Deletion mapping of moloney type C virus: polypeptide and nucleic acid expression in different transforming virus isolates.

Copyright ©)1976 American Society for Microbiology PrintedinU.SA.

Deletion

Mapping of Moloney Type C Virus:

Polypeptide

and

Nucleic Acid Expression

in

Different

Transforming

Virus

Isolates

WADE P. PARKS,* RICHARD S. HOWK, ANTHONY ANISOWICZ, AND EDWARD M. SCOLNICK

Laboratory of Tumor Virus Genetics, National Cancer Institute, Bethesda, Maryland 20014,* andMeloy

Laboratories,

Rockville,

Maryland

20850

Received for publication12December 1975

The viral

polypeptides and viral RNA present in cells transformed by various

replication-defective type C viruses derived from Moloney

murine leukemia

virus were

examined. Different portions of the Moloney type C viral genome

were

retained

in

the different transforming viruses, thus providing an

opportu-nity

for deletion mapping of the Moloney type C genome. DNA

transcripts were

prepared that are complementary to three distinct nonoverlapping portions of

the Moloney

viral genome. Based on an analysis of the polypeptides produced in

the different transformed cells, one complementary DNA apparently represents

sequences

coding for Moloney gp7O; one complementary DNA represents a

region

of the

Moloney genome common

to

all of the transforming viruses

examined, and one complementary DNA represents the sequences for

p30, p15,

p10,12.

A

partial map of the

different

replication-defective transforming viruses

is

suggested.

Mammalian type C

viruses

that are

capable

of fibroblast transformation

in

tissue

culture

(FT>)

have been

isolated

from

murine,

feline,

and primate hosts (13,

14,

16,

41,

45).

Among

the

FT+

murine

viruses, three

have been

de-rived

by passage of the Moloney

murine

leuke-mia

virus

(Mo-MuLV)

in

various

rodents. First,

the

Harvey sarcoma

virus

(Ha-SV)

was

isolated

by

passage

of Mo-MuLV

in rats

(18).

Second,

the

Moloney

sarcoma virus

(Mo-SV)

was

iso-lated

by passage of the Mo-MuLV

in

BALB/c

mice (27).

Third,

the

Abelson

virus was

isolated

by passage of Mo-MuLV

in a

BALB/c

mouse

treated with

a

glucocorticoid

(4).

All

mammalian

FT+

viruses

examined

to

date

have been shown

to

be defective for

repli-cation;

thus, they

can

be

isolated and studied

in

transformed nonproducer

cells free of

replicat-ing

helper

virus

(2, 5,

6). Nucleic

acid

hybridi-zation

experiments

utilizing

such

nonproducer

cells have

indicated that both Mo-SV and

Ha-SV

are

recombinants

composed

of

two

distinct

classes of

nucleic

acid sequences

(35).

Mo-SV

is

arecombinant between

part

of Mo-MuLV and

additional

sarcoma-specific

sequences

(36).

Similarly,

Ha-SV is

composed

ofa set

of

Mo-MuLV

sequences

and

an

additional

set

of

sar-coma-specific

sequences

derived

fromrats

(38);

the

sarcoma

virus-specific

sequences

in

Mo-SV

and Ha-SV

are

distinct from each other.

Al-though several important

biological

studies

have been reported (32, 33, 40), analysis of the

Abelson viral genome and associated proteins

has been more

preliminary. Abelson virus also

consists

partly of Mo-MuLV genetic

informa-tion, but

additional sequences not present in

Mo-MuLV

have not yet been

demonstrated in

the

Abelson-transforming virus. Nevertheless,

neither the

sarcoma-specific sequence of Mo-SV

or

Ha-SV

is

contained

in

Abelson

virus-trans-formed cells

(36).

In current

studies,

we

have

focused

not on

the

sarcoma-specific

sequences

of

each of these

FT+ viruses

but

on

the Mo-MuLV

portion

of

each virus. Since each of the genomes

responsi-ble for fibroblast transformation appears

to

be

a

recombinant with part of the genome

Mo-MuLV,

we

undertook experiments

to attempt to

correlate

the Mo-MuLV nucleic

acid

se-quences remaining

in

each of these FT+ isolates

with

the Mo-MuLV

structural

polypeptides

synthesized

in

transformed

heterologous

non-producer cells. To

measure

the Mo-MuLV

nu-cleic

acid sequences, viral

complementary

DNA

(cDNA)

was

fractionated

by

hybridization

to

RNA

from different

nonproducer

cells.

Our

re-sults

suggest

that

different

polypeptides

and

different portions of the

Mo-MuLV

genome

are

present

in

transformed

nonproducer cells,

that

deletion

mapping of Mo-MuLV

is

possible,

and

that, by correlating

the

proteins

synthesized

with

the

RNA

expressed,

cDNA

probes

repre-491

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senting different

portions

of theMo-MuLV ge-nome can

be

prepared.

MATERIALS AND METHODS

Cells and viruses. All cells were grown in the Dulbecco-Vogt modificationofEagleminimal

essen-tial medium with high glucose containing either

10%calfserum(Colorado Serum Co., Denver,Colo.) or10%fetal calfserum(Gibco, Long Island, N.Y.).

Cells were grown in humidified Wedco incubators (Wedco, Silver Spring, Md.) in an atmosphere of

10%CO2orindisposable glass bottles(Bellco Glass,

Inc., Vineland, N.J.) on a roller deck apparatus. Viruses were recovered from supernatant fluids

from roller bottle cultures and purified in 20-liter amounts by continuous flow centrifugation in su-crosedensitygradients. The cells andvirus strains employed in the present studyand their relevant

characteristics are provided in Table 1. The cells

used for thepropagation of these viruses were as

follows: NIH 3T3 cells (21) and normal ratkidney (NRK) cells (12) from George Todaro, Bethesda, Md.; mink lung fibroblasts (CCL64), rabbitcornea

cells (CCL60), and a canine kidney cell, MDCK

(CCL34), from theAmericanType Culture

Collec-tion; and the Sc-i wild mouse cell permissive for

replication of N-orB-tropic murine type C viruses (17)from Janet Hartley, Bethesda, Md.

Synthesis of viral-specific [3H]DNA. The

endoge-nous reverse transcriptase reaction from sucrose

densitygradient-banded viruseswasusedto synthe-sizethe[3H]deoxycytidine-labeled cDNA's; the

con-ditions have been fully detailedinearlier publica-tions (37, 39). Optimal detergent concentrations

wereexplored for eachviruspreparation for

synthe-sis ofprobes and variedfrom 0.01 to 0.02% Triton

X-100.The specific activityof thecDNA'swas

approxi-mately2x107 counts/minperjug.The cDNA homol-ogoustoMoloney leukemia viruswaspreparedfrom

Mo-MuLV grown inNIH 3T3 cells. The cDNA

ho-mologoustoMo-SVwasprepared fromavirus

popu-lation containing RD-114 and the D56(S+L-) strain ofMo-SV replicating in a canine kidney cell (31;

unpublished data). The portions of theMoloney leu-kemia virusgenome represented in the Mo-MuLV

cDNAand the fractionsthereofaredetailed below.

Isolation of viral and total cellular RNA and cellular DNA. Total cellular RNA wasisolated by

the method of Glison et al. using cesium chloride centrifugation (15). Concentrations of RNAwere

de-terminedby their absorbanciesat260rn,and puri-fied RNAsolutionswerestoredat-20 C. 32P-labeled

60-70S MoloneytypeC viral RNAwasisolated by

velocity sedimentation in linear 10to30% sucrose

gradients. Cellular DNAwasprepared essentially asdescribed (10).

Hybridization procedures. The procedures for DNA:RNA and DNA:DNAhybridization have been described and are provided in the legends to the figures and tables herein. Hybridization was

ana-lyzed by S1 nuclease digestion or cesium sulfate

centrifugation as previously described (8). Cesium

sulfatewaspurchased from Henley Co., New York.

AllCrt values have beencalculated by the method of Birnstieletal. (9) and correctedto0.18M

monova-lentsalt concentration (10).

Cot

valueswere

calcu-latedasdescribed (10).

Fractionation of [3H]DNA probestoproduce

spe-cific DNA fragments. The fractionation of DNA transcriptshas been describedinarecent publica-tion (36)and is similartoprocedures also used for the preparationof cDNA's fromRoussarcomavirus

(D.Stehelin, J. M. Bishop, and H. Varmus,,personal

TABLE 1. Virus strains and cells employed

Virus Relevantproperties Sourceand/or

refer-ence Producer cells

Mo-MuLV/NIH NIH 3T3 cell line producingMoloney leukemia virus 2, 21 Mo-MuLV/NRK NRK cell line producing Moloney leukemia virus 36, 12 X-MuLV/Sirc Rabbit cornea cell line producing a xenotropic type C 7, 11

virusfrom BALB/c 3T3 cell line

G-MuLV/Sc-1 (N-tropic) Wildmouse cell line(Sc-i)producing Gross MuLV with 17; J. Hartley,

N-tropic host range personal

com-munication

G-MuLV/Sc-i

(B-tropic)

Sc-i

producing Gross MuLV with a B-tropic host range

V-NRK NRK cell line producingendogenous rat type C viruses 37,11, 12

Nonproducer cells

D56(S+L-) NRK NRK cell line transformed by D56(S+L-) strain of 1, 5, 6, 12

Mo-SV

Mo-NRKp- NRK cell line transformed byHT-1 strain of Mo-SV 20, 12

Ha-NRK NRK cell line transformed byHa-SV 38, 12

Ab-NRK NRK cell line transformed byAbelson virus 36, 12

Ki-Mink Mink cell line (CCL 64) transformed by Kirsten sar- 22, 19 comavirus

Ann-1 NIH 3T3 cell line transformed by Abelson virus 34, 21

Ha-MDCK Caninekidney cell line transformed by Ha-SV Unpublished data

D56(S+L-)MDCK Canine kidney cell line transformed by D56 strain of 30

Mo-SV

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[image:2.503.72.466.423.655.2]

communication). The general method is detailed herein and specific details are given in the appropri-atelegends. [3H]cDNAtranscripts werehybridized tototal cellular RNA from the variousnonproducer cells to aCrt of5 x 103 to 1 x 104 mol/s per liter. The hybridization reactions were then processed by hy-droxylapatite chromatography in the following manner. A

water-jacketed

hydroxylapatite column wasprepared with a bed volume of 1 g of hydroxyl-apatite resin permilligram of RNA in the hybridiza-tionreaction. Thehybridization mixture was loaded onto acolumn at 24 C in buffercontaining 0.025 M sodiumphosphate (pH 6.8)-0.6 M NaCl. After wash-ing with 5 to 10 column volumes of the starting buffer, the temperature was raised to 64 C and the column was washed with asecond buffercontaining 0.14 Msodium phosphate, pH 6.8, with 0.6 M NaCl. Thesecond buffer wash was monitored by radioac-tivity measurements on fractions eluted from the column with thiswash until less than 1% of the peak of radioactivity was eluted. The remaining radioac-tivity waseluted from the column with 0.46 M so-diumphosphate (pH 6.8)-0.6 M NaCl. The cDNA in the 0.14 and 0.46 M sodium phosphate washes was dialyzed for2to 3 hagainst 0.01 M Tris-hydrochlo-ride, pH 7.2, treated with 0.5 N NaOH for 5 h at

37 C, dialyzed against 0.01 M Tris-hydrochloride, pH 7.2,andlyophilized prior to use.

Further purification of the fractionated cDNA transcripts was carried out to select for the cDNA portionsthat formed more stable hybrids. Hydrox-ylapatite-cycled cDNA transcripts were hybridized to saturating levels of appropriate nonproducer total cell RNA and, at the completion of the reac-tion,the reaction mixture was then treated with S1 nuclease to digest unhybridized cDNA. After S1

digestion, the hybridization reaction was extracted

with a 1:1 mixture of redistilled phenol (buffered with 0.1 M Tris-hydrochloride, pH 8.0) and

chloro-form-isoamyl alcohol (24:1). After hydrolysis, the

deproteinized hybrid solution was dialyzed to

re-move phenol and was again treated with 0.5 N NaOH for 5 h at 37 C. After alkali hydrol-ysis, the cDNA fractions were dialyzed exten-sively against 0.01 M Tris-hydrochloride, pH 7.2,

and lyophilized prior to use. These steps were

as-sociated with some loss of cDNA and recoveries are given below.

As a third fractionation procedure, particularly forthe final purification of thesarcoma virus-spe-cific sequences containedinMoloney sarcoma virus, cesiumsulfate densitygradient centrifugation was

employed. The sarcoma virus-specific cDNA

ob-tained by procedures previously described using hydroxylapatitechromatography washybridizedto

the RNA of a NRK nonproducer cell

nonproduc-tively transformed by a strain ofMo-SV isolated from the HT-1 hamster cell (20) and referred to herein as the HT-1 strain ofMo-SV, or Mo-SV p-(36) (see Table 1). After S1 nucleasedigestionofthe

probe, as detailedabove, theprobe wasstill found

tocontainapproximately 5to 10%ofsequences

ho-mologous to Mo-MuLV.To remove theseMo-MuLV sequences, the cDNAwashybridized to Mo-MuLV 60-70S viralRNAandfractionated intohybridized

and nonhybridized regions by cesium sulfate

den-sity gradient centrifugation. Details of the frac-tionation are given in theresults. After purification in cesium sulfate, thesarcoma-specific cDNA was dialyzed andlyophilized prior to use.

Immunological assays.Immunoassays of the pro-teins from Moloneyleukemia virus were performed by double-antibody competition radioimmunoas-says. The major viral glycoprotein, gp7O, was iso-lated using a slight modification of the procedures of Strand and August (42). Briefly, purified, con-centrated viruspreparations (approximately 25 mg) were made1% in Triton X-100 in 0.2 M KC1 contain-ing 0.01 MTris-hydrochloride, pH 7.8, with 0.001 M EDTA. After 30 minofincubation at 24 C with occa-sional shaking, the solubilized virus was centri-fuged at 104,000 xg for 1 h. The supernatant was removed, extracted three times with 10 volumes of diethyl ether to remove the detergent. The extracted solubilized viral protein was then dialyzed against two changes of 0.01 M BES (Sigma Chemical Co., St. Louis, Mo.), pH 6.5, with 0.001 M EDTA. After dialysis at 4 C, the virus was applied to a column (1 by 15 cm) ofphosphocellulose p-11 (Whatman)

equilibratedin0.01 MBES, pH 6.5. Following

appli-cationof the sample at4C, the column waswashed until no detectablematerial at an absorbance at 280 nm was detected. A linear 200-ml gradient of 0 to

0.5 MKClinBES was thenapplied; 2-ml fractions were collected and monitored by conductivity, ab-sorbance at 280 nm, and, where indicated, by

so-diumdodecylsulfate-polyacrylamide gel

electropho-resis. A periodic acid-Schiff-staining polypeptide

with amolecular weight of 70,000 elutedat 0.15M

KCl. A polypeptide(s) with comparable staining

andmolecularweight characteristicswasalso noted in the phosphocellulose flow-through fractions at

concentrations4to 10 timeshigherthan thateluted

at0.15MKCl.Onlyasingle bandwasnotedeluting

at0.15MKClincontrasttothe doublebandtermed gp69,71byStrand and August (42).

Thegp7Owas furtherpurified bySephadexG-150

chromatography. Briefly, fractions from

phospho-cellulose chromatography were

pooled, dialyzed

against 0.01 M

Tris-hydrochloride, pH

7.6, con-centrated 10-fold by lyophilization, and

dialyzed

again. This material

(approximately

0.5 mg) was

appliedto acolumn (3.0 by 90cm)

equilibrated

in

0.01 M Tris-hydrochloride, pH 7.6, with 0.001 M

EDTA, and run at 16 ml/h. Fractions were moni-tored by sodium dodecyl

sulfate-polyacrylamide

gels;fractionscontaining

gp7O

and free of other

de-tectablepolypeptidebandswere

pooled,

lyophilized,

and used forimmunological assays. The

Sephadex

G-150-purified

gp7O

migrates as a

predominantly

70,000polypeptide, althoughsome

lower-molecular-weight bandsarenoted

(unpublished

data). Isolatedgp7O, iodinated with chloramineT, was

precipitable with immune but not with control

se-rum and demonstrated

predominantly

group- and

type-specific reactivities. Thedetails ofthe

immu-noassayproceduresasgiveninthe

legend

toFig. 1 aresimilartothose

previously

described fromthis

laboratory (29). Theprocedures for the

immunoas-sayof theMoloney

p30

andtheMoloney

low-molecu-lar-weightpolypeptides

(p10,12

and

p15)

have been

described in detail in a previous

publication

(29).

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494 PARKS ET AL.

In some cases, precipitate counts were determined inan LKB gamma counter equipped with a beryl-lium crystal, which gives a 70% efficiency for 125I determinations. A single pool (IS-166) ofpolyvalent goat anti-Mo-MuLV supplied by Roger Wilsnack, Baltimore, Md., wasused forall assays at the fol-lowing concentrations: gp70, 1:1,600; p30, 1:6,400; p15, 1:200; andp10,12, 1:400.

RESULTS

Viral polypeptide expression in

nonpro-ducer cells. By appropriate isolation

proce-dures it is possible

toinfect and transform

non-mouse cells

with

different transforming viral

genomes

derived from the Moloney

leukemia

virus (35). It is thus

possible

toevaluate mouse

type

C viral

polypeptide expression

in

such

cells in

the absence of expression of endogenous

mouse type

C virion

polypeptides

(28).

Initially,

a

series of cells infected

witheither Mo-MuLV or

the

replication-defective

transforming

viral

genomes was

assayed

for the different

Moloney

polypeptides. The results

are

shown

in

Fig.

1.

As shown

in

Fig. 1A, a gp7O was detected

at a

level of

5 to 10

gg

of

cellular protein

in NRK

cells infected with Mo-MuLV; complete

compe-tition

with the labeled gp7O from

the

Mo-MuLV-infected NKR cells

was

obtained

at 100

[image:4.503.269.459.54.339.2]

lig

of protein.

D56(S+L-)

NRK,

even

at

100

ug

of protein,

was not

found

to contain

detectable

Mo-MuLV

gp7O. However, since D56(S+L-)

NRK cells contain

10to 20

times

less viral RNA

and other viral proteins

(Table

2)

than

Mo-MuLV

in

NRK cells, the

D56(S+L-)

canine cell,

which

has p30

levels comparable to Mo-MuLV,

was

also used

to assay

gp7O expression. When

cell

extracts

from the

D56(S+L-)

canine

cell

were

used

as

competing protein,

no

significant

displacement

of

labeled

gp7O

viral

polypeptide

was

noted,

even at

concentrations

of

protein

that exceeded those required for detection of

gp7O

in

producer levels by 50-fold. Also,

no

significant

displacement

was

noted with

Mo-NRK p-,

Ab-NRK,

or in

NRK

cells

producing

endogenous

rat

viruses,

V/NRK.

Ha-NRK (not

shown)

was

also negative.

Addition of

purified

gp7O

to

negative

extracts

resulted

in

complete

displacement

curves, suggesting that

the

nega-tive

results

were not

due

to

interfering

sub-stances

(unpublished data).

We,

therefore,

con-clude that

none

of

these four strains of

trans-forming

virus

expresses significant amounts of

Mo-MuLV

gp7O

in

nonproductively

trans-formed

heterologous cells.

In

Fig. 1B,

p30

competition

immunoassays

with

the

same extracts are

shown

in an

assay

which

specifically

reacts

with

mouse

type C

p30

and

will not measure rat

type C

p30.

As has

previously

been

reported,

D56(S+L-) NRK (not

R90

~90

90

50-10D.

001 0.01 01 0 0 00

MICROGRAMS PROTEIN PER ASSAY

FIG. 1. Competition radioimmunoassay analysis of Mo-MuLV polypeptides in producer and nonpro-ducer cell extracts. Extracts (20%, vollvol) of

Mo-MuLVINRK(LO), D56(S+L-) MCDK (A), Mo-NRK

p- (V), Ab-NRK (x), uninfected NRK (-), and MCDK (0D) were prepared for immunoassay by scraping cells from monolayer cultures, pelleting by centrifugationat 1,000 x g for 5min,and solubiliza-tion in 0.1 MNaCl-0.01 M Tris-hydrochloride, pH 7.8, containing 1.0% Triton X-100. Protein concen-tration was determined by the method ofLowry et al. (24) using bovine serum albumin as a standard. Antigen extracts were tested indouble-antibody

imn-munoassay reactions as previously described (28), except that in the assays forp10O,12andp15 approxi-mately 50,000 counts per assay were employed and precipitate counts after one wash in 0.1 M NaCl-0.01 M Tris-hydrochloride, pH 7.8, with 0.5% normal rabbit serum were measured as describedin the text. In A through D, displacement curves with purified unlabeled polypeptides (0) are shown to indicate assaysensitivity. (A) gp7O. The specific activity of labeled antigen was 6,uCi of

12C'I/pg

of protein. (B) p30. (C) p15, Moloney. (D) p10,12, Moloney. The specific activity of the latter three labeled polypep-tides waspreviously reported.

shown) by complement fixation and

radioim-munoassays contain detectable levels of MuLV

p30 (1). In contrast, no detectable murine p30

could be detected in either Mo-NRK

p-,

Ha-NRK cells, or Ab-Ha-NRK. The absence of p30 was not previously reported in Abelson

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DELETION MAPPING OF MOLONEY TYPE C VIRUS 495

TABLE 2. Comparison of levels of Moloney leukemia virus RNA and polypeptides in producer and

nonproducer NRK

ceilsa

Cellstudied RNA levels Immunoreactiveproteins'

1/2

Crt Final_tentex-

plo,

12 p15 p30 gp7O

Mo-MuLV/NRK 23 100 625 580 2,400 360

D56(S+L-)NRK 200 44 75 90 75 <20

Mo-NRKp- 27 28 <5 <5 <2 <20

Ab-NRK 160 36 550 500 <2 <20

Ha-NRK 18 16 <5 <5 <2 <20

a For RNAand protein levels in nonproducer cells, each hybridization reaction was incubated at 66 C and

contained in 0.05 ml: 0.02 MTris-hydrochloride,pH7.2, 0.6 MNaCl,0.05%sodium dodecyl sulfate, 5 x 10-5 EDTA, and approximately 3,000trichloroaceticacidcounts of [3H]cDNA perminfrom Mo-MuLV/NRK. The final extent of the reaction was at a

Crt

of104 mol/s per liter. The100% value with the Mo-MuLV/NRK RNA was approximately 2,600counts/min. Reactions were analyzed withS1nuclease.

bThe levelsof immunoreactive proteins expressed as nanograms per milligram of total cellular protein weredetermined as described in the legend to Fig. 1.

ducers

because heretofore heterologous

nonpro-ducer

cells

have

not

been available, an

impor-tant

prerequisite since

normal

mouse

cells

con-tain a

significant basal level of immunoreactive

p30 that can be measured by

radioimmunoas-say

(28).

Results

of competition immunoassays, for

p10,12 and p15

are

shown

in Fig.

iD and

C,

respectively. Importantly, Ab-NRK reacts at

protein levels

comparable

to cells

infected with

and

producing Mo-MuLV. In other studies

not

shown,

the genetic

stability

of

this expression

has

been

demonstrated by showing that NIH

cells also transformed by the same strain of

Abelson

virus, Ann-1 (34)

(provided by C.

Scher, Boston, Mass.), had

similar

expression

of the

type-specific

low-molecular-weight

Molo-ney

polypeptides,

whereas Ha-SV-transformed

NIH

3T3 cells were negative.

D56(S+L-)

canine

cells also

express

both

p10,12 and p15,

consistent

with

earlier studies

which this genome has been

reported

to

contain

sequences

for

p12

and

p30 expression

(43).

Thus, it is clear that Abelson virus and D56

(S+L-)

Mo-SV both express the

Moloney

low-molecular-weight polypeptides

but

that

Abel-son

virus-transformed

nonproducer

cells fail

to

produce immunoreactive

p30.

No

detectable

re-action in

either the

p1O,12

or

the

p15

assay

were

observed

with either

Ha-NRK

or

Mo-NRK

p-,

indicating that they do

not

contain

significant

levels of

gp7O,

p30, p10,12,

or

p15.

Correlation

of levels

of

Moloney

virus

poly-peptides and

Moloney

viral-specific

RNA in

different NRK cell.

To further evaluate the

patterns of

polypeptide

expression

in

NRK cells

infected

with thevarious

Mo-SV

strains,

cellu-lar RNA from the transformed

nonproducer

cells and from cells

producing

Moloney

leuke-mia virus were

examined for levels of

viral-specific Moloney RNA. The kinetics of reaction

between the cell RNA and

a

cDNA prepared

from

Moloney leukemia virus

are

summarized

in

Table 2. NRK

cells

producing

Moloney

leu-kemia virus had

a

1/2

Crt of

23

mol/s per

liter,

a

value that was almost identical

to

the 1/2 Crt

levels

in

the

Ha-NRK cells

and the Mo-NRK

p-cells. Thus,

in

the

case

of

the Mo-NRK

pa

and

Ha-SV NRK-transformed

cells,

even

though

no

Moloney structural proteins are detected

in

such cells in the

competition

radioimmunoas-says,

the concentrations of RNA

homologous

to

Moloney

leukemia virus

are

comparable

to

the

RNA

found

in

the NRK cells

producing

Molo-ney

leukemia virus. The difference noted is

in

the final

extent

of the reaction with the

Molo-ney

cDNA

probe

and

not

the concentrations of

RNA

homologous

to

the

Moloney

leukemia

ge-nome. In

fact, the

D56(S+L-)

NRK

and

Abel-son

NRK cells had somewhat lower absolute

amounts

of the

Moloney

leukemia

virus-specific

RNA,

even

though

the viral

polypeptides

dis-cussed

in

the

preceding

section

were

readily

demonstrated.

Thus,

the

inability

to

detect

any

viral structural

proteins

orthe

varying pattern

of expression

seen in

various

transformed

non-producer

cells does

not

correlate

simply

with

a

decrease in the absolute levels ofRNA

homolo-gous

to

Moloney

leukemia virus. These

results,

however,

do

not

exclude

translational

interfer-ence

with

polypeptide synthesis.

Fractionation of DNA

representing

various

parts

of the Mo-MuLV genome. To further

assess the differing polypeptide patterns

ob-served

in

the

nonproducer

transformed cells

and

to

determine whether the

apparent

absence

of

polypeptide

expression

in

certain

Mo-SV

strains was

due

to

transcriptional

or

transla-tional

controls,

Mo-MuLV cDNA

was

fraction-ated

by

hybridizing

it

with the RNA contained

on November 10, 2019 by guest

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[image:5.503.57.449.88.177.2]

in

the

NRK cell transformed by the

D56

(S+L-)

strain

of Mo-SV (fraction

2A,

Table

3). The

portion

that hybridized

to the

S+L--positive

RNA

was

further

hybridized

a second time to

the

same

RNA and

was treated with

S1

nu-clease,

as

detailed

above. This

constitutes,

as

shown in

Table

3,

fraction

2B of the

Mo-MuLV

genome.

This fraction (2B) of

the

Mo-MuLV

cDNA

which did hybridize

to the S+L- genome was

further

subfractionated into

a

portion

thatwas

contained in the Mo-NRK

p-cells anda

portion

that

was not

contained

in

the

RNA of

those

cells.

This fractionation

was

performed with

hydroxylapatite

chromatography,

and

these

portions

represent

fractions

4 and 5 ofthe

Mo-MuLV

genome

(Table 3).

In

addition, fraction

6 was

prepared

by

hy-bridizing the whole Mo-MuLV

cDNA

directly

to

the Mo-NRK

p-

RNA and

treating

the reaction

with

S1 nuclease.

The

hybridization properties

of

the six fractions

of the Mo-MuLV cDNAare

summarized

in

Table

4. All

hybridization

re-sults indicate the final

extentof the

hybridiza-tion

reactionat

Crt

values

of 104mol/sperliter

and

are

normalized

to

100%,

based on the

ex-tent

of

hybridization

with

Mo-MuLV.

Fraction

2B

hybridized

well

to

the

RNAs from all of the

NRK nonproducer cells

transformed by the

dif-ferent

strains oftransforming virus.

In contrast

to

whole

Moloney cDNA (fraction 1), the final

extent

of

the reaction with each

nonproducer

RNA

is

higher with fraction 2B relative

to

the

final extent with

the

RNA from

Mo-MuLV

NRK

producer cells. As controls, the final

ex-tent

of reaction

relative

to

Mo-MuLV

was

com-parable with

the RNA of either

a

xenotropic

MuLV

virus

growing

in

Sirc

cells

or an

N-tropic

or

B-tropic

strain of

Gross virus

growing

in

SC-1

cells.

Fraction

3,

which

represents

a

portion

of

Mo-MuLV

genomenot

contained in the

S+L-

NRK

RNA, hybridizes

appreciably

to

the

Mo-NRK

p-RNA.

Fractions

4

and

5,

which

represent the

further

subfractionation

of

fraction

2B,

also

re-veal

some

important

differences

notseen

with

either the

Mo-MuLV fraction 1

or

2B

probes.

The results with fraction 4 indicate that this

portion

of

the

Mo-MuLV

genome

is

homologous

to

both the S+L- D56 and

virtually

equally

homologous

to

the

Mo-NRK

pa

RNA. With

frac-tion

4,

a

higher

final

extent

of

hybridization

with each

nonproducer

RNA

relative

to

that

with

the

whole

Mo-MuLV

probe

is

detected in

the

Harvey

NRK and Abelson NRK cells.

Equally important,

the

Ki-Mink

nonproducer

cell

(Table 1) gives

the

highest

final

extent

of

hybridization

with this

specific portion

of the

Mo-MuLV

genome.

Fraction

5,

which

represents

the portion of

the

Mo-MuLV

genome

that is contained in

S+L- NRK but

not

contained

in

Mo-NRK

p-,

reveals

an

important finding

that

correlates

well with the

pattern

of

protein expression

pre-viously

shown in

Table

2.

Fraction

5

hybridizes

appreciably

to

the

Abelson-transformed NRK

RNA but

not to

either the

Mo-NRK

p- or

Harvey

NRK RNA. As

a

control

for

fraction

4,

fraction

-5

failed

to

hybridize

to

the Ki-Mink

cellular

RNA. As

a

further control,

fraction

6,

which

represents

the

cDNA

fragment

homolo-gous to

Mo-NRK

p-

RNA, gives

a pattern

of

hybridization

similar

to

the results with

frac-tion 3

cycled cDNA probes. The results

indicate

TABLE

3.

Summary ofpurification of

Mo-MuLV

cDNA

Fractional

Trichloroa-probedesig- Properties of cDNA Method ofpurification cetic acid

mint

nation

counts/min

1 Homologoustowhole Mo-MuLV 10 x 106 100

2A HomologoustoD56(S+L-)NRK Fraction 1 cDNA + D56 2.6 x 106 26

RNA -.

hydroxylapa-tite,0.46 Msalt wash

2B Homologousto

D56(S+L-)

NRK Fraction 2A + D56 RNA, 1.3 x 106 13

S1 nuclease resistant

3 NothomologoustoD56(S+L-)NRK Fraction 1 cDNA + D56 2.4 x 106 24

RNA -+

hydroxylapa-tite,0.14 Msaltwash

4 cDNAcommon toD56(S+L-)NRK and Fraction 2B + Mo-NRK 0.2 x 106 2

Mo-NRKp- p- RNA--

hydroxylap-atite, 0.46 M salt wash

5 cDNAhomologoustoD56(S+L-)NRK, Fraction 2B + Mo-NRK 0.3 x 106 3

nothomologoustoMo-NRKp- p- RNA-. hydroxylap-atite, 0.14 M saltwash

6 HomologoustoMo-NRKp- Fraction 1 cDNA + Mo- 1.7 x 106 17

NRK p- RNA, S1

nu-clease resistant

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DELETION VIRUS 497

TABLE 4. Hybridization with different fractions ofMo-MuLVcDNA withcellularRNAsa

Finalextents of hybridization of fraction: RNAsource

1 2B 3 4 5 6

Mo-MuLV/NRK 100 100 100 100 100 100

D56(S+L-)NRK 44 100 6 97 91 61

Mo-NRK p- 28 40 22 94 10 91

Ha-NRK 16 26 4 49 5 52

Ab-NRK 26 52 3 67 31

X-MuLV/Sirc 40 40 41 29 36

G-MuLV/Sc-1 (N-tropic) 38 38 39 25 30

G-MuLV/Sc-1 (B-tropic) 32 38 38 25 28

Ki-Mink 5 10 3 19 3

V/NRK 7 3 3 5 3

Mink,uninfected 2 3 3 3 3

aForhybridization of cDNA fractions to total cellular RNAs, reactions were carried out in a volume of

0.05 ml at 66 C to a Crt value of 104 mol/s per liter with an input of approximately 1,000 to 3,000 trichloroacetic acid counts of cDNA per min and 250 ,ug of total cellular RNA. The conditions of hybridiza-tion are asdescribed in footnoteatoTable 2. Mo/MuLV NRK total cellular RNAhybridized maximally to all the cDNA's tested and therefore was considered 100%; all other values are normalized to this. The actual final extents ofhybridization were as follows: whole Mo-MuLV cDNA hybridized66%;S+L--positive cDNA fraction 2Bhybridized 74%; S+L--negative cDNA fraction 3 hybridized 53%; S+L--positive, Mo-NRK-positive cDNA fraction 4 hybridized 76%; S+L--positive, Mo-NRK-negative cDNA fraction 5 hybridized 68%; Mo-NRK p- fraction 6 cDNA hybridized 78%. Each value represents the average of three determinations that reached the final extentof hybridization. The average standard deviation is 5% of the value given (range, 1to10%). Blanksindicate values not determined.

that,

by the fractionation

procedure outlined,

the Mo-MuLV

cDNA

can

be

purified

into

at

least three distinct

nonoverlapping

portions

(fractions 3, 4,

5)

and that

hybridization with

these fractions reveals various

types

of RNA

in

transformed

nonproducer

cells that

cannot

be

detected

with

the whole Mo-MuLV cDNA

probe.

Percentage

of Mo-MuLV

genome

repre-sented

by

different

portions

of the

cDNA.

To

evaluate what

proportion

of the Mo-MuLV

ge-nome was

represented

by

the various cDNA

fractions,

an

experiment

was

performed

in

which

increasing

amounts

of

the

fractionated

[3H]cDNA's

were

hybridized

to

limiting

amounts

of

32P-labeled

60-70S Mo-MuLV viral

RNA. The

results

are

shown

in

Fig.

2.

With

increasing

quantities

of the

[3H]cDNA

fraction

1, up to 57%

of the

input 32p

RNA

was

pro-tected. The shape of the

curve

indicates that the

whole

Mo-MuLV

genome is not

represented

uniformly

in the cDNA transcript. The 57%

hybridization

valuewasachieved withatwo-to

threefold

molar excess of cDNA. When

frac-tions 2B

and

3were

hybridized

totheMo-MuLV

60-70S

RNA,

each

fraction

clearly

represented

less of the

Mo-MuLV

genome

than the

starting

fraction

1.

At

approximately

a

twofold

excessin

each

case,

about

25%

of the

genome was

pro-tected

from

nuclease

digestion.

Again,

when

fractions

4

and

5were

hybridized

in a

similar

fashion,

a two- to

threefold

excess

protected

roughly

15 to 18%

of

the

32P

viral RNA. Because

of limited quantities of the

fractionated

[3H]cDNA

probes

and the fact that only

a two-to

threefold molar

excess

could be

added, final

extents

of

hybridization could

not

be

achieved.

However,

the results

are

consistent

with the

results

obtained with

[3H]DNA

and

excess

RNA experiments cited above

and

indicate that

the

differing cDNA fractions

represent

smaller

fractions

of Mo-MuLV

genome

than

repre-sented

in

the whole cDNA

probe.

Relative

concentrations of

sarcoma-specific

and

MuLV-specific

RNA in

transformed

non-producer

cells. To

assess

the

relative levels of

MuLV-specific and

sarcoma-specific RNA

in two

strains

of

Mo-SV,

it was

first

necessaryto

further

purify

the

sarcoma-specific

cDNA (36)

to

free

it

completely

of MuLV

sequences.

To

do

this,

the

sarcoma-specific

DNA (as

outlined

above)

was

hybridized

to

Mo-MuLV

60-70S

RNA

and the

hybridization

reaction

was

frac-tionated

in cesium

sulfate

as

shown

in

Fig.

3.

A

small

peak

of

hybridized

cDNA

was

obtained

in

the region of the

gradient

witha

buoyant

den-sity of

approximately

1.62

g/cm3.

Fractions 16to

22were

pooled

to

obtain

a

"sarc"

cDNA

homolo-gousto

Moloney

sarcoma

virus, which

was

com-pletely free of Mo-MuLV

sequences.

This

sar-coma-specific cDNA and

a

Mo-MuLV cDNA

(fraction

1,

Table

3) were

hybridized

either

to

cellular

RNA of

D56(S+L-)

NRKcellsorto

the

RNA of Mo-NRK

p-

cells,

and the results

were

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[image:7.503.46.444.80.225.2]

4~u

C

0.5 10

ngcDNA

FIG. 2. 32p protection studies with fractionated

cDNA probes. Each 0.05-mi reaction mixture was

incubated at 66 C for 36 h andcontained 250

trichlo-roaceticacid counts Of32p~60-70S RNA permmnwith a specific activity of 5 x 105 trichioroacetic acid

counts/mmnper pg. Eachreaction mixture contained ionic components as indicated in footnote a toTabke2 and was hybridized to increasing amounts of

f3H]cDNA fractionated as indicated in Table 3 and

4. The specific activity of the wholestarting DNA

probe was 2 x 107trichioroacetic acidcounts/mmnper pg. Thus, for an input of 250 trichioroacetic acid

counts of32p~per min, one would expect 10,000 3H counts to protectall of the 32p counts if the cDNA

transcriptcontained a 1:1 distribution of cDNA.

Hy-bridization was analyzed with the use of Si nuclease

aspreviously described (8). Symbols: *, whole Mo-MuLV cDNA; U,fraction 2B; O.fraction 3; *, frac-tion 4; A, fraction 5; fraction 6 (not shown) was

similar to hybridization pattern noted with fractions

4and 5.

analyzed by RNA association kinetics. The re-suits are shown in Fig. 4. In Fig. 4A, the reasso-ciation of the Mo-NRKpa RNA is shown with the sarcoma-specific cDNA and the Mo-MuLV cDNA. The results indicate that the rate of hybridization with each cDNA was equal with the two cDNA fractions; the 1/2 Crt for each probe was approximately 3 x 102 mol! s per liter. In Fig. 4B, the similar experiment was performed with the D56 S+L- strain of NRK RNA. The rate of association was slower with this cellular RNA (as shown in Table 2) but again was identical with both cDNA probes; the 1/2 Crt value with each probe was approxi-mately2 x 102 mol/s per liter. Thus, in both the Mo-NRK pa RNA and the S+L- NRK RNA, although the absolute values for the viral RNA differ in the two cells, the kinetics of the reac-tion for the MuLV portion and the

sarcoma-specific portion

are

identical in each

case

for the

twosarcoma

virus

strains.

DNA:DNA

hybridization

with

purified

cDNA

fractions.

Employing fractions 1,

2B,

and

3,

itwas

possible

to

directly

test

whether

at

high

Cot

values

heterologous nonproducer cells

contained

as

much

Mo-MuLV DNA

as

producer

cultures. The results shown

in

Table

5

indicate

that both

fraction

1

and

2B

hybridize

to

both

Mo-MuLV/NRK

DNA and

D56(S+L-) NRK

DNA. In

contrast,

fraction

3

hybridizes

to

Mo-MuLV NRK DNA but

notto

D56(S+L-) NRK

DNA.

Thus, the

samepattern

of results

is

ob-tained

with DNA:

[3H]DNA studies

as

noted

in

RNA:

[3H]DNA studies. Thus, the results with

these

two

fractions of the Mo-MuLV probe

are

consistent with the

hypothesis

that

S+L-

Mo-SVrepresentsadeletionmutantof part of

the

Mo-MuLV

genome.

Expression of MuLV cDNA fractions

in sar-coma

virus

nonproducer cells

superinfected

with

heterologous helper viruses. To

assess

with

yet

another

experimental

approach

whether the

differing

patterns

of RNA

expres-sion

seen

in

sarcoma

virus-transformed

nonpro-ducer cells

represented

in

part

transcriptional

blocks

to

expression, various

sarcoma

virus-transformed nonproducer

cells were

superin-fected with

heterologous helper

type

C

viruses

torescue

the

sarcomaviruses contained in

the

cells.

The

RNA from such a

productively

in-fected cell

was

then tested

against the different

portions of the Mo-MuLV

cDNA's

to see

whether

sequences would be

expressed

in

the

rescued

genome not

contained in

the

nonpro-ducer cell.

Correspondingly,

proteins of

the

Mo-MuLV virus

were

also analyzed

in

the

nonpro-ducer cell superinfected

with

the

heterologous

helper

viruses to see

whether other

proteins

would also be expressed. In each case, FT+

viruses

were

isolated and the

ratio of

FT+

virus

to

helper

was

determined.

RD114-pseudotyped

FT

viruses

had a 1:1 ratio or a

fourfold FT+

virus excess.

The

Wo-LV-pseudotyped

FT+

vi-ruses

had

ahelpertoFT+ virusexcessof five-to

tenfold

(unpublished data). The results of the

hybridization

and

polypeptide

analysis from

these cells

are shown in

Table

6. The results

indicate that the

final

extent of

hybridization

with the

various

cDNA's does

not

change

in

nonproducer cells

after infection with

heterolo-gous

helper

virusin thepresence

of

the

heterol-ogous

helper

virus.

Similarly,

the proteins

de-tected by

radioimmunoassay

are

identical

to

those

profiles detected

inthe nonproducer cells,

asindicated in

Fig.

1.

The

heterologous

viruses

used in

these

experimentsdonot cross-reactin

the nucleic

acid

hybridization

orimmunoassays

used.

The

results indicate

that the absence of

I

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[image:8.503.68.264.56.263.2]

600-

1.6~

E

14

4 8 12 16 20 24 28

Bottom FRACTION NUMBER Top

FIG. 3. Cesium sulfatepurification of the Mo-SV-specific[3H]DNA.Partially purified Moloney sarcoma-specific [3H]DNA (200,000 trichloroacetic acidcounts/min) washybridized with 60-70S Moloney leukemia virusRNA to aCrtof4.3x101mol/perliter usingreaction conditions described previously (36). The hybrids wereseparated from the sarcoma-specific [3H]DNA byCs2SO4density gradient centrifugation in polyallomer tubes (8). One-half of the reaction mixture was mixed with 11.0 ml of

CsSO4

(p =1.52

g/cm3)

(Gallard-Schlesinger,New York) in 0.01 MTris-hydrochloride(pH 7.2),0.001 M EDTA, and 0.01%(vol/vol)sodium

laurylsarcosinate. Thegradients were centrifuged in a Beckman 65 angle rotor for 72 h at 15 C at 44,000

rpm. Gradients were collected by puncturing the bottom of the tube and collecting 20drops/fraction. The buoyant density was calculatedfromtherefractive index of every

fifth

fraction. Recovery ofcounts per minute wasgreater than 95%.

expression

of the RNA

in

the various

nonpro-ducer cells is

not

due

to a

block

in

the

transcrip-tion

of part of the MuLV genome

contained

in

the

transformed cell.

DISCUSSION

The

availability

of three different

fibroblast

transforming

(FT+)

viruses

derived

from

Mo-MuLV

provides

the

opportunity

for

an

analysis

of both the

sarcoma-specific

sequences

associ-ated with fibroblast

transformation

as

well

as

the sequences

responsible

for

polypeptide

expression.

We

recently

compared

the

sarcoma-specific

sequences

of

Mo-SV,

Ha-SV,

and

Abel-son

virus

(36),

and the

current

studies

were

undertaken

to

correlate Mo-MuLV nucleic

acid

sequences

and the

polypeptides

expressed

in

cells

transformed

by

three

different FT+

vi-ruses.

The

different FT+

viruses

have

been

found

to

produce

different Mo-MuLV

polypep-tides

and to

express

different

portions of

the

Mo-MuLV

genome

in

association with their

re-spective

transforming

functions.

Froma

com-bined

analysis

of the

expression

of

polypeptides

and nucleic acids

in

the

different

FT+

viruses,

certain

facts have

emerged. First,

replication-defective,

transforming

viruses

produce

neither

Mo-MuLV gp70,

as

shown

herein,

nor,

as

previ-ously

shown,

viral

reverse

transcriptase

(23,

31).

Thus,

the

expression of these

two

proteins

would seem

to

be

least

closely

linked

to

the

expression

of

transforming functions

and/or

to

have

been

deleted. Second, the

p10,12

and

p15

can

be

expressed

in

the absence

or

presence

of

p3O

in

the

different

FT+

isolates.

Thus,

expres-sion

of

low-molecular-weight

polypeptides

ap-pears

to

be

more

closely

linked

to

expression

of

the

transforming

functions than

p30

and

more

distantly

linked

to

the

expression

of the

gp7O

and

reverse

transcriptase. These results with

the

low-molecular-weight

proteins

are

consist-ent

with the data

on various

woolly monkey

sarcoma

virus-transformed

nonproducers

re-cently

reported by

Aaronson

et

al. (3).

Third,

in

some

strains

of

FTP+

viruses

(Mo-NRK

p-

and

Ha-SV),

none

of the known structural

proteins

of Mo-MuLV is

produced.

Because

of the differing patterns of

polypep-tide

expression,

we

analyzed the transformed

nonproducer cells for

RNA

homologous

to

Mo-MuLV

and,

in correlation with the

protein

data, noted that different portions

of the Mo-MuLV

genome

were

expressed

in these

cells.

Thus,

wewere ableto

prepare

cDNA

fractions

by

hybridizing

the whole Mo-MuLV

cDNA

with the RNA from the cells

transformed by

499

on November 10, 2019 by guest

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[image:9.503.102.393.50.253.2]

10 a

z

a:)

m

z

6 4

10

6

4

2

lo' I0z

Crt(mfdes-sec/L)

10'

FIG. 4. Hybridization ofwhole Mo-MuLV cDNA andpurifiedsarccDNA to Mo-NRK-p-and

S+L--NRKtotal cellular RNA. Thereactionswerecarried outinavolumeof0.05 ml withaninputof

approxi-mately 4,000trichloroaceticacid countsofMo-MuLV cDNAperminor1,000trichloroaceticacid countsof

cDNAsarcperminand 50pgoftotal cellularRNA. The conditionsof hybridization areasdescribed in thetext.(A) HybridizationtoMo-NRKp-total

cellu-larRNAwith wholeMo-MuLV cDNA(E)andcycled

"sarc" A cDNA(U).At thefinalextentof hybridiza-tionthe Mo-MuLV cDNAhybridizedto1,150counts! minand the cDNA "sarc"hybridizedto875 counts! min. These valuesaretakenas100%forcalculation ofCrt value. (B) Hybridization to S+L--NRK total cellularRNAwith whole Mo-MuLV cDNA(0)and

cycled sarc "A" DNA (O). At the final extent of hybridization, the Mo-MuLV cDNA hybridized to 2,000trichloroacetic acidcounts/minand the cDNA

sarc hybridized to 878 trichloroacetic acid counts! min.These valuesaretakenas100%forcalculation ofCt values.

different FT+ viruses.

Hybridization

with these

probes

provided

several interesting

observa-tions. First,

a portion of the murine type C genome ispresent in all the

replication-defec-tive murine FT+ viruses,evenwhennomurine

typeC polypeptidesareproducedinsuch cells.

This

commonregionseemstobecloselylinked

tothe

transforming

functions andrepresentsas

aminimum estimateatleast15%of theMuLV

genome. The

results

suggest that the

recom-binationeventleadingtothe formation of FT+

viruses

is occurringatacommonregionof the

murinetypeC viralgenome, even

though

this

event

has ledtotheacquisition of

heterologous

sarcoma-specific

sequences

derived

fromratsor mice.

Secondly,

anexcellent correlationwasfound

between the final

extents

of

Mo-MuLV RNA

expressed

and the number of

structural

poly-peptides

produced.

For example,

Abelson-transformed

cells contained RNAsequencesnot present in Mo-NRK p- cell RNA but

fewer

RNA

sequences

than

were

contained in D56

(S+L-)

NRK cells.This

correlated well

with

the

fact that Ab-NRK contained

low-molecular-weight MuLV proteins, Mo-NRK

p-

contained

no

MuLV

structural

proteins,

and S+L-

NRK

contained

low-molecular-weight

proteins

plus

MuLV

p30.

These results suggest that the

cDNAprobes prepared

by

cycling against

dif-ferent nonproducer RNAs represent at

least

partof thesequences

coding

forp30 and/or the

low-molecular-weight

proteins.

Thus,

infuture

studies it should be possible to compare the

cross-reaction between the cDNApresumed to

encode for p30 from other mammalian type

C

viruses.

It

should also

now be

possible

to

ex-plore

the genetic basis for the apparent

group-specific

reactivities of the

p30

and the

type-specific

immunoreactivity

of the

low-molecular-weight proteins (29, 43) by comparison with other murine type C viruses.

The

third

observation

from these studies is

that the patterns of RNAor

protein expression

didnot

change

inthe thetransformed

nonpro-ducer cells, evenwhen

they

weresuperinfected

with

heterologous helper

type C virusesto

pro-duce infectious

sarcomavirus.

These

data,

cou-pled

tothe observations of Maiseletal.

(25, 26)

that the

replication-defective

Ha-SV and

Mo-SV contain asmallergenomethan

Mo-MuLV,

indicate that these F+ viruses are deletion

mutantsand that the RNA andprotein

expres-sion patterns noted are due to

differing

dele-tions of the Mo-MuLVgenomeand not to

tran-TABLE 5. Hybridizationwithdifferent fractions of

Mo-MuLV cDNAwithcellular DNAa Finalextentsofhybridization

CellDNAsource Fraction Fraction Fraction

1 2B 3

Mo-MuLVNRK 100 100 100

D56(S+L-) NRK 20 29 <4

V/NRK <1 <1 <1

a For DNA:DNA reassociation, reactions were

carriedoutinavolume of0.05ml for100hat66Cto a Cot value of >2.4 x 104 mol/s perliterwith an

input of approximately 1,500 trichloroacetic acid

countsperminute of cDNA and 100,ug ofcellular

DNA. Final extent values arenormalized to 100% basedon Mo-MuLV/NRK. The actual final extent ofhybridizationwith Mo-MuLV/NRKwith fraction

1 was 45%, with fraction 2B was 44%, and with fraction3was24%.

zc

-

,

io 0 )0

30

30

0 .

,-

-0-2,

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http://jvi.asm.org/

[image:10.503.73.265.50.278.2] [image:10.503.276.464.486.556.2]

[image:11.503.44.439.82.200.2]

DELETION MAPPING OF MOLONEY TYPE C VIRUS 501

TABLE 6. RNA and protein expression in nonproducer cellssuperinfectedwithheterologousviruses

Proteins'

Cell Fraction 2B Fraction 3 Fraction 5

p10,12 p30

Mo-NRK p- 40 22 10 <10 <2

+ Wo-LV 42 23 12 <10 <2

Ha-MDCK 26 4 5 <10 <2

+ RD-114 26 4 5 <10 <2

D56(S+L-)MDCK 100 6 91 2,600 4,000

+ RD-114 100 5 92 3,200 4,500

Ab-NRK 52 3 31 3,000 <2

+ Wo-LV 48 3 33 3,200 <2

Mo-MuLV/NRK 100 100 100 3,500 5,250

aHybridization reactions were

carried

out to a final

CAt

value of 104

mol/s

per liter as described in the footnote to Table 4. Wo-LV indicates a type C virus isolated from a woolly monkey (45).

b The levels of immunoreactive proteins expressed as nanograms per milligram of total cellular protein weredetermined as described in the legend to Fig. 1.

1 D56(S'L-)Mo-SV |

2 Mo-NRKpA

3. HarveyMo-SV _

4.Abelson Mo-SV ..xxxX ..XXXXXXXXX...X.. I "JNT" pl0,12p30

p15

FIG. 5. Proposedsequenceof different

Mo-MuLV-derivedfibroblast transforming viruses. The differ-entsarcoma-specific sequences ofD56(S+L-)Mo-SV

(-) and Harvey Mo-SV (000) are identified by

letter symbols. The postulated transforming se-quencesof Abelson Mo-SV (xxx) arealso shown. Indicatedlengths ofgenomesshownareapproximate although there is evidence that. Ha-SV and D56(S+L-) Mo-SV contain smaller RNA subunits than Mo-MuLV (26; unpublished data). For com-parative purposes, the gene sequence ofRous sar-comavirusproposed by Wongetal.(44) ispresented.

scriptional

or translational blocks.

Thus,

the

protein

and

RNA

expression

patterns

noted

earlier

can

be

transposed

to a

partial

genetic

map as

shown

in

Fig.

5. Fromourpresent data

an internal insertion of the "sarc" sequences

cannot be

excluded; further,

it is not

possible

totranspose the order of

the

defectivesarcoma

genomestothe whole Mo-MuLV untilmore is

known

about

the

specific

mechanism of

recom-bination involved in sarcomavirus formation.

However, Fig. 5summarizes earlier discussion

points and emphasizes: (i) the common joint

region, (ii) the relative

linkage

of the MuLV

sequences with respect to transforming

func-tions, and (iii) the

heterogeneity

of the sarc

sequences.

No

attempt is madeto order these

functions withrespect to either the 5' or 3' end

of

the

virus, as has been

elegantly

done

re-cently by Wang et al. for avian sarcoma

vi-ruses (44).

However, if

the

mechanism

of formation of

all

fibroblast-transforming

isolates isthe same

and

if

the

geneorder is similar for mammalian

and avian

viruses,

then the 5'-3' order ofthe

linear RNA genome

S+L-

would be

5',

p30,

p12,

p15, sarc, common-3'.

For

the Abelson and

HT-1 strains

of Mo-MSV

and

Ha-SV,

increased

deletions from the circular proviral

DNAwould

result

in

the

sarcsequences

becoming

closerto

the

5'

end of the

linear RNA sarcoma virus

genome. Since translation

begins

at the 5' end

of

mRNA, the relevant

implications

of this

model

is

that

invitro translation of the

defec-tive

sarcomavirus genomes would favor

trans-lation

of the

transforming

protein.

ACKNOWLEDGMENT

This workwassupported byacontractfrom the Virus

CancerProgram.

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