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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).
7711
RI
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)
1323
DraI'JLT 1443
~ ITAAAZ1XAACGAA JGA~GAA _TTAA~CI 1563
1683
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).
Biochemistry: Nishizawa et al.
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7714 Biochemistry: Nishizawa et al.
A B1 23456 1 23456
car*. kb.. 2 o l
OFe 9.4
t* * , _ --4.4-m
'A$*2.O3
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
.4 :
2,Y-
O.56-~
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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