Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.

0022-538X/86/100167-10$02.00/0

Copyright

© 1986, American

Society

for Microbiology

Site-Directed Mutagenesis

of the

gag-myc

Gene of Avian

Myelocytomatosis

Virus

29:

Biological

Activity

and Intracellular

Localization of

Structurally

Altered

Proteins

MARK L. HEANEY,' JACALYN PIERCE,2 ANDJ. THOMAS

PARSONS'*

Department of Microbiology, UniversityofVirginia School of Medicine, Charlottesville, Virginia22908,1 andLaboratory

of

Cell and Molecular

Biology,

National Cancer

Instituite,

Bethesda,

Maryland

202052

Received 24February1986/Accepted 17 June 1986

Transfection ofchicken embryo cells with pMC29, aplasmidvectorcontainingthesequencesfor theacute transforming virus MC29, and a cloned transformation-defective helper virus, pAMst, resulted in

morpho-logical transformation, the synthesis of P11gag-mYc(theproduct ofthegag-myconcogene),and the production

of infectiousvirus.MC29mutantsbearing site-directed deletions within the gag-specificsequences orwithin the

middle portion of themyc sequencesefficiently induced transformation of chicken embryo cells in culture. However, variants containingdeletions ofsequencesin the amino-terminal halforcarboxy-terminalportion of

themyc gene were defective fortransformation. Thegag-myc proteins encoded by these variants efficiently

localizedto the cell nucleus. Premature termination mutantswere isolated which encodedgag-myc proteins

lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the

cytoplasm. Deletion ofasfewas 11residueswithin themiddleof the myc-specificsequences (residuesIle-239 toGlu-249) significantlyreducedtheefficiencyof chicken hematopoietic cell transformation.

The acute avian retrovirus MC29 contains the oncogene v-myc which is required for the induction of carcinomas, sarcomas, and, most commonly, acute leukemia in infected

animals (5, 35). Alterations in the expression of the normal

cellularhomolog,c-myc wereimplicated inneoplastic

trans-formation ina number ofspecies (27). In the case ofavian

leukosis virus-induced B-celllymphomasin chickens,

inser-tion of retrovirus transcriptional elements at or near the

c-myclocus (8, 37, 39)appears to alterc-myc gene

expres-sion. Cell lines derived from murine plasmacytomas(44) and

human Burkitt

lymphoma

(3, 4) possess a characteristic

chromosomal translocation whichmay in some cases result

inthe transcriptional activation ofthec-myc gene. In

addi-tion, cell lines derived from humantumors contain

amplifi-cations ofthe c-myc gene whichresult in elevated levels of

c-myc RNAtranscripts (12, 14, 32).

Infection of avian cells withMC29 results in the

expres-sion ofavirus-encoded 110-kilodalton(kDa) fusionprotein,

Pfl0gag-myc,

containing gag-specific and myc-specific se-quences(9, 26).The

P110yw9-mYc

proteinis aphosphoprotein

(10) that binds to double- and single-stranded DNA and is

associated withthenuclearmatrixin MC29-infected cells (1,

15, 17). In cultured cells, expression of the

p110g(g-n1'

protein results in the efficient transformation ofboth avian

embryo fibroblasts and hematopoietic cells of macrophage

origin (7, 20, 23).

The amino acid sequence of the MC29-encoded

pllOag-my'

reveals several

provocative

structured motifs

(Fig. 1A). The amino-terminal half ofthe

myc-specific

se-quence contains several

regions

rich in

proline

residues,

particularly residues Pro-155 to Pro-170, in which 10of 15

residues are proline. The carboxy-terminal

portion

ofthe

gag-myc protein is rich in

arginine

and

lysine

residues and

may account for the DNA

binding

properties

of thegag-myc

protein (2). The center

portion

of the v-myc sequence

contains a

glutamic

acid-rich

region

in which 17 of 35

* Corresponding author.

residuesareeitherglutamic acidor asparticacid. This region

is encoded by a sequence which corresponds to the 5'

boundary ofthe secondcodingexonofthe c-myc gene and is

conserved among myc proteins ofchicken, mice, and man

(6, 13, 45-47). As pointed out by Ralston and Bishop (40),

this acidic region has homologous counterparts in the

nu-cleus-associated proteins encoded by the ElA and v-myb

oncogenes.

Conventional genetic analysis of the v-myc gene has

yielded little informationregardingthestructurallyand

func-tionally important regions of the v-myc gene product.

Ramsayetal. (41) isolatedthreespontaneousMC29mutants

containingdeletions rangingfrom 200 to 600 nucleotides in

themyc gene (10,42).These mutantstransform chickenand

quail fibroblastsbutexhibitsignificantlyreduced efficiencies

for transformation ofchicken cells ofhematopoietic origin

(41). The viral mutations were mapped to the

carboxy-terminal half ofP11W9aR-mYc (18).

Inthis paper we describe theconstruction and

character-ization of site-directed deletion mutations within the

gag-myc geneof MC29. Analysis ofthe

biological

properties

of these MC29 mutants revealed that deletion ofas few as 11 amino acids within a

particularly

acidic domain of the gag-myc protein greatly reduced the

efficiency

of chicken

macrophagetransformation but notthatof chicken

embryo

cells. Nuclear localization studies showed that the gag-myc

proteins encoded by a numberof

large

deletion mutations

localizedtothecell

nucleus,

with the

exception

ofpremature termination mutations that encoded truncatedgag-myc pro-teinslackingthe

carboxy-terminal

halfof the

protein.

These

structurally altered

proteins

werefound in both the nucleus

and the cytoplasm of transfected cells. The MC29 mutants described in this paper further define a

region

of the myc

gene required for efficient

macrophage

transformation. In

addition, they

provide

useful reagents for

defining

the

im-portant structural and functional domains of the myc gene

product.

167

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168 HEANEY ET AL.

3NT

* plY plU Ap;zl

ATG

,gag

P.. -.r-h Acidic Basic Region Region Region

myc

Proline-richRegionl

B

wild 1,

tgpe t p19 plO &p27 ATG

Agag

Size of Deletion

Pro-rich Acidic Basic

Region Begion Region

myc

45 79 145 170

PPAPSEDIWKKFELLPMPPLSPSRRSSLAAASCFP....RQEGGPAAASRPGPPPSGPPPPPAGP

CH201

CH202 AcidicRegionEM:

228 262

DSEEQEEDEE IDVVTLAEANESESSTESSTEASEE

BasicRegionD :

300 359

kRLKLDSRGVLKQ SNNRKCSSPRTLDSEENDKRRTHNVLERQRRNELKLRFFALRDQ P

360 415

EVANNEKAPKVVLKKATEYVLSLQSDEHKL AEKEQLRRRREQLHNLEQLRNSRA

CH203

11a0

ii

t1 CH204

CH205

t

CH206 CH207 t

CH208

CH301 t

11 13'NT 20aa LE1111111113'NTI 84aa

1l111111111111111113NT1 60aa

-EM

122aa

_

I1||||||||||||g||3

NT 182a0

b

Ic

FIG. 1. Structure of thegag-mnyc geneandP110'"'"'Ycprotein. (A)Schematicdiagramof thegag-mnycprotein.Aminoacidsequences are deduced from the nucleotidesequencedata(2, 46). (B) Mapsof deletion mutations. The isolation and characterization of individual mutations

wereperformedasdescribed in Materials and Methods.a, Aminoacid deletion followedby54 aminoacids unrelatedtomIvc; b,amino acid deletion followedby 10 amino acidsunrelatedto mvc;c,frameshift deletion of 54carboxy-terminalamino acids followedby9 unrelatedamino acids. 2, denotes the additionof unrelated amino acidsequencesderived from sequences3' tothemycgene(see text).

MATERIALS AND METHODS

Cells, viruses, and plasmids. Chicken embryo and quail embryo cell cultures were prepared and maintained as describedpreviously (11). COS-1 cells(22) weremaintained in Dulbecco modified Eagle medium plus 10% fetal calf serum.Viral DNAs used in the construction ofpMC29were obtained from the following sources: prnvc (29) from T. Papas, National Cancer Institute, and pJD100 and pSVO10 (20) from G. Gilmartin. A plasmid, tdPr-RSV-B, bearing a permuted copy of tdPrB-RSV (16) was obtained from J. Coffin. Chicken embryo cells were transfected by the cal-cium phosphate precipitation method of Graham and Van der Eb(24)asmodifiedby Bryantand Parsons(11). Optimal transfection with the replication-defective pMC29 or mutagenized pMC29 was attained when these DNAs were mixed(ina4:1 ratio) with pzvMst helpervirusDNA, usinga total of 1 .tgofDNAper60-mmculture dish. Wild-type and

mutant virus stocks were prepared from supernatants of transfected cultures 7 to 14 days posttransfection. COS-1 cells were transfected by calcium phosphate precipitation with theglycerol shock modification (34) by using 10 ptg of plasmid DNAper100-mmculture dish. Invitro transforma-tion of bone marrow cells was carried out essentially as

described by Beug et al. (7).

Isolation of deletion mutations in the gag-myc gene. The

deletion mutationspCH201, pCH203,andpCH207containa

deletion ofmyc-specific sequences flanking the Clal site in

themycgene.DNAofthe plasmid pMC29waslinearized by

partial Clal digestion and subjected to BAL 31 digestion (0.05 U/ml at 37°C) for 30, 60, or 90 s. The samples of

digested DNA were combined, the staggered ends were

repaired with Klenow fragment of Escherichia (cli DNA polymerase,andthe DNA wasligated and transformed into competent E. coli HB101. The resulting colonies were

screened for the loss of the Clal site. pCH101, pCH202, pCH204, pCH205, pCH206, and pCH301 were constructed by either complete or partial digestion with the restriction endonucleases designated in the text. Partial restriction

R

I B B R

Quail gag env

E

FIG. 2. Construction of the plasmids pMC29 and pAvMst. The cloningstrategyusedtogeneratepMC29 and pAMst is described in detail in Materials and Methods. The RSV-derived sequences (B) delineatedbytheBamnHI siteinthegaggeneand theBgllI site in the

src gene were replaced by the BamnHI fragment from the MC29 proviral clone pmnc (A) (29). A Hindlll fragment containing the

complete MC29 recombinant genome was cloned into a plasmid

vector(D)containing the SV40 origin of replication(19)yieldingthe plasmid pMC29 (E). The pAvMst helper-virus plasmid (C) was

isolatedby deletion of thesequencesbetween thetwoMstIlsites in thesrcgeneofpJDIOO (B).

A

CH101 t _".4

-".- 1'. I

'. ...

t ---- ----I

'NTI

AIL ,-..A- d1111111111111111113

X,7,S,M,9-,--,v-|EMlg3,>.-.Hllll t J. VIROL.

-Z'n

u .

C C

.F 5

-6 E-UTI

co I-z

eZ e a] CD

-ImmM.-z-g.IuLLL---J

R.", .. .-..

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FIG. 3. Light microscopy of infected and uninfected quail embryo cells. (A) Uninfected quail cells. (B) Cells infected with pMC29

(pAMst). (C) Cellsinfected with CH207 (pAMst). (D) Soft-agar colony of cells infected withCH201(pAMst). Magnification, x140.

digestswere carried out by varying the time or temperature

ofdigestion or both. After digestion, the DNA wasligated

with T4 DNA ligase and used to transform E. coli HB101.

The boundaries of each deletion were determined by direct

DNA sequencing(33).

Celllabeling, immunoprecipitation,andgelelectrophoresis.

COS-1 cellswereincubated inmethionine-free media for30

min before the addition of labeling medium containing 100

,uCi of

[35S]methionine

per ml. Cells were labeled for2 to4

h and harvested as described by Bryant and Parsons (11). Cellextracts wereprepared bythe method of Abramset al. (1), and theimmunoprecipitation was performedasfollows. Cell extracts were incubated with 5 ,ul ofpolyclonal rabbit

anti-gagoranti-myc for60 minat4°Cfollowedby incubation

withFormalin-fixed Staphylococcusaureusforanadditional

60minat4°C. Immunecomplexeswerewashedsequentially

with RIPA buffer (150 mM NaCl, 1% deoxycholate, 1%

Triton X-100, 0.1% sodium dodecyl sulfate, 50 mM Tris

hydrochloride [pH 7.2]), high-salt buffer (2 M NaCl, 0.5%

deoxycholate, 1% NonidetP-40, 10mM Trishydrochloride

[pH 7.5]), RIPA buffer, 1M

MgCI2,

and 10 mM Tris

hydro-chloride (pH 7.5). The immune complexes were boiled in

sample buffer for 3 min, and equal samples were then

electrophoresedin 10.5%

polyacrylamide

gels (28)and

auto-radiographed.

Indirect immunofluorescence stainingof cells. COS-1 cells were seeded on cover slips and transfected with plasmid

DNA 12 to 18 h later. At 48 h after transfection, the cells were fixed with cold methanol for 5 min followed by

treat-mentwith coldacetonefor 3 min(31). Thefixed cells were incubated with 100,ul of eithera mousemonoclonal

antibody

to

p279"l

(38)orarabbit

antibody

tothe myc

protein (36)

for

30 min at room temperature. The

binding

of the

primary

mouseantibodywaslocalized

by

incubation ofthecellswith

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170 HEANEY ET AL.

goat F(ab')2 anti-mouse immunoglobulinG (10

Vxg/ml

(Jack-son Immuno Research Laboratories, Inc.) for 30 min fol-lowed by incubation with fluorescein-conjugated rabbit F(ab')2 anti-goat F(ab')2 (10 Fig/ml) (Jackson) for an addi-tional 30 min. Localizationofprimaryrabbit antibody bind-ing was carried out in a similarmannerby using rhodamine-conjugated mouse F(ab')2 anti-rabbit F(ab')2 (10 pLg/ml). Between incubations with antibody, the cover slips were washed three times with phosphate-buffered saline. The stained cells were examined and photographed with a Leitz fluorescence microscope (Leitz/Opto-MetricDiv. ofE.Leitz Inc.) equipped with epi-illumination.

1

2

3

4

5

6 7 8

9

101112

9

7.4.

dmpm Q,wlo'm

.A.-f-ow

sup

_up

-_

68*

RESULTS

Isolation ofan infectious MC29 plasnmid vector. To intro-ducedefined mutations in the gag-myc geneofMC29 andto facilitate the biological analysis of the resulting mutations, weused standard methodsof molecularcloningtoconstruct a recombinant plasmid containing an MC29 provirus and a

second plasmidtoprovidehelper-related functions. Inbrief,

aplasmid,pmyc,containingapartial MC29 provirus (29)and

5'-flanking host quail DNA was used as the source of

myc-specific sequences. Exploitingthehigh degreeof

nucle-otide sequence homology among the avian retrovirus gag structural genes, we inserted a partial gag-myc fragment from the plasmid pmyc into the gag region of pJD100, a nonpermuted proviral clone of Rous sarcoma virus (RSV) inserted into pBR322 (48). Intheresultant construction, all of theRSV-specific pol andenvgenes andmostof RSV

gag-and src-specific sequences were deleted (Fig. 2). The

pBR322 vector sequence was then replaced with that of

pSVO10, which containsthesimian virus 40(SV40)originof

replication (21). The resultant plasmid pMC29 is a

nonpermuted clone of the MC29 provirus that encodes a gag-mycfusion protein identical in sequence toMC29 gag-mycandcontains the SV40 and pBR322 origins of replication

(Fig. 2).

Because MC29 is a replication-defective virus, a helper

virus plasmid, pAMst, was constructed by deletion of

virtu-ally all of the src genefrom pJD100 (Fig. 2). Transfection of

chicken embryo cells with a mixture of pMC29 and the

helper plasmid pAMst DNA yielded characteristic

MC29-1

2

3

4

116-

92--p 1 1ogag-myc

i.

*, _{_},

66-

45-FIG. 4. pMC29 directs thesynthesis ofP110j"gg-"m. Infectedand

uninfected quail cells were labeled with [35S]methionine,

im-munoprecipitated with rabbitantiserumtothe RSVgagproteinsand

analyzed by sodium dodecylsulfate-polyacrylamide gel electropho-resis as described inMaterials and Methods. Lanes: 1, uninfected cells; 2, helper-virus-infected cells; 3, pMC29-(pAMst) virus-infectedcells; 4, MC29-transformed quail cell line, Q8.

43.

FIG. 5. Immunoprecipitation of wild-type and mutantgag-myc

proteins. COS-1 cellsweretransfected withtheindicatedDNAsand labeledwith[35S]methionineasdescribedin Materials andMethods.

Cell extracts wereimmunoprecipitated with rabbit anti-gag serum

(lanes 1,2,and 4 to 12)orrabbitanti-mnyc sera(lane 3). Lanes: 1, mock-transfected COS-1cells; 2, pMC29; 3, CH101;4, CH201; 5, CH202; 6, CH203; 7, CH204; 8,CH205;9,CH206; 10,CH207; 11, CH208; 12, CH301.Thepositionsof the markerproteins phosphor-ylaseb(97.4kDa), bovineserumalbumin(68kDa),andovalbumin

(43kDa) are shown at the left.

liketransformation in 7 to 14days. Culture media harvested from transformed chicken embryo cultures induced stable transformation when used to infect either chicken orquail embryo cells (Fig. 3B). These cultures were

indistinguish-ablein morphology from chickenorquail cells transformed

with wild-type MC29 virus stocks obtained from several different sources (data not shown). These experiments es-tablished the biological activityof the MC29plasmidvector and thetransforming activityof the cloned v-myc sequence. Metabolic labeling of pMC29-transformed quail embryo cells with [35S]methionine andimmunoprecipitation of gag-relatedproteins with either rabbit anti-gag rabbit serum (Fig. 4, lane 3) or anti-p271("i monoclonal antibody (not shown) revealeda 110-kDaprotein that comigrated with P110gag-my( of the MC29-transformed quailcellline andQ8 (Fig. 4, lane 4),aswellasPr76and thebreakdownproducts of Pr76. The

P1109"'g-n'C was absent from helper-infected cells (Fig. 4,

lane 2), indicatingthat P110""' expression resulted from

theexpressionof theMC29-specific sequences. Transfection

of COS-1 cells (an SV40-transformed CV-1 monkey cellline) with pMC29 yielded high-level, transient expression of

P110'g-f7l9'

(Fig. 5, lane 2)within 48 to 72 h posttransfection. Intracellular localization of P110"g9g-m'v in pMC29-transfected COS cellswas ascertained by indirect immuno-fluorescence with a monoclonal antibody to p279a'. The observed nuclearfluorescence(Fig. 6B) was consistent with previous reports that P110g"eIgn'' is a nucleus-associated protein (1, 15). These experiments clearly documented the efficient expression of biologically active P11V'9-mv" protein encoded bypMC29.

Isolationand characterization of site-directed mutations in gag-myc. In aninitial attempt to determine which regions of P 109"s'-n"'I wererequired for MC29-induced transformation of chicken embryo cells, we introduced site-directed

dele-tions into the gag-myc gene of pMC29. Two methods of

mutagenesis wereused. Deletions wereeither generated by

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FIG. 6. Immunofluorescence localization of wild-type and mutantgag-myc proteins. COS-1 cells were transfected with the indicated DNAsand examinedbyindirectimmunofluorescence,as described inMaterials and Methods, by using either a monoclonal antibody top279'ag

(A, B, andD to H) orrabbitanti-myc serum (C). (A) Mock-transfection. (B) pMC29. (C)CH101. (D) CH201. (E)CH202. (F) CH203. (G)

CH204. (H)CH205. (I)CH206. (J)CH207. (K)CH208. (L)CH301.

BAL 31 exonuclease or were isolated by the fusion of

restriction sites within the gag-myc gene. The size and

position ofthese deletions aresummarized in Fig. 1B.

Both in-frame and out-of-reading-frame mutations were

isolated (Fig. 1B). The in-frame deletions included pCH201,

an 11-amino-acid deletion within the acidic region of the

gag-mycprotein, and

pCH203,

an84-amino-acid deletion in the center of the myc gene that lacks the same region and

approximately 30 flanking amino acids. The out-of-frame

mutant, pCH207, contains a 57-base-pair deletion and en-codes a protein containing 1l1 of the gag-specific amino

acids,230aminoacidsof theaminoterminusofmyc,and 54

unrelated amino acids. A second frameshift mutant,

pCH208, wasgenerated byan incomplete repairof the Sall

siteoftheplasmid pCH207.Thisproduceda+2alteration in

thereadingframefrom the Sall siteto thepointof deletion

in pCH207 and a +1 shift in the readingframe thereafter.

The net result was q mutation that, due to a termination codon in the +1reading frame,encodesaprotein containing

219 myc-related

famino

acids and only 10 unrelated amino acids.

The restriction enzyme site fusion deletions ranged from

20 amino acids to 182 amino acids. The smallest deletion,

pCH202, is afunction ofthe Sall and ClaI sites in the myc

genefollowing thefilling in with T4 DNA polymerase. This

mutationcontainsaglutamine residue as a result of the filling

in and deletes 9 of the 17 glutamic acid and asparagine residues within the acidic region (Fig. 1). Deletion of the sequences between each of the three HincII sites in myc

generated deletion mutants lacking 60 amino acids

(pCH204), 122 amino acids (pCH205), and 182 amino acids

(pCH206). pCH204 contains adeletion that overlaps thatof

pCH202 and extends the deletion 40additionalamino acids

toward the carboxy terminus. The deletion within pCH206

overlaps those inpCH205 andpCH204. pCH301 containsa

345-base-pair deletion of the 3' end of the myc

coding

sequencesandnontranslatedregion, resultinginthedeletion of54amino acids of thecarboxyterminusof the

P110gi`-my'c

andthe addition of 9 unrelated amino acidsbefore

termina-tion. pCH101 encodes a protein with a 170-amino-acid

deletion, resulting in the removal of

approximately

75% of

the p27-gagsequences

(amino

acids Thr-258to

Ile-427).

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172 HEANEY ET AL.

Biological properties of the MC29 mutants. Chicken em-bryo cells werecotransfectedwith plasmidDNAcontaining

each of the respective mutations and pAMst DNA. The morphological alterations of the cellswereassessed after 14

to21 days, and the culturesupernatants were usedtoinfect

quail embryo cells. The in-frame myc deletion mutants

CH201, CH202, CH203, and CH204, and the in-frame gag-deletion, CH101, induced thetransformation and growth in softagarof chicken and quail embryo cells. Themorphology

of these mutants (Fig. 3) andtheir growth in soft agar were indistinguishable from thatof wild-typeMC29. Thein-frame

deletion mutants CH205 and CH206 were transformation defective, as were both frameshift mutants CH207(Fig. 3C)

and CH208.

Toestablisharapidandefficient methodfor the analysisof

expression of structurally altered gag-myc proteins, COS-1

cells were transfected with individual mutagenized plasmid

DNA and the transient expression of the encoded gag-myc

proteins was monitored by immunoprecipitation. In each case, alabeled myc-relatedprotein ofanappropriate

molec-ular weight could beimmunoprecipitated with eithergagor

myc antiserum (Fig. 5). Thus, weestablished that the trans-formation-defective mutants were capable of producing a stable myc-specific gene product under conditions of

tran-sient expression. The detection of structurally altered

gag-myc proteinsrin COS-1 cells permitted us to determine the intracellularlocalization of these proteins byindirect immu-nofluorescence. Each of the transformation-competent

mu-tants encoded a protein that efficiently localized to the cell nucleus (Fig. 6C, D, E, F, and G). In addition, the

transfor-1 2 3 4 5 6

...

1

6-9

2-

66-_:r.~

-p 1 1

ogag-myc

-p

88gag-myc

FIG. 7. Intracellularlocalization of truncatedgag-mycproteins.

COS-1 cells were transfected with pMC29 or pCH207 DNA and

labeled with[35S]methionineasdescribed in Materials andMethods.

Nuclear andcytoplasmicfractions werepreparedbyamodification

ofthe procedure ofAbrams et al. (1) asdescribed by Morganand

Parsons (36). Nuclear fractions (lanes 1 to 3) and cytoplasmic

fractions (lanes 4 to6) were immunoprecipitated with rabbit

anti-inyc serum, and immune complexes were subjected to

polyacryl-amidegel analysisasdescribed in thelegendtoFig.5. Lanes: 1 and 4,mock-transfectedcells;2and5,pMC29-transfectedcells;3 and6,

pCH207-transfectedcells.ThepositionsofpMC29-encoded

P110g"g-and pCH207-encoded

P88g"a-"my"

are indicated at the right.

Molecularweight markers areindicated at the left.

TABLE 1. Transformation of chicken bone marrow cellsby wild-type andmutantMC29 viruses

Virus(helper Approximate Colonies formed/106 virus) titer(FFU)a nucleatedcellsb

MC29(MAV) 104 75,77

103 8, 14

102 1,2

pMC29(tdPrB) 104 50, 35

103 3,3

102 0, 0

CH101(tdPrB) 104 15

l03 3

102 0

CH201 (tdPrB) 104 0

CH202(tdPrB) 104 0, 0

CH203 (tdPrB) 104 0,0

CH204(tdPrB) 104 0,0

a The transforming titer(focus-formingunits)of the virus stocksonchicken embryocells wasdeterminedby end-pointdilution.

bBonemarrow cells were preparedasdescribedbefore (7). Two values representduplicate assays.

mation-defective in-frame deletion mutations pCH205 and

pCH206,aswellaspCH301,encodedproteins that localized

to the nucleus (Fig. 6H, I, L). In contrast, the truncated

proteins

encoded

by

thetransformation-defective frameshift

mutations pCH207 and pCH208 were found both in the

cytoplasmandin the nucleus(Fig. 6J and K). To confirm the

dual localization of the truncated gag-mycprotein encoded

by pCH207 (P88g(19-'""), COS-1 cells transfected with

pCH207DNAwerelabeled with[35S]methionine and nuclear

andcytoplasmic fractions were prepared. Immunoprecipita-tion with gag antiserum revealed that pMC29 encoded p1102"2'-.rn which was foundprincipally in the nucleus (Fig. 7, lanes 2 and 5). In contrast,

P88gag-my',

encoded by pCH207, was observed in both nuclear and cytoplasmic fractions

(Fig.

7,lanes 3 and6). In summary, it appears that none of the in-frame deletions, including the carboxy-terminaldeletion present inCH301, remove sequences nec-essary for nuclear transport.However, the dual localization of pCH207- and pCH208-encoded proteins suggests that sequences within the carboxy-terminal half of the myc protein play somerole in stable nuclear association.

Identification ofmyc deletion mutants with altered trans-forming properties. Ramsay et al. (41) described mutantsof MC29containing deletions within the 3' half of the gag-myc genethat exhibita reduced abilityto transform hematopoi-etic cells but not fibroblast cells invitro. These mutants also showed a greatly reduced pathogenicity in chickens (19).

The transformation-competent MC29 deletion mutants

CH101, CH201, CH202, CH203, and CH204 as well as

wild-type molecularly cloned MC29 (pMC29) were used to

transform chicken hematopoietic cells in culture. Culture supernatants from chicken embryo cells infected with pMC29 (and atdPrB helpervirus) were used toinfect both chickenembryocells andchicken bone marrow cells (Table

1). Wild-typeMC29 and the p27gagdeletion mutant (CH101)

readily induced characteristic transformation of bone mar-row cells, yielding transformed cells in liquid culture and transformed colonies in semisolid agar (Fig. 8). Wright-Giemsa-stained preparations of transformed bone marrow cells revealed thetypicalmorphology of MC29-transformed macrophages (Fig. 8). Inaddition, the phagocytic property of the transformed cells was established by their ability to

phagocytize latex beads (data not shown). In contrast, the

mutantsCH201, CH202, CH203, and CH204, which readily J. VIROL.

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a6

-::.

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FIG. 8. Light microscopy of infected and uninfected chicken bone marrow cells. Bone marrow cells were infected as described in MaterialsandMethods withpMC29 (1)orCH101 (2)or wereuninfectedcontrols (3). (a) Liquid bonemarrowcultures(magnification, x140). (b)Semisolidagarcolonies(magnification, x140). (c) Wright-Giemsa-stained preparationsoftransformed cells(magnification, x1000).

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174 HEANEY ET AL.

TABLE 2. Summaryoftheproperties of MC29mutants Celltransformation

Mutant Amino acids deleteda Bone

Growth

in Intracellularlocalization

Embryo

Boesoft

agarb

marrow

CH101 Thr-258gag. . .Ile-427gag + + + Nucleus

CH201 Ile-239.. .Glu-249 + - + Nucleus

CH202 Asp-220.. .(Glu).. .Ile-239 + - + Nucleus

CH203 Arg-205.. .(Pro).. .Ala-288 + - + Nucleus

CH204 Asp-220.. Val-279 + - + Nucleus

CH205 Asn-98... Val-219 - NDC - Nucleus

CH206 Asn-97...Val-279 - ND - Nucleus

CH207 Glu-231... .(n54) - ND - Nucleus andcytoplasm

CH208 Asp-220...

.(n,O)

- ND - Nucleus andcytoplasm

CH301 Ala-362 ...

(ng)

- (-)d - Nucleus

aResidues in parentheses representamino acids insertedas aconsequence ofmutagenesis.Thenvalue inparenthesesis the addition of unrelated amino acids

(Fig. 1).

bGrowth of transformed embryo cells in soft agar. ' ND, Not determined.

dInfection of bone marrow cells with a virus of unknown titer.

transformed chicken embryo cells, exhibited a significantly

reduced efficiency ofchicken bone marrowcell

transforma-tion (Table 1). CH301, which did not transform chicken

embryo cells, also was defective for bone marrow cell

transformation. Immunoprecipitation of extracts prepared

from

[35S]methionine-labeled

transformed bonemarrowcells

(transformed with either pMC29 or CH101) with anti-myc

serum confirmed the presence ofwild-type or structurally

altered gag-myc proteins (data not shown). We conclude

from these experiments that a deletion of a myc-specific

sequence as small as 11 amino acids (residues Ile-239 to

Glu-249) within the gag-myc gene product

significantly

re-duces theefficiency by which thisproteinmediates

hemato-poietic celltransformation.

DISCUSSION

Tofacilitate theinvitro genetic analysis of the myc gene,

we constructed the plasmid pMC29, which contains the v-myc sequencesof the avian retrovirus MC29.Transfection

of chicken embryo cells with pMC29 DNA and a plasmid

DNA containing either a tdPrA-RSV or tdPrB-RSV helper

virus genome yielded virus stocks containing defective

transforming virus and a replication-competent,

nontransforming helper virus. These transforming virus

stocks inducedtransformation of chicken and quail embryo cells and bone marrow cells in culture. Cells transformed with recombinant MC29 (pMC29) were indistinguishable from cells infected with wild-type MC29 obtained from several sources. Chicken or quail embryo cells transformed with either the transforming variants or with wild-type

pMC29 exhibited analteredmorphology and readily formed

colonies in soft agar. Bone marrow cells transformed with pMC29 or CH101 (a variant without

p279`lr)

were

morpho-logically indistinguishable from cells transformed with

wild-type MC29 viruses. Wright-Giemsa staining as well as the

phagocyticactivity of these cells indicated that they were of

the macrophage lineage. Inaddition, as described by Leutz et al. (30), establishment of cell lines from transformed colonies induced with either pMC29 or CH101 was

depen-dent upon the addition of chicken myelomonocytic growth factor.

Deletion of a substantial portion (approximately 75%) of

the

p27`(lx

sequences did not alter the ability of the mutant

virus (CH101) to transform either chicken or quail embryo

cellsorbone marrowcells (Table 2). Recentlyweextended thisanalysistoinclude the characterization of additional gag deletion mutations, the largest of which retains only the carboxy-terminal 36 amino acids of p27g"l'. The mutants with these deletions transform both chicken embryo cells and bonemarrowcells inculture, indicating that thegag-specific

sequences do not play a major role in MC29-mediated transformation in vitro (M. L. Heaney, J. H. Morgan, J. H. Pierce, and J. T. Parsons, manuscript in preparation). Re-cently, Shawetal. (43) showed that in vivo-derived variants of MC29 containing deletions of gag sequences arecapable oftransforming chicken embryo cells.

Examination of the amino acid sequence of the v-myc protein reveals several interesting structural features. Sev-eral clusters of proline residues reside within the amino-terminal half of themolecule, whereas thecarboxy-terminal portion is lysine and arginine rich. Large deletions which impinge upon or delete either of these regions yielded transformation-defective viruses. In both cases, the deletion ofamino acid sequence did not appear to alter the localiza-tion of the gag-mycprotein to the nucleus(Table 2). This is particularly interesting with respect to the mutant CH301,

which contains a deletion ofthecarboxy-terminal 50amino acids, including the deletion ofsequences particularly rich in lysines and arginines (Fig. 1). Ourresults indicate that none of theregions of themyc protein removed by thesedeletion mutations specifies an amino acid sequence necessary for nuclearlocalization, such as the nucleartransport signal of the SV40 Tantigen (25). Thepremature-terminationmutants CH207 and CH208, however, didexhibit an altered pattern of localization, being present in both the nucleus and the cytoplasm. Since the deletions in CHC207 and CH208 are quite extensive, it isdifficult to assessthe roleofthedeleted amino acid sequencesin stable nuclearassociation. Amore detailedanalysis of this region, however,is warranted.

Deletion ofas many as84amino acids within the middle

portion of the myc-specific sequences did not substantially

reducethe ability of the mutant virus (CH203) to transform chickenembryocells(Table 2). However,deletion of as few as 11 amino acids within this same region (CH201) greatly reducedtheefficiency of bone marrow cell transformations. Our results agree with and extend the previously reported results of Ramsey et al. (41, 42), who reported that large deletions within the 3' portion of the myc gene sequences greatly reduced theefficiency of hematopoietic cell transfor-J. VIROL.

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http://jvi.asm.org/

mation. The deletion of11 aminoacids in the mutantCH201

resulted in the removal of residues within a particularly

acidic region of the miyc protein (16 glutamic and aspartic acid residues within a sequence of 35 amino acids; Fig. iB). Itis unclear how such a change mayinfluencetheinteraction of the gag-rnvc protein with hematopoietic cell-specific

factors required for transformation. However, several

inter-esting possibilities can be entertained. First, such sequence

changes could alter the stability of the mnc protein in hematopoietic cells. Second, such sequence changes could selectively alter the interaction of the

g(ig-rnvc

protein with specific hematopoietic cell transcription factors (proteins) required for gene expression. Finally, myvC sequence alter-ations may reduce the efficiency ofinteraction with specific

DNA transcriptional regulatory elements.

The unique structural features contained within the

g(ig-myc protein arelikelytobeintimatelyinvolved inregulating

its biological and biochemical functions. The studies pre-sented here show that the alteration ofoneofthese features, namely the glutamicacid-rich regionof the protein, appears toalter thebiological activityof the resultant MC29 virus, in that it exhibits diminished capacitytotransform

hematopoi-etic cells. The additional mutants

analyzed

in this

study

showthat several other structural featuresare necessaryfor

gag-rnvc function. However, the extensive nature of these

mutations precludes us from commenting on the possible

consequences of such changes. Nonetheless, these mutants offer a starting point for the continued structural and func-tional analysis of the gag-mzy( gene product.

ACKNOWLEDGMENTS

Wethank B.Creasyfor the excellenttechnicalassistance.Weare

indebted to S. Parsons. J. Morgan. and D. Bryant for providing antibodiesand useful discussionsduringthecourseof thestudyand to S. Farinaand Mac forhelp in the preparationoffigures.

J.T.P. is a recipient of a Faculty Research Award from the American Cancer Society. This work was supported by Public Health Service grants CA29243 and CA 27578 from the National

CancerInstitute.

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