A. Horisberger, and D. Patterson, manuscript in preparation) in the region pathogenic for Down's syndrome. The gene is induced by type I interferon (IFN-a/,B) and by some viruses. Furthermore, its expression is modulated by biological response modifiers involved in viral infection, inflammation, and immune response (12). However, the real function of p78 is still unknown. 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 IFN inducibility (21, 22). The intracellular localization of p78 implies that the protein is involved in cytoplasmic functions (21). p78 may be inhibitory for influenza virus, as is the IFN-induced mouse protein Mx (33). In contrast to the mouse Mx system, however, there might be no strict correlation between the induction of the p78 protein and the antiviral activity of IFN. Thus both IFN-a and gamma IFN (IFN-y) protect human cells against influenza virus infection, whereas only IFN-a is a potent inducer of p78 protein in vitro (12, 19). IFN-y-primed cells, however, are able to accumulate p78 protein during viral infection, suggesting that IFN--y programs cells to full antiviral activity upon virus
infection (12). We have no evidence that p78 gene expression plays a pathophysiological role in specific diseases. All individuals tested so far are positive for the protein p78 after exposure to IFN (36). However, p78 is a sensitive marker for biological activity of IFN, including acid-labile IFN in diseases such as systemic lupus erythematosus (36; D. Jakschies, H. K. Hochkeppel, M. A. Horisberger, H. Deicher, and P. von Wussow, J. Biol. Response Modif., in press) or acquired immunodeficiency syndrome (P. von Wussow, D. Jakschies, B. Block, I. Schedel, M. A. Horisberger, H. Hochkeppel, and H. Deicher, AIDS, in press). Expression and analysis of cDNAs may help to define the function of the p78 protein. In this report, we describe the cloning of two types of full-length cDNAs which are homologous to the mouse Mx cDNA. Structural analysis of proteins encoded by these clones revealed the presence of three consensus sequence elements with distinct spacing which could confer a guanine nucleotide-binding domain to human p78 and p78-related proteins and mouse Mx protein. MATERIALS AND METHODS Abbreviations. The human proteins p78 and p78-related protein (old nomenclature) could be named Hu-Mxl and Hu-Mx2, respectively, and the corresponding genes MX1 and MX2. To avoid confusion between the abbreviations Hu-Mx and Mu-Mx (murine protein Mx), we have kept the old nomenclature throughout the report. Cells, viruses, infections, and IFN. Human embryonic lung cells (HEL, Flow Laboratories 2002) were grown in minimal
* Corresponding author. t Present address: Institute of Experimental Dermatology, University of Munster, Munster, Federal Republic of Germany.
1171
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Eagle medium-Earle salts supplemented with 10% fetal calf serum. They were infected with 1 PFU per cell for the studies on virus replication. Working stocks of vesicular stomatitis virus and of influenza A virus, strain fowl plague (Rostock/34/H7N1), were prepared from the allantoic cavity of 10-day-old embryonated eggs. Titers of viruses were determined on primary cultures of calf kidney cells by the procedure for plaque assay as previously described (19). Recombinant human IFN-cxB (rHuIFN-cx8 in the numeric designation; >95% pure) was produced in yeasts and purified by affinity chromatography with monoclonal antibodies. IFN-a2c was from Boehringer Ingelheim. Escherichia coli strain, plasmids, and gene library. cDNA was synthesized from cytoplasmic poly(A)+ RNA that was isolated from human embryonic lung cells treated for 6 h with 1,000 IU of rIFN-otB per ml (12, 23). The cDNA was cloned into EcoRI-cleaved arms of Xgtll as previously described (28). Positive plaques were detected with an oligo-labeled cDNA probe comprising upstream noncoding sequences and coding sequences for p78 mRNA (clone B1.1 [23]). Positive cDNA inserts were subcloned into the EcoRI site of Bluescript M13 vector (Stratagene, San Diego, Calif.) for sequencing. Both strands of positive clones were sequenced by the dideoxy method (Sequenase; U.S. Biochemical Corp.). In vitro synthesis of mRNAs. A typical in vitro reaction mixture 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, Madison, Wis.), 1 p.g of template, and 10 U of T7 RNA polymerase (Stratagene) in a final concentration of 40 mM Tris hydrochloride (pH 8), 8 mM MgCl2, 2 mM spermidine, and 50 mM NaCl. Incubation was performed at 37°C for 60 min. DNase I (1 U) was then added, and the mixture was incubated at 37°C for 10 min. The reaction mixture was phenol extracted, and RNA transcripts were precipitated with 0.6 volume of isopropanol containing 0.3 volume of 3 M sodium acetate (pH 6). In vitro synthesis of proteins. About 1/10th to 1/15th of an in vitro transcription reaction was used in a typical translation reaction. The rabbit reticulocyte lysate (micrococcal nuclease digested; Amersham International, Amersham, United Kingdom) was used at an 80% concentration and supplemented with 7 ViCi of [35S]methionine per 6 1J of reaction. The reaction mixture was incubated at 30°C for 60 min.
Immunoprecipitation. The in vitro-synthesized proteins were treated with 1% sodium dodecyl sulfate and diluted in 10 mM Tris hydrochloride (pH 7.5)-50 mM NaCl. They were incubated for 3 h at 4°C with the different antibodies (see Results) and then incubated for two more hours at 4°C with protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology) as a 50% suspension in buffered phosphate saline containing 0.5% bovine serum albumin. Immunoprecipitates were washed as described previously (31) and analyzed by
polyacrylamide gel electrophoresis (26). NH2-terminal amino acid sequence. Protein p78 was purified as described elsewhere (21) and its amino acid sequence was analyzed in a Beckman 8906 sequencer as described by Chang et al. (6). Isolation of genomic clone containing p78 (MX1) gene promoter and fusion of two derived fragments with the cat reporter gene. A chromosome 21-Hindlll library (American Type Culture Collection-National Institutes of Health human chromosome-specific libraries, 1988) was screened with the partial cDNA clone B1.1 (23). A positive clone also
J. VIROL.
hybridizing with an oligodeoxynucleotide (5'-CAGTGCTG GAGTGCGGCCTCCGCTC-3') corresponding to the most upstream region of the cDNA sequence (see Fig. 4) was sequenced and subcloned to fuse it, in toto or in part, with the cat reporter gene derived from a pRSVCAT plasmid (American Type Culture Collection-National Institutes of Health repository of probes and cloned genes, 1988). Transfection and chloramphenicol acetyltransferase assay. After two rounds of CsCl-ethidium bromide gradient purification, 6 pLg of the various plasmid-cat constructs were transfected with 106 human L-132 cells (ATCC CCL 5) in the presence of 12 Fig of herring sperm carrier DNA by the calcium phosphate precipitation method (15). To eliminate a major bias caused by variability in transfection efficiency from one dish to another, we introduced, as an internal marker, plasmid pCH110 (16), which expresses ,B-galactosidase under the control of the simian virus 40 early promoter. Each cat construct to be tested was cotransfected with pCH110 in a 3:1 molar ratio. A colorimetric assay based on the use of O-nitrophenyl-3-D-galactopyranoside was used to measure the P-galactosidase activities in cytoplasmic extracts from transfected cells (16). After 48 h, the cells were either treated or not treated with 3,000 IU of IFN-cx2c per ml for 16 h. The chloramphenicol acetyltransferase assay was performed as described previously (14).
RESULTS Cloning of cDNAs encoding the entire p78 protein. To isolate cDNAs encoding the human p78 protein, we constructed a Agtll library of cDNAs prepared from mRNAs isolated from human embryonic lung cells which had been induced with IFN-ot. The library was screened with a partial cDNA clone (referred to as the B1.1 clone in reference 23) encoding 5' untranslated sequence and NH2-terminal sequence of the p78 protein. A total of 1.5 x 105 recombinants gave 22 positive bacteriophage clones, of which 10 were randomly selected and 9 contained an EcoRI insert of 2,600 base pairs (bp) (an open reading frame of approximately 2,100 nucleotides would suffice to encode the entire p78 protein). These inserts were subcloned into the EcoRI site of Bluescript vector DNA, and plasmid DNA was amplified in bacteria. Restriction analysis of the purified DNA indicated that two types of cDNAs had been cloned. The partial restriction map of a group of seven cDNA clones was consistent with that of the partial cDNA clones described earlier (23) encoding part of the p78 protein. These clones were named p78 cDNA (Fig. 1). The partial restriction map of the other group composed of two cDNA clones differed extensively from the p78 cDNA group (Fig. 1). These clones were designated p78-related cDNAs because they hybridized very strongly with the other group of cDNAs. Proteins encoded by p78 and p78-related cDNAs. Previously, we have shown that the partial p78 cDNA B1.1 clone used to screen the Xgtll cDNA library coded for the p78 protein by using the hybrid selection procedure followed by immunoprecipitation of the translation product (23). Since the cDNAs cloned in the present work were long enough to encode the p78 protein, we transcribed mRNA in vitro for translation in the reticulocyte lysate system. By this procedure, clone 2-8b cDNA (representative of the p78 cDNA group) directed the synthesis of a polypeptide with the same apparent molecular weight as the p78 protein synthesized in vivo (Fig. 2A). Clone 1-1 cDNA (representative of the p78-related cDNA group) directed the synthesis of a polypeptide with a slightly faster migration rate, indicating that
VOL. 64, 1990
~1
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS 1
0
kilobases
2
1173
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 the conventional 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 by approximately 2 kilodaltons (Fig. 2A). Two-dimensional gel analysis of in vitro translation products showed that the p78 protein was more acidic than the p78-related protein (Fig. 2B). The p78 protein synthesized in vitro had the same antigenic properties as the p78 protein isolated from IFNinduced cells since it was immunoprecipitated by specific monoclonal antibodies (Fig. 2C). The p78-related protein reacted with polyclonal antibodies (data not shown) or with monoclonal antibodies directed against the p78 protein, demonstrating that the two proteins shared common antigenic determinants (Fig. 2C). In vivo expression of p78 and p78-related proteins. The in
A 1
2
3
4
-N
"
B
5
i_
vivo expression of protein p78 is well documented (21, 23). The expression of p78-related protein in human cells has not yet been described. We addressed the question of in vivo expression of p78-related protein since we knew that the corresponding mRNA was present in IFN-induced cells. In vitro translation studies (see above) showed that mRNAs encoding p78-related protein could be translated into protein which was recognized by antibodies directed against p78. We therefore analyzed total extracts of HEL cells induced with 5,000 IU of rIFN-aB per ml for the accumulation of p78-related protein. Western blotting (immunoblotting) of two-dimensional gels with polyclonal antibodies revealed only one strong spot corresponding to protein p78 (Fig. 3).
C 1
K
92.5 69
2
3
4
5
6
7
NEPHGE basic
acidic
1
-
46 C 2
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30
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3
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FIG. 2. In vitro synthesis and characterization of proteins encoded by p78 and p78-related cDNAs. mRNAs were transcribed from cDNA clones 2-8b and 1-la and translated in the reticulocyte lysate with [35S]methionine as a radioactive marker. (A) One-dimensional gel analysis of radioactive polypeptides synthesized in vitro with mRNAs from clone 2-8b (p78 protein) (lane 1), or clone 1-la (p78-related protein) (lane 3); 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 polypeptides synthesized in vitro with mRNAs from clone 2-8b (p78 protein) (panel 1) or 1-la (p78-related protein) (panel 2); panel 3, mixture of samples from panels 1 and 2. Only the region of the film showing p78 and/or p78-related proteins is shown. First dimension; Nonequilibrium pH 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 immunoprecipitates 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) immunoprecipitation. Lane 7, Same as lane 3, but with a shorter film exposure. Arrowheads point to the immunoprecipitated p78-related and p78 proteins.
1174
HORISBERGER ET AL.
J. VIROL.
---
NEPHGE
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acidic
b
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FIG. 3. In vivo expression of p78 and p78-related proteins. Monolayers of HEL cells were treated or not treated with 5,000 IU of rIFN-cXB ml for 20 h in the presence of [35S]methionine in the medium. Proteins from total cell extracts were separated by two-dimensional gel electrophoresis, and they were then transferred onto nitrocellulose. (a, c, and e) Pieces of nitrocellulose were assayed by an enzyme-linked immunosorbent assay with mouse polyclonal antibodies raised against the pure p78 protein (21). (b, d, and f) The same pieces were then exposed 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 and b 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. Arrowheads depict the position of the p78 protein. per
p78-related protein was not detected, even when the gel was overloaded (Fig. 3c), indicating that it is not accumulated in vivo in HEL cells. Structure of cDNAs encoding p78 or p78-related proteins. The composite sequence of the cDNA for p78 mRNA is shown in Fig. 4. Two partial cDNA clones, B1.1 and C'l, which we have described earlier (23), comprise upstream noncoding sequences and coding sequences for p78 mRNA. The B1.1 clone spans from nucleotides 1 to 720, and clone C'1 spans from nucleotides 615 to 1680. The cDNA clone 2-8b extends from the EcoRI site in position 137 to the 3' end (2788). The three cDNA clones were completely sequenced in both directions, and no single-nucleotide difference was
found in the regions of overlap. The upstream noncoding sequence contains an alternative exon (249 to 324) which was present in clone B1.1 but absent in clones C'1 and 2-8b. The absence of this exon created a Hindlll site at the junction AAG.. .CTT. The alternative exon was present in 5 of 10 cDNA clones analyzed. The mouse Mx cDNA also contains an optional exon resulting from an alternative splicing in its upstream noncoding region (24), but the presence of this exon seems to be less frequent than in the cDNA for p78. The cDNA clone 2-8b had a polyadenylation site (AATAAA) at position 2768. Nucleotide sequence analysis of cDNA clone 2-8b revealed an open reading frame of 662 residues with the
L_
Bl. 1
*
10
_-CrA GCa 50
30
70
90
AGAGCGGAGCCGCACTCCAGCACGCGCAGGGACCGCCACCGGACCC0GCCAGGCATCCCAGTGTCACGGTGGACAC GCCrCCC 130 110 170 _- 2-8b 150 190 CTGTCCCC :A TTGCCGCCCACCTGCTCACC_ _TCGTCTT 230 210 270 tiO 290
CCCTGAAC 310
ACGCCGCCC 330 350 370
cCC
CCACT
390
TATTTGAAGGAACGTATA
CACC
MetValValS.rGluValAspIleAlaLysAlaAspProAlaAlaAlaS.rHisP
410
450
430
470
490 CTCTATACTGAATGGAGATGCTACTGTGGCCCTGTTGCAGCCAGTATGAGGAGAAGGTGCGCCCCTG
roLouLeuLouAsnGlyAspAlaThfirVaLAlaGlnLysAsnProGlySerValAlaGluAsnAsnLeuCysS-rGlnTyrGluGluLysValArgProCy
510 530 550 570 590 CACACTCATCTCr_CG_N_
sIleAsprAuIleAspSerLouArgAlaLeuGlyValGluGInAspL*uAlaL*uProAlaIlvAlaVal1l GlyAspGlnSorS-r,GlyLysS-rSer 630 650 F-.- C'1 GCACTGTAGGTCCTCC_CAGAGACGACTACG
610 GTTG
670
690
C
VaLouGluAlaL.uSerGlyValAlaLeuProArgGlyS.rGlyIleValThrArgCysProLeuValL.uLyszauLysLysLeuValAsnGluAspL 710
B1.1 ..-
730
750
770
790
AGTGGAGAGCCAAGGTCAACC AACAATAAAGCCATCGCCGG ysTrpArgGlyLysValSerTyrGlnA,spTyrGlull Glull-SerAspAlaS-rGluValGluLysGlulleAsnLysAl&GlnAsnAl&Il-AlaG1 810
830 850 AAT~~~~~~~CTCcc5%ArACAGCTcCCCGAGATGTC
_
870
890
yGluGlyM.tGlyIleSerHisGluLeuIleThrLuGluIleSorSerArgAspValProAspLeuThrL.uIl.ASpL.uProGlyIleThrArgvaI 910
930
950
970
990
GCTGTGGGCATCAGCCTG
CCCA
AlaValGlyAsnGlnProAlaAspIleGlyTyrLysIleLysThrL.uIleLysLysTyrIl*GlnArgGlnGluThrIleSrL.uValValValProS 1010
1030 1050 1070 1090 _ _ _ _ _ _CGAGTCCGC~GCCGGG~GCCCAGGGCAGCCATcCGAATCFrGACGAAGCCTGATCTGGT
*rAsnValAspIleMaThrThrGluAlaLouSerM?tAlaGlnGluValAspProGluGlyAspArgThrIllGlyIl.L.uThrLysProAspL.uVa 1110
1130
1150
1170
1190
lAspLysGlyThrGluAspLysValValAspValValArgAsnL*uValPheHisL.uLysLysGlyTyrM.tIl.ValIaysCysArgGlyGlnGlnGlu 1210
1230
1250
1270
1290
ATCCAGGACChGCCAGMCCTGTCGACCGAAAAAAC71YAACACAArCGGTYCGAGGAAGCA
IleGlnAspGlnLuSerLeuSerGluAlaLeuGlnArgGluLysIlPhePh.GluAsnlHisProTyrPheArgAspLuL.uGluGluGlyLysAlaT 1310
1330
1350
1370
1390
hrValProCysLuAlaGluLysLeuThrS.rGluL uIllThrHisIleCysLysSorLuProL.uLuGluAsnGln1leLysGluThrHisG1nAr 1410
1430
_
A
=
1450 C
A
1470 G
1490 A ~~~~~~~~~~~~~~~~~~~CAGA
G
gIleThrGluGluLIuGlnLysTyrGlyValAspIllProGluAspGluAsnGluyqsM.tPh.PheLeuIllAspLysIllMnAlaPhAsnGlnAsp 1510
1530
_
A
~
TPGGG
1550 ACTcG
C
1570 cA
1590
CLAJG
ACAATAAT;
IloThrAlaLuMstGlnGlyGluGluThrValGlyGluGluAspIl.ArgL.uPh.ThrArgL.uArgHisGluPheHisLIsTrpSerThrIleIleG 1610
1630
1650
luAsnAsnPh GlnGluGlyHisLysIl-LouS-rArgLysIl GlnLysPh 1710 1730 1750
1670
C'1
-f
1690
GluAsnGlnTyrArgGlyArgGluLsuProGlyPh ValAsnTyrAr 1770
1790
gThrPheGluThrIleValLysGlnGlnlleLysAlaLeuGluGluProAlaValAspMstL uHisThrValThrAspM.tValArgLzuAlaPhbThr 1810
1830
1850
1870
1890
AspValSorIleLSysAsnPh GluGluPh-PheAsnLouHisArgThrAlaLysS*rLysIl GluAspll*ArgAlaGluGInGluArgGluGlyGlulL 1930 1910 1950 1970 1990
ysduIrlArgLauHisPheGlnM.tGluGlnlleValTyrrysGlnAspGlnValTyrArgGlyAlaL.uGlnLysVa1ArgGluLysGluL.uGluG1 2010
2030
2050
2070
2090
uGluLydsLysLsLysSerTrpAspPhoGlyAlaPh*GlnS-rS-rSerAlaThirAspS rS-rMotGluGlull-Ph GlnHisL*uMetAlaTyrHis 2110
2130
2150
2170
2190
GlnGluAlaS*rlysArgleSorS-rHislleProLeulalellGlnPh PhotLouGlnThrTyrGlyGllnGlLoeuGlnLysAlaMetLouGlnL 2210
2230
2250
2270
2290
*uLauGlnAspLysAspThrTyrS.rTrpL.uLauLysGluArgS.rAspThrSerAspLysArgLysPheL.uLysGluArgLauAlaArgL.uThrGl 2310
2330
2350
2370
2390
2430
2450
2470
2490
2530
2550
2570
2590
nAlaArgArgkrgLeuAlaGlnPh.ProGlyEnd 2410 2510 2610
2710
_
2630 2650 _ _ _ TACc~mCTA:TrrAT
2730
2750
2670 2690 TGCCcrCACAAA
2770
2-Sb -l
FIG. 4. The nucleotide sequence of p78 cDNA and the predicted amino acid sequence of p78 protein. The composite cDNA sequence shown was obtained from three clones, B1.1, C'l, and 2-8b. The presumed cap site nucleotide corresponds to the A* (residue 1). The overline indicates the position of the oligodeoxynucleotide used to screen a genomic library for isolation of the promoter sequence. Vertical arrows show 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 in close agreement with the apparent molecular weight of p78 (Mr 78,000) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the purified natural p78 protein (21). Moreover, the NH2-terminal sequence of the natural purified p78 protein, namely, VVXDIAKA (X was an amino acid not determined), corresponded exactly to the amino acid 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 open reading frame extending from nucleotides 2 to 2062. The precise nature of the NH2 terminus of p78-related protein remains uncertain. A tandem of AUG codons in position 44 is not likely to serve as initiation site since these codons are not in an ideal context for translational initiation (25). Furthermore, the corresponding 77-kDa protein of 673 amino acids (larger than the p78 protein) was not synthesized in the reticulocyte lysate (Fig. 2A). The AUG at position 164 could serve as an initiation codon. Since there is an open reading frame upstream of this codon, it is possible that translation is initiated further upstream, beyond the boundary of the clone. Nevertheless, because a G and a C residue occur at positions -3 and -2, respectively (with respect to the first base of the triplet) while G occurs at the +4 position, it is reasonable to assume that this AUG acts as the initiation codon, giving rise to a 72,500-kDa protein of 633 amino acids. This would be in close agreement with the Mr of the recombinant polypeptide synthesized in vitro and is slightly smaller than that of protein p78 (Fig. 2A). Moreover, the homology with the mouse Mx protein starts precisely at the methionine coded by this AUG in position 164. It has been proposed that AU-rich regions, particularly those with the motif AUUUA, play a role in controlling mRNA stability (5, 32). It is interesting that the sequence ATTTA is present once within the alternative exon (5' noncoding region) and once within the 3' nontranslated region of the p78 cDNA (positions 278 and 2469 on Fig. 4). The same motif is present in four positions in the p78-related cDNA, namely, three times in the coding region and once in the 3' nontranslated region (positions 1667, 1855, 2003, and 2556 in Fig. 5). These sequences may regulate gene expression at a posttranscriptional level by making the mRNA unstable. Protein sequence analysis. The p78 protein has a 34amino-acid segment at its NH2 terminus which was not found in p78-related protein or in Mu-Mxl protein (Fig. 6). p78 and p78-related protein had 63% homology in their common sequence, whereas they had 67 and 58% homology, respectively, with the Mu-Mxl protein. The calculated amino acid composition of p78 was in very good agreement with that experimentally determined previously with the natural p78 protein (21). p78 had a slight excess of acidic residues (94 Asp plus Glu versus 88 Arg plus Lys), whereas p78-related protein had almost equimolar amounts of acidic (80) and basic (81) residues, confirming that p78 was more acidic than p78-related protein (Fig. 2B). The Mx-related proteins had the highest homologies in their NH2-terminal halves (Fig. 6). p78 contained seven cysteines (positions 42, 52, 105, 280, 322, 336, and 533) which are highly conserved in the mouse Mxl protein. The Mu-Mxl protein contained four extra cysteines which were all located in the second half of the protein. p78-related protein had eight cysteines, but only four occupied positions common with the p78 protein (positions 52, 105, 280, and 533). Five differences in cysteine position occurred in the NH2-terminal half of p78-related protein (positions 42, 115,
J. VIROL.
218, 322, and 336), indicating that the folding of p78 may be quite 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 GTPbinding site (10). The three consensus sequence elements and corresponding sequences in p78 and p78-related proteins are shown in Fig. 7. The general features of these domains are discussed in detail below. Structural and functional analysis of p78 gene promoter. Primer extension analysis indicated that the p78 cDNA sequence (clone B1.1) is complete at its 5' end and that the presumed cap site nucleotide corresponds to the A* (residue at position 1) in Fig. 4 (data not shown). Transient transfection with the 1,800-bp human genomic fragment containing the putative p78 (MX1) gene promoter (Fig. 8A) fused to the cat reporter gene (pHHcat) showed that this fragment suffices to confer the IFN response to the cat reporter gene (Fig. 8B). To further localize the functional element responsible for this activity, we fused a limited region containing only 83 nucleotides upstream from the presumed cap site and the 5' -27 first nucleotides of the first exon (pNFcat) to the cat reporter gene. This short fragment was apparently as active as the entire genomic fragment described above (Fig. 8B). In vivo levels of p78 protein do not correlate with antiviral state. We examined whether p78 protein had an antiviral action by comparing its intracellular levels with the antiviral state at various times after IFN treatment. Human embryonic lung cells were treated with 100 IU of rIFN-aoB per ml. The medium containing IFN was removed from the cultures after 24 h of incubation, and the cultures were further incubated in medium lacking IFN. At the times indicated in Fig. 9, cell cultures were either extracted for measurement of p78 content or infected with virus. Virus yields were measured in medium harvested at 20 h after infection, and they were taken as a measure of the antiviral state at the time of infection. The p78 protein was rapidly induced and reached maximum levels 16 to 24 h after IFN treatment. The protein levels were very stable thereafter, and they decreased to one-third of the maximum level at day 7 after induction by IFN (Fig. 9). These late p78 levels represented full-size protein as detected on a Western blot (data not shown). The stable levels of p78 protein were in contrast with the antiviral states against influenza virus and vesicular stomatitis virus, which had reverted to preinduction levels within 4 to 7 days. We therefore did not observe a strict correlation between the antiviral state and p78 protein levels in human fibroblasts treated with IFN. DISCUSSION In the course of cloning cDNA encoding the p78 protein which we had purified and characterized earlier (21), we isolated a cDNA encoding a protein closely related to p78, sharing extensive amino acid sequence homology and antigenic determinants. Sequence analysis revealed that the two corresponding mRNAs cannot originate from the same gene but are derived from two separate but closely related genes. Evidence for the existence of two Mx-related genes in human cells has already been described by others (1). The presence of more than one Mx-related gene has been identified in mice (34) and rats (27). Moreover, two distinct Mx-related proteins exist in cattle (17) and in several other mammals (18). The unambiguous assignation of cDNA clone p78 to the
~CATG CTA G CT
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS
VOL. 64, 1990
1177
90 70 50 30 10 CTTcCAGCAAcAGCcAcCAcCATTcGGCACAGTGccAcCACAAACGATGTTTcCTccAAAcTGGCAGGGGGCAGAGAAoGACGCTGCITTcCTcGccAAG
PheGlnGlnGlnProProProPheGlyThrValProProGlnM.tHtPhProProAsnTrpGlnGlyAlaGluLysAspAlaAlaPhOLuAlaLys 110
150
130
170
190
GACTTCAACTTTCTCACTTTGAACAATCAGCCACCACCAGGAAACAGGAGCCAACCAAGGGCAA7GGGGCCCGAGAACAACCTGTACAGCCAGTACGAGC
AspPheAsnPheLouThrLeuAsnAsnGlnProProProGlyAsnArgS-rGlnProArgAlaMbtGlyProGluAsnAsnLouTyrS-rGlnTyrGluG 210
250
230
270
290
AGANGGTGCGCCCCTGCATTGACCTCATCGACTCCCTGCGGGCTCTGGGTGTGGAGCAGGACCTGGCCCTGCCAGCCATCGCCGTCATCGGGGACCAGAG
lnLysValArgProCysIleAspLeuIleAspSerLuArgAlaLeuGlyValGGluGlnAspLIuAlaLuProAlaIleAlaValIleGlyAspGlnSe 310
330
CTCGGGCAGAGCTGCTGT
T
390 370 350 CCCAGAGGCAGCGGAATCGTAACCAGGTGTCCGCTGGTGCTGAAACT&AAAAAG
rSerGlyLysSerSerValL.uGluAlaLeuSerGlyValAlaLeuProArgGlyS@rGlyIleValThrArgCysProLeuValLuLysLuLysLys 490 470 450 430 410 CAGCCCTGTGG=GCATGGGCCGGAAGGATCAGCTACCGGAACACCGAGCTAGAGCTTCAGGACCCTGGCCAGGTGGAGAAAGAGGDATAAAAAGCCCAGA GlnProCysGluAlaTrpAlaGlyArgIleSerTyrArgAsnThrGluL uGluIAuGlnAspProGlyGlnValGluLysGluIleHisLysAlaGlnA 590 570 550 530 510 ACGTCATGGCCGGGAATGGCCGGGGCATCAGCCAGAGCCATCAGCCTGGAGATCACCTCCCCTGAGGTTCCAGACCTGACCATCATTGACCTTCCCGG snValM.tAlaGlyAsnGlyArgGlyIleSerHisGluLouIleSerLouGluIleThrSerProGluValProAspLeuThrIleIleAspLeuProGl 670 690 650 630 610 TCAAGAAGTACATCCAAGACGCGATCAACTTG CATCACCAGGGTGGCTGTGGACAACCAGCCCCGAGACATCIGGACTC GATCAAGGCCTC
yIleThrArgValAlaVaLAspAsnGlnProArgAspIllGlyleuGlnIlLysAlaLeuIleLysLysTyrIleGlnArgGlnGlnThrIleAsnLeu G
770 790 750 730 710 CTTG TTCDIAACCGTGGACATTCCACCACGGAGGCGCTGAGCATGGCCCATGAGGTGGAcCCCGAAGGGGACAGGACCATCGGTATCCTGACCA
ValValValProCysAsnValAspIleAlaThrThrGluAlaLeuSerMetAlaHisGluValAspProGluGlyAspArgThrIleGlyIleLeuThrL 810
830
850
870
890
AACCAGATCTAATGGACAGGGGCACDCAGAAAAGCGTC.ATGAATGTGGTGCGGAACCTCACGTACCCCCTCAAGAAGGGCTACATGATTGTGAAGTGCCG
ysProAspLeuMetAspArgGlyThrGluLysSerValM.tA,snValValArgAsnLouThrTyrProLouLysLysGlyTyrMOtIleValLysCysAr 910
930
950
970
990
GGGCCAGCAGGAGATCACAAACACTGAGCTTGGCAGAGWCAACCAAGAAAGAAA ACATTCTTTCAACACATCCATATTCAGAGTTCTCCTGGAG
gGlyGlnGlnGluIleThrAsnArgLeuSerLeuAlaGluAlaThrsLsysGluIleThrPhePheGlnThrHisProTyrPh*ArgValLuLuGlu 1010
1030
1050
1070
1090
GAGGTCAGCCACGGTTCCCCGAcCTGCAGAAAGACTTACCACTGAACTCATCATGCATATCCAAAAATCGCTCCCTGAAGAAAAGG
GluGlySerAlaThrValProArgLeuAlaGluArgLeuThrThrGluLeuIleMbtHisIllGlnLysSerLeuProLeuLeuGluGlyGlnIl-ArgG 1110
1130
1150
1170
1190
AGAGCCACCAGAAGGCGACCGAGGAGCTGCGGCGTTGCGGGGCTGACATCCCCAGCCAGGAGGCCGA=AsAGAGTTCTTTCAATGAGAAACAAGAT
luS-rHisGlnLysAlaThrGluGluLeuArgArgCysGlyAlaAspIleProS-rGlnGluAlaAspLysMbtPh*Ph-LouIllGluLysIleLysMe 1210
1230
1250
1270
1290
GTMrAATCAGGACATMAAAAGAT=G tPheAsnGlnAspIleGluLysL.uValGluGlyGluGluValValArgGluAsnGluThrArgLOuTyrAsnLysIleArgGluAspPhOLysAsnTrp 1390 1370 1350 1330 1310 GTAGGCATACTTGCAACTAATACCCAAAAAGTTAAAAATATTATCCAGAAGTAAAAG ACAGTATCGAGGCAAGGAGCTTCTGGGAT ValGlyIleL.uAlaThrAsnThrGlnLysValLysAsnI1lOIlHisGluGluValGluLysTyrGluLysGlnTyrArgGlyLysGluLOuLuGlyP 1410
1430
1450
1470
1490
TGTCAACTACAAGACATTAGATCATCGTGCATCAGTACATCCAGCAGcGGTGGAGCCCGCCC'TTAGCATGCTCCAGAAAGCCATGGAAATTATCCA h.ValAsnTyrLysThrPh.GluIllIleValHisGlnTyrIl@GlnGlnLOuValGluProAlaL.uSerMetLuGlnLysAlaMetGluIleIleGl 1510
1530
1550
1570
1590
GTC A GAGCA GCAAGCTTTCATTAACGTGGCCAAAAAACATGGCGAATTCAACCTAAC nGlnAlaPheIleAsnValAlaLysLysHisPheGlyGluPhePh AsnL uAsnGlnThrValGlnSerThrIl GluAspIleLysValLysHisThr
1670 1690 1650 1630 1610 GCAAAGGCAG AAAACATGATCCAACTTCAGTTCGA GACGTGTTTGCAAGATCAGATTTACAGTGTTGTTCTGAAGAAAGTCCGAGAAG
AlaLysAlaGluAsnMetIleGlnLeuGlnPheArgMetGluGlnM4etValPheCysGlnAspGlnI leTyrSerValValL uLysLysValArgGluG 1710
1730
1750
1770
1790
AGATrTTTAACCCrTGGGGACGCCTrCACAGAATATGAAGTTGAACTCTCATTTTCCCAGTAATGAGTCTTCGGTTCCTCC rACTGAAATAGGCAT luIloPheAsnProLeuGlyThrProSerGlnAsnMetLysLeuAsnSerHisPhOProSorAsnGluSerSerValSerSerPheThrGluIleGlyIl 1810
1830
1850
1870
1890
CCACCrGAATGCCTACTTCTGGAAACCAGCAAACGTCTCGCCAACCAGATCCCATTATAATTCAGTATTTATGCrCCGAGAGAATGGTGACTCCTTG
eHisLeuAsnAlaTyrPheLeuGluThrSerLysArgLeuAlaAsnGlnIleProPheIleIleGlnTyrPheMetLeuArgGluAsnGlyAspSerLou 1910
1930
1950
1970
1990
CAGAAAGCCATGATGCAGATACTACAGGAAAAAAATCGCTATCCTGGCTGC1CTCAAGAGCAGAGTGAGACCGCTACCAAGAGAAAATCCTTAAGGAGA
GlnLysAlaMetMetGlnIleLeuGlnGluLysAsnArgTyrSerTrpLeuLuGlnGluGlnSerGluThrAlaThrLysArgArgIleLeuLysGluA 2010
2030
2050
2070
2090
GAATTTACCGGCTCACTCAGGCGCGACACGCACTCTGTCAATTCTCCAGCAAAGAGATCCACTGAAGGGCGGCGATGCCTGTrGTI TCITGTGCGT rgIleTyrArgLeuThrGlnAlaArgHisAlaLuCysGlnPheSerSerLysGluIleHisEnd 2110
2130
ACTCCArTTCTAAAGGGGAGTCGGTGCAGGATGCCGCTTCTG 2210
2230
2310
2330
2190 2150 2170 GGGGCCAAACTCTTCTGTCACTATCAGTGTCCATCTCTACTGTACTCCCTCAG 2250 2270 2290 CTACA7GAWCTCCATCTGGGTCC 2370 2390 2350
CGTAGCACACAGTTACAGTGTCCTAAGATACTGCTATCATTCTTCGCTAATTTGTATTTGTATTCCCTTCCCCCTACAAGATTATGAGACCCCAGAGGGG 2410
2430
2450
2470
2490
AACGAATGAG CAGCACCrGCAGCAC _ACTCACTGAAOG GAAGGTCTGGGTCAAATrCCTCACTGTGTAGTC
2510
2530
2550
2570 ---
2590
TGCTGTGTAAGTGATGGAGATACCTGAGGCTATTGCTCAAGCCCAGGCCTTGGACATTTAGTGACTGTTAGCCGGTCCCTTTCAGATCCAGTGGCCATGC 2610
2630
2650
2670
2690
CCCTGCTTCCCATGGTTCACTGTCATTGTGTTTCCCAGCCTCTCCACTCCCCCGCCAGAAAGGAGCcTGAGTGATTCTCTTrCTTCrGTTTCCCTGA 2710
2730
TTATGATGAGCrTCCArTGTCrCTGTrAAGTCTrTGAAGAGG FIG. 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.
1178
HORISBERGER ET AL.
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MVSEVDIAKADPAAASHPLGDATVAQKPGSVAENN1CSQYEEKVRPCIDIDSLRIA1VEQDLAL MGP Y Q MD5V-RH-T
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DMMHTVTE*VRLAFTDVSIKNFEEFFNHRTAKSKIEDIRAEQEREGEK
A
526
551 516
625 592
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EEEKKKKSWD. .FGAFQSSSATDSSME EIFQHUM4YHQEASKRISSHIPLIIQFFMRQYGQQLQKAMLQLLQDKDT NPLGTPSQNMKNSH-P-NESSV-FT. FT-GI-N-FL-T-LAQ-F-Y-REN-DS !4-I-E-NR -K-T-ALIN. PAT-NN-QFPQKG Lr-K-Y--CRN-GRQ--KY-I-K-F-EEIE-M ----TSK
626
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662
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HA-C-SSKEIH -QQ-E-AT-RI-IY C-F-E-Q-RE-K-R-L-DE--QK-K-SD
633
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470 435 436
RFQMEQIQQVYCQDQVYRGALQKVREKEL
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E-V-R-NET-YNKI-ED-KN-VG-LAT-T-KVKN-IHEEVE-Y-K..K-L--K-I-H-Y-00-V-L
-AQ--S-G-S-KK-NN-A-DDH-EY-EIDSPEVQStE
D-KA-S-I-KRV
S-QKAMEIIQQ--IN-AK-HGNQ VT-mOTAKAR FQQ N-RR-K---QT-VKILSND
I-SVV-K-EIF 525
...
694
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 solid overlines. Sequence of Mu-Mxl was taken from reference 33.
cellular gene product p78 relies on several parameters such as molecular weight, pl, amino acid composition, and identity of the NH2-terminal amino acid sequence determined chemically on the purified protein with that predicted from the cDNA clone. cDNA clones encoding the p78-related protein were estimated to be fivefold less abundant than
those encoding protein p78, reflecting most likely a corresponding lower steady state of transcripts. These genes are regulated at the transcriptional level, and a preliminary functional analysis of the p78 (MX1) gene promoter showed that a very short sequence suffices to confer inducibility by IFN-ox. Other constructions are already available that will
GTP-binding protein G
consensus
HuMxl (p78) HuMx2 MuMxl
Fish Mx ras
EF-Tu GscL SRP54 SRPreceptor
77 43 43
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G K S G K S G K S G K S a g gv G K S h vd h G KT G ages G K S G I qgs G K S CG vn gv G K S
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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 in comparison 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.
VOL. 64, 1990
CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS
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J. VIROL.
HORISBERGER ET AL.
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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 IU of IFN-a-B per ml. One day later, the culture medium was removed, cells were washed, and fresh medium lacking IFN was added for further incubation 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 are given. Virus yields are expressed as the percentage of yields in control cultures not treated with IFN. The levels of p78 in parallel cultures were determined on Western blots as described elsewhere (21).
permit a more detailed dissection of the functional domains of this promoter (J. Dellis and J. Content, manuscript in preparation). The p78-related gene may also be regulated at a posttranscriptional level in vivo since we were unable to detect p78-related protein in diploid fibroblasts treated with IFN, using a very sensitive immunoassay (21). Therefore, we assume that it is the p78 which is the functional protein in this 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) correlating most likely to the rate of decay of Mx protein, which is a very stable protein with intrinsic anti-influenza virus activity (20, 33). This has not been found in human diploid cells. Moreover, there was no correlation between protein p78 levels and the antiviral state induced by IFN-a. Thus, 30 to 50% of maximum levels of p78 were still present at a time when cells had regained full susceptibility to both viruses tested. These observations suggest that the p78 protein is not involved in a specific antiviral mechanism against influenza virus and that its function is more general than just participating in the antiviral activity of IFN. p78/Mu-Mx proteins have their cysteines conserved at their NH2 termini, which may indicate that a functional domain is in this region. We discovered three consensus sequence elements defining a putative GTP-binding domain precisely in this region of strongest sequence homology. The exact sequence of the three elements is conserved in the family of p78/Mu-Mxl (Fig. 7) as well as in a fish gene with homology to MX (35). There is a mismatch in the third element with the strict consensus sequence, which is TKxD instead of NKxD (10) and which is found, for instance, in the elongation factor, in the ras protein or in G-binding proteins (Fig. 7). However, the N->T deviation has been very recently found in two proteins involved in the vectorial transport of proteins, namely, the 54-kDa protein of the signal recognition particle (SRP54) and the a-subunit of the SRP receptor in the endoplasmic membrane which has been
shown to bind GTP (4, 8, 30). We therefore infer that p78 and its homologs belong to a distinct subgroup of guanosine nucleotide-binding protein (4). The N--T deviation in the third consensus element, which is the guanine-binding domain (10), may alter the affinity and/or the strict specificity of this domain for GTP and may indicate a low affinity of Mx homologs for nucleotide binding which might be compensated by the abundance of the protein in IFN-induced cells (21). Alternatively, it may indicate a specificity for bases related to guanosine such as xanthosine or inosine. Base aberrations in nucleic acids may be more common than previously thought. For instance, a double-stranded-RNA-unwinding activity is capable of converting adenosine residues to inosine residues, which have chemical properties similar to those of guanosine residues
(3).
Guanylate-binding proteins of Mrs 67,000 and 56,000 are induced by IFN in human cells, and they have been isolated by GMP-agarose affinity chromatography. These proteins, however, do not seem to correspond to p78 or to Mx homologs because they differ in Mr and because they are also induced in mouse cells defective in MX gene expression (7). Several IFN-induced proteins and IFN-regulated enzymes react with either nucleotides or nucleic acids (9). Other proteins with the GxxxxGS/T domain react with nucleic acids, namely, unwinding proteins or viral replication proteins (13). We are currently testing the hypothesis that protein p78 binds a nucleotide or a nucleic acid. We plan to express p78 or a truncated form of it in E. coli to use in in vitro studies. A major task may be the expression of an active form of protein p78 since this protein seems to have a very low solubility. ACKNOWLEDGMENTS We 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
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CLONING AND FUNCTIONAL ANALYSIS OF HUMAN Mx PROTEINS
from the Fund for Medical Scientific Research (Belgium) and by the Cancer Research Foundation of the Belgian General Savings and Retirement Fund. M.G.W. is research assistant of the National Fund for Scientific Research (Belgium). J.D. is supported by a grant from the Institute for encouragement of Scientific Research in Industry and Agriculture (Belgium). LITERATURE CITED 1. Aebi, M., C. E. Samuel, H. Arnheiter, 0. Haller, and C. Weissmann. 1987. Isolation and expression of cDNA clones encoding human Mx and Mx-related protein. J. Interferon Res. 7:719. 2. Arnheiter, H., and 0. Haller. 1983. Mx gene control of interferon action: different kinetics of the antiviral state against influenza virus and vesicular stomatitis virus. J. Virol. 47: 626-630. 3. Bass, B. L., and H. Weintraub. 1988. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 55:1089-1098. 4. Bernstein, H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, and P. Walter. 1989. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition 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 nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:1670-1674. 6. Chang, J. Y., H. Herbst, R. Aebersold, and D. G. Braun. 1983. A new isotype sequence (VK27) of the variable region of K-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Biochem. J. 211:173-180. 7. Cheng, Y.-S. E., R. J. Colonno, and F. H. Yin. 1983. Interferon induction of fibroblast proteins with guanylate binding activity. J. Biol. Chem. 258:7746-7750. 8. Connolly, T., and R. Gilmore. 1989. The signal recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide. Cell 57:599-610. 9. Content, J. 1986. Biochemical aspects of interferon action, p. 163-189. In R. Perez Bercoff (ed.), The molecular basis of viral replication. Plenum Publishing Corp., New York. 10. Dever, T. E., M. J. Glynias, and W. C. Merrick. 1987. GTPbinding domain: three consensus sequence elements with distinct spacing. Proc. Natl. Acad. Sci. USA 84:1814-1818. 11. Gilman, A. G. 1987. G proteins: transducers of receptor-generated 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, the human 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 triphosphatebinding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination. FEBS Lett. 235:16-24. 14. Gorman, C. 1985. High efficiency gene transfer into mammalian cells, p. 143-190. In D. M. Glover (ed.), DNA cloning, a practical approach, vol. 2. IRL Press Limited, Oxford, England. 15. Graham, F. L., and A. J. Van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus S DNA. Virology 52:456-467. 16. Herbomel, P., B. Bourachot, and M. Yaniv. 1984. Two distinct enhancers with different cell species specificities coexist in the regulatory region of polyoma. Cell 39:653-662. 17. Horisberger, M. A. 1988. The action of recombinant bovine interferons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells. Virology 162:181-186.
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18. Horisberger, M. A. 1988. Homologs of the IFN-induced mouse Mx protein in 14 animal species. J. Interferon Res. 8:S102. 19. Horisberger, M. A., and K. de Staritzky. 1985. Sensitivity of influenza 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 and stability of the Mx protein in different tissues of mice, in response 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 induced human 78 kD protein: purification and homologies with the mouse Mx protein, production of monoclonal antibodies, and potentiation effect of IFN-y. J. Interferon Res. 7:331-343. 22. Horisberger, M. A., P. Staeheli, and 0. Hailer. 1983. Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc. Natl. Acad. Sci. USA 80:1910-1914. 23. Horisberger, M. A., M. Wathelet, J. Szpirer, C. Szpirer, Q. Islam, G. Levan, G. Huez, and J. Content. 1988. cDNA cloning and assignment to chromosome 21 of IFI-78K gene, the human equivalent 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 characterization of its interferon- and virus-inducible promoter. Mol. Cell. Biol. 8:3065-3079. 25. Kozak, M. 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196:947-950. 26. Laemmli, U. K. 1970. Cleavage and structural proteins during the 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 encodes cytoplasmic and nuclear proteins in rat cells. J. Virol. 62:2386-2393. 28. Nielsen, P. J., G. K. McMaster, and H. Trachsel. 1985. Cloning of eukaryotic protein synthesis initiation factor genes: isolation and 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 element of 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 signalrecognition particle, docking protein and two E. coli proteins with 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 a monoclonal antibody directed against an interferon-induced 56,000-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 from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667. 33. Staeheli, P., 0. Haller, W. Boll, J. Lindenmann, and C. Weissmann. 1986. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44:147-158. 34. Staeheli, P., and J. G. Sutcliffe. 1988. Identification of a second interferon-regulated murine Mx gene. Mol. Cell. Biol. 8:45244528. 35. Staeheli, P., Y.-X. Yu, R. Grob, and 0. Haller. 1989. A double-stranded RNA-inducible fish gene homologous to the murine 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-homologous protein in mononuclear cells from patients with systemic lupus erythematosus. Arthritis Rheum. 33:1-5.
