Proc. Nati. Acad. Sci. USAVol. 86, pp. 7711-7715, October 1989Biochemistry

v-maf, a viral oncogene that encodes a "leucine zipper" motif(avian retrovirus/transformation/DNA binding protein)

MAKOTO NISHIZAWA*, KOHSUKE KATAOKA*, NAOAKI GOTOt, KOSAKU T. FuJIWARA*,AND SADAAKI KAWAI*t*Department of Tumor Virus Research, The Institute of Medical Science, University of Tokyo, 4-61, Shirokanedai, Minato-ku, Tokyo 108; and tDepartmentof Veterinary Pathology, Faculty of Agriculture, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Communicated by Hidesaburo Hanafusa, July 5, 1989 (received for review May 31, 1989)

ABSTRACT We have molecularly cloned the provirus ofthe avian musculoaponeurotic fibrosarcoma virus AS42.Nucleotide sequence analysis of a biologically active clone ofAS42 showed that this virus encodes a viral oncogene, maf. Thededuced amino acid sequence of the v-maf gene productcontains a "leucine zipper" motif similar to that found in anumber ofDNA binding proteins, including the gene productsof the fos, jun, and myc oncogenes. However, unlike theseoncogenes, the cellular maf gene was not transcriptionallyactivated by growth stimulation of cultured cells.

0 1 2 3 4 5kb

EcdSadNcolSOBamHI1BstEHb11 ~~~I IDral

BallPstl

EcoRI

I II I

To date, more than 40 oncogenes have been identified.Among these, the myc, myb, ski, jun, and fos genes aretermed "nuclear oncogenes" as they encode proteins that aretargeted to the cell nucleus (1). It has been shown recentlythat the v-jun oncogene is derived from a cellular geneencoding a major component of transcriptional trans-activator AP-1 (2-4). Interestingly, a major fraction of thecellular jun/AP-1 protein forms heterodimers with the c-fosprotooncogene product and the formation of this proteincomplex is essential for both jun/AP-1 and fos-encodedproteins to act as transcriptional regulators (5-10). In both ofthese proteins, periodic repeats, containing a leucine everyseven residues, form an a-helical structure believed to playa key role in protein dimerization (8, 9). This structure,termed the "leucine zipper" (11), has also been found inmyc-encoded proteins and in the products of four oncogene-related genes-junB (12), junD (13), fra-1 (14), and fosB(15)-as well as in the yeast GCN4 gene product (16), the ratenhancer binding protein (C/EBP) (17), and the cyclic AMP-responsive enhancer binding protein (CREB) (18).

Recently, we isolated a transforming retrovirus, AS42,from a naturally occurring musculoaponeurotic fibrosarcomaof a chicken. This virus induced transformation offibroblastsin culture and tumors that were pathologically indistinguish-able from the original tumor from which this virus wasderived. Unique pathological features of the tumors inducedby this virus suggested to us that the viral oncogene of AS42might be an unusual one. Indeed, an analysis of the structureof a molecular clone of the genome of this virus showed AS42to contain an oncogene whose deduced amino acid sequenceincludes leucine zipper-type repeats and unique repetitivestretches of glycine and histidine residues.

MATERIALS AND METHODSMolecular Cloning. The AS42 provirus was cloned from a

partial genomic library constructed from DNA ofAS42 trans-formed, nonvirus-producing cells. Using a gag-specific 1.35-kilobase (kb) BamHI fragment of a molecular clone of theRous sarcoma virus genome (19) as a probe, a 4.7-kb EcoRI

LTR Agag maf-IAsflv

FIG. 1. Restriction endonuclease map of the AS42 provirusclone. This clone contains 5'-flanking sequences (wavy line) and partof the AS42 provirus. Shaded bars represent the remaining gag andenv gene sequences; double-headed arrow indicates the transducedsequence found in the AS42 virus genome. The putative oncogene-encoding open frame is indicated by a solid bar. LTR, long terminalrepeat.

fragment derived from the transforming virus genome wasdetected by Southern blot analysis of DNA from the trans-formed cell clone (data not shown). To enrich for this frag-ment, an EcoRI digest of the DNA was size-fractionated bypreparative agarose gel electrophoresis. The excised 4.7-kbEcoRI fragment was ligated to EcoRI-cleaved and dephos-phorylated AgtlO DNA and was packaged into phage particlesin vitro. After sequential screening of the partial library withthe gag-specific probe, we obtained a recombinant phagecontaining the provirus-derived 4.7-kb fragment. The EcoRIfragment was subcloned into pUC-9 plasmid and was used forstructural analysis. Nucleotide sequences were determined bythe dideoxynucleotide sequencing method (20).§

Transfection Assay. Conditions for the growth and main-tenance of chicken fibroblast cells have been described (21).As described below, the cloned AS42 provirus genomicfragment seemed to contain all of the transduced sequencebut did not contain all of the virus genome. To assay thebiological activity of the isolated sequence, the viral se-quences missing from the insert-i.e., the 3' part of the envsequence and the 3' long terminal repeat-were supple-mented by ligation with a 1.5-kb EcoRI/BamHI fragmentexcised from a replication-competent retrovirus vector,pRV-2, which we have recently constructed from Roussarcoma virus DNA. The procedure for the construction ofthis vector will be published elsewhere. The ligation productwas cotransfected with DNA from a helper virus clone,

Abbreviation: PMA, phorbol 12-myristate 13-acetate.tTo whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M26769).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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7712 Biochemistry: Nishizawa et al. Proc. Natl. Acad. Sci. USA 86 (1989)

pYAV-e (22), by the Polybrene/dimethyl sulfoxide method guanidine thiocyanate method (25). Stimulation of serum-(23). starved resting cells by phorbol 12-myristate 13-acetate

Blot Hybridization Experiments. pSae-1 plasmid, from (PMA) was performed as described (26). The 1.0-kb chickenwhich the 0.7-kb DNA fragment used as an AS42-specific v-fos-specific probe was excised from the NK24 virus ge-probe was excised, was constructed as follows: A 1.0-kb Bal nome as described (27).I/BamHI fragment containing only the coding sequence ofthe putative viral oncogene was subcloned into the polylinker RESULTSof pUC-9. Then, a highly G+C-rich portion found in thecoding sequence was deleted from this subclone by double Genomic Structure ofthe AS42 Virus. Physical mapping anddigestion with BssHII and BstEII, treatment with the Klenow partial sequence analysis of the molecularly cloned AS42fragment of DNA polymerase I, and self-ligation with T4 proviral fragment revealed that this fragment lacked the 3'DNA ligase. For blot analyses of genomic DNAs, both part of the viral genome but contained all of the sequencestringent and relaxed conditions ofhybridization and washing believed to be transduced (Fig. 1). The biological activity ofwere used as described (24). Total RNAs to be analyzed by this cloned fragment was confirmed by recovery of trans-Northern blot hybridization were prepared by the CsCl/ forming virus from cells transfected with the cloned fragment

SaciI _c|afg_TT~~~~~~~~~~~~~~~~~~~~~~ATGGAGACAATAGPGATGIle.IleLysTyrValLeuAspArgGlnLysmhrAlaProLeumhrAspGlnGlyI leAlaAlaAlaMetSerSerAlaIleGlnPror euValMetAlaValValAsnArgGluArgMet

(1)BalI NcoI

GCAMTIGAACGGTGAGCGTCGC 1;AlaSe~rGluLeTuAlaMetSerGlySerAspLeuPromhrSerProTeuAlaMetGluTyrValAsnAspPheAspLeubletLysPheGluVal TysLysGluProValGlumhrAspArg

3

.23

BglI NotI PstI243

IleIleSerGlnCysGlyArgLeuIleAlaGlyGlySerLeuSerSerThrPraMetSerThrPraCysSerSerValProProSerProSerPheSerAlaProSerPraGlySerGly(50)

ACCE A 1 363ThrAspGlnLysmhrHiIseuLeup~r~r~etThrGlyTyr proGlnGlnLeuAsnProGluAlaT uGlyPheSerProGluAspAlaValGluAlaLeuI leAsnSerSer

(100)PvuII SacII NotI NaeI NaeI NotI

CACCACCGC3GG3CGCCTICGATGGCTATGC GGC GGCG _ 483HisHisProLeuPraGlyAlaPheAspGlyTyrAlaArgGlyGlnGlnLeuAlaAlaAlaAlaGlyGlySerValPraAlaGluGluMetGlySerAlaAlaAlaValValSerAlaVal

(150)SacII BglI BssHII BssHII

603IleAlaAlaAlaAlaAlaGlnGlyGlyAlaProHisTyrHisHisHisHisHisHisProHis~iisGlyGlyGlyGlyGlyGlyGlyGlyHisProHisGlyA]laAlaPraGlySerAla

.-.~~~~~~~~~~~~~~'~~~~~~~~~~------- (200)SacII

C:CGOC£CTTCG(:CICTCCC 723ProProSerSerAlaSerSerSerAlaAlaGlySerGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyAlaGlyGlyLeuHisHisProHisHisGlyGlyGlyGlyGlyGlyGlyGlyLeu

PvuII BstEII PvuII EcO0109CACTICGACGACCGCTIU1UCGIT W(250HisPheAspAspArgPheSerAspGluGlnLeuValmhrMetSerMetArgGluI~euAsnArgGlnLeuArgGlyVal SerLysGluGluValIleArgLeuLysGlnLysArgArgThr

(250)

843

PvuII PstI Bg1IITAAAGCAGGATCTOC 963

LeuLysAsnArgGlyTyrAlaGlnSerCysArgPheLysArgValGlnGlnArgHisValLeuGluSerGluLysAsnGlnLeuLeuGlnGlnValGluHisLoeuLysGlnGluI leSer(300) * - *----*----------__ *--------------

BamHIAGGCTG= 1083ArgLeuValArgGluArgAspAlaTyrLysGluLysTyrGluLysl~euValSerAsnGlyPheArgGluAsnGlySerSerSerAspAsnProSerSerProGluPhePheMetT'yrPro

(350)Bsu36I

1203ArgG1uSerSerThrThrVaNMetTRR

(369)

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~ ITAAAZ1XAACGAA JGA~GAA _TTAA~CI 1563

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T~mAATTAAG=WGPCATT~rCKAA~rCAGATAGCATGGASTA_ m:TrA 1803ClaI

ATAATCTITA A = _ P_ 1923SphI

AMGCATATXGGCTACATTTCATATAA 2043HpaI

TAAA 2163

FIG. 2. Nucleotide sequence of the v-maf gene of the AS42 virus. A comparison of the AS42 sequence with sequences of other avianretroviruses (28, 29) revealed that the whole of the pol gene sequence and parts of gag and env genes were replaced by a 2-kb AS42-specificsequence. The numbering of the nucleotides (right end of each line) or amino acids (in parentheses) begins from the putative 5' recombinationsite. The leucine zipper structure is indicated by asterisks and dotted lines. Amino acid repeats of glycine and histidine are also emphasizedby dotted lines. Two ATTTA sequences, possibly involved in the selective degradation of the mRNA (30), and adenine clusters found in the3' noncoding sequence are underlined.

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Proc. NatL. Acad. Sci. USA 86 (1989) 7713

ligated to a DNA fragment containing essential viral se-quences missing from the clone and helper virus DNA. Therecovered virus was indistinguishable from AS42 virus in itstransforming activity, indicating that the cloned sequence,which was flanked by partial gag and env sequences, wasresponsible for the transforming activity of AS42 virus.Therefore, this region was subjected to nucleotide sequenc-ing.

Nucleotide Sequence of the Viral Oncogene of AS42. Asshown in Fig. 2, the gag-encoding region present in thedefective AS42 virus is fused to an open reading frame of 1.1kb, which is consistent with our immunoprecipitation of agag fusion protein of 100 kDa from lysates ofAS42-infectedcells (data not shown). A computer search failed to findsignificant homology between the transduced putative onco-gene sequence and any known gene. This gene was namedmaf, after musculoaponeurotic fibrosarcoma.The maf coding sequence was followed by -0.9 kb of

A+T-rich sequence, including two adenine clusters consist-ing of 24 and 30 adenine residues. A putative mRNA desta-bilizing signal, ATTTA (30), possibly derived from the 3'noncoding region of the c-maftranscript, was found twice inthe 3' noncoding region of the v-mafgene. In contrast to thenoncoding sequence, the coding sequence of the maf genewas characterized by its high G+C content, particularly in aregion of the gene encoding long repeats of glycine andhistidine residues.The deduced amino acid sequence of the mafgene product

contains, in its carboxyl-terminal region, a periodic repeat offour leucine residues similar to that proposed to form aleucine zipper structure (11). A prediction of protein confor-mation by the system ofChou and Fasman (31) indicated thatthis region of the v-maf-encoded protein was highly helixpermissive (data not shown). A domain rich in basic aminoacid residues that precedes the periodic leucine repeats in anumber of leucine zipper-containing proteins has been im-plicated in DNA binding (9, 11). The deduced amino acidsequence of the corresponding basic region of the v-mafgeneproduct shares 20-30% homology with this region of theDNA binding proteins shown in Fig. 3, with the exception ofthe yeast GCN4 protein and members ofthe myc family. Thissuggests that the maf gene product may be a DNA bindingprotein.

v-mafv-fosfra-1fosBv-iun

-un-_nD

N -rnZcN-mycL-mncC/EBPCREBGCN4

Blot Analysis of the c-maf Gene and Its Transcript. Toconfirm that the v-mafgene was derived from cellular DNAsequences, DNAs prepared from uninfected chicken cellsand human peripheral blood cells were digested with restric-tion enzymes and subjected to Southern blot analysis. Toavoid nonspecific hybridization, a 0.7-kb v-maf-specific frag-ment, lacking the A+T-rich 3' noncoding sequences and theparticularly G+C-rich portion of the coding sequence, wasexcised from the pSae-1 plasmid and used as a hybridizationprobe. As shown in Fig. 4, cellular sequences hybridizing tov-maf (potential c-maf sequences) were detected in both thechicken and human genomes, even under stringent hybrid-ization conditions. Low stringent hybridization allowed thedetection of possible c-maf-related genes.

It is well known that expression of the c-fos, c-jun, andc-myc protooncogenes are rapidly induced by growth stim-ulation of quiescent cells (1, 37, 38). Similar transcriptionalactivation has also been observed in the case of other relatedgenes encoding leucine zipper motifs-namely, junB (12),fra-J (14), and fosB (15). This prompted us to examineexpression of the c-maf gene in growth-stimulated cells.Contrary to our expectation, the level of the 3.2-kb c-maftranscript was the same after phorbol ester treatment ofcultured cells as it was in serum-starved resting cells (Fig. 5Left). Treatment of cells with the phorbol tster PMA did,however, induce transcription of the c-fos gene as shown inFig. 5 (Right). Transcription from the c-maf gene was alsounaffected by serum stimulation or treatment of cells withdibutyryl cyclic AMP (data not shown).

DISCUSSIONWe have identified a viral oncogene and have named it maf.The deduced amino acid sequence of the transduced v-mafgene contains a leucine zipper motif in its carboxyl terminus.The region of the maf-encoded protein sequence adjacent tothe periodic leucine repeats was weakly homologous to thecorresponding DNA binding regions of other leucine zipper-containing proteins, suggesting that the maf-encoded proteinmight be a transcriptional trans-activator. It should be notedthat a few amino acid residues are strictly conserved amongthe proteins containing leucine zippers. For example, asshown in Fig. 3, the amino acid residues located 5 residuesamino-terminal to the beginning of the leucine repeats are,

leucinezipper

-30 -20 -10

IR'LKQKRRi NRGYA 2SC RFKRVQQRHV| LEEEERERIIR NKM RNRRRELTDT LEEEERRR RtR ER L RNRRKELTDF LEEEEKRRRJJR ERN A RNR RELTDR LQERIKRAERKR MRNRjIA1ASRKS RKRKLERIAR LQERII VERRR R RLA K KRKLERIAR LQERI KAEKR R A RKLERISR LALRDEIPEVA NNEKAPKVVI LKRATEYVLS LTLRDHVPELV KNEKAAKVVI LKRATEYVJS LALRDQVPTLA SCSKAPKVVI LSRALEYLQA L

dRRERNNIAV RII§JDKAKQR NVETQ&JVLE LEAA RIREVEL MKNREAAREH RRKKEYVKC LESSDPAALKR ARINTEAARRSR IARKLJRMKQ L

Homologywith v-maf

7/306/307/307/309/309/302/303/302/306/309/304/30

FIG. 3. Comparison of the amino acid sequence of the putative DNA binding domain of the v-maf-encoded protein with those of proteinscontaining a leucine zipper motif. Amino acids that are identical in the v-maf-encoded protein and other proteins are boxed. The standardsingle-letter amino acid code is used. The amino acid sequences are numbered from the first leucine residue of the leucine zipper structure ofeach protein. The deduced amino acid sequences of the DNA-binding proteins are cited from the following refs: FBJ murine osteosarcoma virusv-fos (32),fra-1 (14),fosB (15), avian sarcoma virus 17 v-jun (33), junB (12), junD (13), MC29 avian myelocytomatosis virus v-myc (34), humanN-myc (35), human L-myc (36), C/EBP (17), CREB (18), GCN4 (16).

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7714 Biochemistry: Nishizawa et al.

A B1 23456 1 23456

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FIG. 4. Southern blot analyses ofhuman and chicken DNAs withthe v-mafprobe. High molecular weight DNAs prepared from humanperipheral blood (lanes 1-3) and uninfected chicken fibroblast cells(lanes 4-6) were digested with EcoRI (lanes 1 and 4), BamHI (lanes2 and 5), or HindIll (lanes 3 and 6). Ten micrograms of DNA fromeach digest was separated by agarose gel electrophoresis and trans-ferred to nitrocellulose. 32P-labeled HindIll fragments of A DNAwere used as molecular weight markers. Stringent (A) or relaxed (B)conditions (24) for hybridization and washing were used.

without exception, glutamine or glutamate residues. Analanine residue is present 14 residues amino-terminal to theleucine zipper in all of the proteins shown, except for the mycfamily members (Fig. 3). These conserved residues may playan important role in protein dimerization or in recognition ofspecific DNA sequences.An additional feature of interest in the deduced sequence

of the v-maf-encoded protein is a region that contains a repeatof six histidines and three tracts of glycine residues. It isnoteworthy that a similar seven-residue glycine repeat isfound in another leucine zipper-containing protein, C/EBP(17).Using a maf-specific DNA fragment to probe Southern

blots of chicken and human genomic DNA, we showed thatthe cellular maf gene is conserved across species. Further-more, under less stringent conditions ofhybridization, South-ern blot analysis of the genomic DNA suggested the presenceof maf-related genes in both the chicken and human genomes.A number of genes related to the leucine zipper-encodingoncogenes fos, jun, and myc have been reported (12-15, 35,36). We have recently isolated cDNA clones of two maf-

M 0 3066120' M 306012d

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probe: v-maf probe: v-fos

FIG. 5. Expression of the c-maf gene is not activated bystimulation of fibroblast cells with PMA. Total RNA (20 pg per lane)isolated from serum-starved chicken fibroblast cells at the indicatedtimes (min) after addition ofPMA were separated electrophoreticallyon 1% agarose/formamide gels followed by blotting to nitrocellulosefilters. (Left) The filter was hybridized to a 32P-labeled v-maf-specificDNA fragment. (Right) The filter was hybridized to a chickenv-fos-specific probe (27). 32P-labeled fragments from a HindIII digestof A phage DNA were used as size markers (lanes M).

related genes from a fibroblast cell cDNA library (unpub-lished data). The maf product may form heterodimers withthe protein products of these maf-related genes or with otherproteins with leucine zipper motifs, prompting specific DNAbinding, as has been shown for the fos- and jun-encodedproteins.

It is known that transcription of the c-fos, c-jun, and c-mycprotooncogenes is rapidly elevated during stimulation of cellgrowth (1, 37, 38). However, at least in fibroblasts, growthstimulation did not transcriptionally activate the c-mafgene.A similar result has been reported with the recently identifiedjun-related gene, junD (13). It is possible that translation ofthe c-mafmRNA is induced or that maf-encoded protein isposttranslationally modified in response to growth stimula-tion. Preparation ofantibody specific to the mafgene productshould be helpful in examining such possibilities, in confirm-ing the nuclear localization of the maf-encoded protein, andin identifying specific DNA sequences recognized by themaf-encoded protein.

We thank H. Shinno-Kohno for her excellent technical assistance,T. Saegusa for plasmid construction, and H. Iba for valuablediscussion. We also thank Dr. H. Hanafusa for critical review of themanuscript. This work was supported by the Foundation for Pro-motion of Cancer Research, The Fujisawa Foundation, research aidof the Inoue Foundation for Science, and a grant-in-aid for cancerresearch from the Ministry of Education, Science and Culture ofJapan.

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