Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

(1)

0022-538X/78/0025-0553$02.00/0

Impaired

Processing of

Precursor

Polypeptides

of

Temperature-Sensitive

Mutants of Rauscher Murine

Leukemia

Virus

W. J. M. VAN DEVEN,* D.VAN ZAANE,t C. ONNEKINK,AND H. P. J. BLOEMERS Departmentof Biochemistry, University of

Nijmegen,

The Netherlands

Received forpublication 24 August 1977

The synthesis and processing of virus-specific precursor polypeptides in

NIH/3T3cellsinfectedatthepermissivetemperature(31°C) with

temperature-sensitive (ts) mutantsof Rauscher murine leukemia viruswas studied in

pulse-chase experiments at the permissive and nonpermissive (39°C) temperatures.

The newly synthesized virus-specific polypeptides were analyzed by sodium

dodecyl sulfate

polyacrylamide

gel

electrophoresis

after immunoprecipitation

with polyvalent and monospecific antisera against Rauscher murine leukemia

virus proteins. In cells infected with ts mutants defective in early replication

steps(the

early

mutantstsl7andts29), andtsmutantsdefective inpostintegration

steps

(the

latemutants ts25 and ts26), the

processing

of the

primary

gag gene

product was

impaired

at the

nonpermissive

temperature. gag-pr75 ofall four

mutants was converted into gag-pr65; however, gag-pr65 accumulated at the

nonpermissive

temperature, and the main internal virion

polypeptide

p30was

not formed.

Therefore,

the

proteolytic cleavage

is blocked

beyond

gag-pr65.

Concomitantly,

theformation of the env

gene-related polypeptide p12(E)

of all

four mutantswasblockedattherestrictivetemperature.In contrast, cells infected with the latemutantts28,which

produced

noninfectiousvirionsat

39°C,

showed

anormalturnoverof the gagandenvprecursor

polypeptides.

Instudiesonthemolecularbiology of type C

RNA tumor viruses, the isolated conditional

lethalmutants of avian (6, 9, 22, 26) and

mam-malian (13, 15, 17,25) RNA tumor viruses

pro-videuseful markers forthe analysisof theviral

genome.Temperature-sensitive (ts) mutants of

murine

leukemia

virus (MuLV) have been

de-rived from the Kirsten (17), Rauscher (15), and

Moloney (25) strains. The mutants of the

Rauscher strain (R-MuLV) have been divided

intothreedifferent classes baseduponthestage

at which virus

replication

is impaired at the

restrictivetemperature (15). Through the

cour-tesy of J. R.

Stephenson,

we have obtained

representatives

of the tsmutants of class I (at

39°C, defective in

replication

functions before

the

synthesis

of viral group-specific antigens),

class II(at

39°C,

defective in functionsbetween

thesynthesisofviralgroup-specificantigens and

production ofvirions), and class III

(noninfec-tious virions

produced

at

39°C).

The virus-specific structural polypeptides of

the RNA tumor viruses are formed through

proteolytic cleavage of precursor polypeptides

tPresent address:DepartmentofVirology,Central Veter-inaryInstitute,Lelystad, The Netherlands.

(for a review see 14). Thus, in the case of

R-MuLV, the

primary

gag gene

product,

gag-pr75,

is converted into gag-pr65, which is

split

into

certain

intermediary

products (with

molecular

weights

between40,000 and

50,000)

and,

subse-quently,

intothe

polypeptides

of the viralcore,

p15, p12,

p30,

and plO (2).

Likewise,

gp69/71

and

p15(E),

the

virus-specific polypeptides

lo-cated in the viral

envelope

as well as in the

plasma membrane of infected

cells,

arederived

from the

primary

env gene

product env-pr82.

Finally,

p12(E),

found in the viral

envelope,

is

presumably

derived from

p15(E) (12,

24; D.van

Zaane, thesis, University of Nijmegen, 1977).

Using

gel

filtration columnsthatdidnotresolve

the two gag precursor

polypeptides

gag-pr75

and

gag-pr65,

Stephenson et al. (18) showed

thattwo ts mutants of

R-MuLV,

ts25 and

ts26,

possessed temperature-sensitive defects in the

proteolytic cleavageof thegaggene-related

pre-cursorpolypeptides. These studieswere under-takentolocalize the defectsmorepreciselyand

toinvestigatewhether the other thermosensitive

replication mutants of R-MuLV also possess

thermosensitive defects in gene

expression.

Therefore, the gene expressions of two

early

(ts17 and

ts29)

and one late (ts28) ts mutants

553

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

(WT) R-MuLV.

MATERIALS AND METHODS Materials.i,-[n'S]methionine (specific activity,380 Ci/mmol) anda'4C-labeled amino acid mixture

(spe-cificactivity,54mCi/matom)wereobtained fromthe Radiochemical Centre, Amersham, England. Anti-gp69/71 and anti-p15sera were the generous gift of M. Strand and J. T. August. Characteristics of these antisera weredescribed by Strand and August (20).

Anti-R-MuLV serum, anti-p30 serum, anti-p15(E),p12(E) serum, andanantiserum raisedagainst bovine serum albumin (anti-BSA serum) were pre-pared and describedbyvanZaaneetal.(24).

Cells and viruses. NIH/3T3 cells (21) were cul-tured as monolayers in Dulbecco's modification of Eagle mediumsupplementedwith 10%fetal calfserum (Grand IslandBiologicalCo.Bio-CultLtd.).

The viruses used included a clonal isolate of WT R-MuLV (WT-248) and five R-MuLV ts mutants, tsl7, ts25, ts26, ts28, and ts29 (15). NIH/3T3 cells were infected with virusasdescribed byStephenson

and Aaronson (15). Infection with ts R-MuLV was performed at31°C,and that with WT R-MuLV was

performedat37°C.

JLS-V9 cells, derived from bone marrow cells of BALB/c mice (4) infected with and producing R-MuLV (V9-R-MuLV), were cultured as monolayers

inEagle minimal essential mediumsupplementedwith 10%calfserum.V9-R-MuLV labeled with

L-[nS]me-thionine was isolated as described byDuesberg and Robinson (5) andwasusedas areference.

Labeling of cells andpreparation of lysates. Subconfluent monolayers of cells growing in plastic

tissue culture flasks (Falcon Plastics, 25 cm2) were starved for 10min inlabelingmedium (Hanksbasic salt solutionsupplementedwith 10%dialyzedcalf se-rumand amino acids except for the radioactive ones). Then, the mediumwasreplaced by labeling medium with aradioactive amino acid(s), and the cellswere

incubated for30min. Afterbeing pulse-labeled, the cells werewashed once, and4 ml of normal culture medium was added; thereafter, the cellseitherwere

lysedimmediatelyorradioactivitywaschased for

var-ious times. Insomeexperimentsthetemperaturewas

shiftedduringthe chaseperiod.Whenindicated,the temperatureshift from 39 to31°Cwasperformed2h afterthebeginningofthe chaseperiod;the tempera-tureshiftfrom31 to39°Cwasperformed immediately

afterpulse-labelingof the cells.

Afterpulsesaswellasafterchases,thecellswere lysed by the addition to the cultures (i.e., cellsand medium)of1ml ofabuffercontaining50mMsodium phosphate (pH 7.2),4.5%sodiumchloride,5.0%Triton X-100, 2.5% deoxycholate, and0.5% sodium dodecyl sulfate (fivefold-concentrated immunoprecipitation

buffer [24]).Afterclearing thelysates by

centrifuga-tion at 220,000 x g for 10min in aTi5Orotor ofa

Spinco L-50 centrifuge, the lysates were stored at -800C.

Immunoprecipitation and polyacrylamide gel

electrophoresis. Detection of R-MuLV-specific

duredescribed byvanZaaneetal. (24). Immunopre-cipitates were analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis according to

Laemmli (10) on 7 to 18% polyacrylamide gradient

slabgels. Radioactivitywasvisualizedbyscintillation

autoradiography(3).

RESULTS

Synthesis and identification of

virus-spe-cific

polypeptides

inNIH/3T3 cells infected

with

WT R-MuLV

(WT-248).

The synthesis

ofvirus-specific polypeptides in NIH/3T3 cells

infected with WT R-MuLV was examined in

pulse-chase experiments at 37°C (Fig. 1A-G).

Cells

were

pulse-labeled

for30minwith a

"C-labeled amino acid mixture, and,thereafter,

ra-dioactivitywaschased for0and20h.Theviral

structural polypeptideswereidentified through

immunoprecipitation

analysis with well-defined

(see Materials and Methods)

monospecific

anti-sera and a polyvalent antiserum against

R-MuLV polypeptides.

Radioimmunoprecipita-tionanalysis of pulse-labeled

cells

with

anti-R-MuLV serum revealed that in addition to the

virus-specific precursor polypeptides gag-pr75,

gag-pr65, and env-pr82

(cf.

references 1, 24),

small amounts of two high-molecular-weight

polypeptides

(indicated with

0)

were

precipi-tated

(Fig.

1A).Uponanalysis with monospecific

antisera it appeared that both

high-molecular-weight

polypeptides

carried antigenic

determi-nantsofthegag-relatedstructuralpolypeptides

p30

and

p15,

butnotoftheenv-related structural

polypeptide gp69/71 (not shown). These

high-molecular-weight polypeptides are therefore

probably virus-specific polypeptides, which in

addition to the antigenic determinants of the

gag-precursorcontainother virus-specific

anti-genic determinants (possibly of reverse

tran-scriptase [8]). They might represent

virus-spe-cific precursor polypeptides whose synthesis is

due to an occasional readthrough at the gag geneterminationsite on aviralmRNA (14).

Moreover, afterpulses as well as after chases, someadditionalhigh-molecular-weight polypep-tides (>90,000) were precipitated with the

anti-seraused,

especially

withtheanti-R-MuLV

se-rum(Fig.

1A-O)

and theanti-p15(E),p12(E)

se-rum(Fig. 1D, indicated with *). Probably, many of these polypeptidesarenot virusspecific and represent cross-reacting host

cell

polypep-tides. However, some of them might be degra-dation products of the high-molecular-weight

precursor polypeptidessynthesizedaccording to a readthrough mechanism. We will not discuss

thesignificanceof these products here, because

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

PRECURSOR POLYPEPTIDES OF ts R-MuLV

pzr-82 _

pr-75,_

c3jl69/71

p-65-

-F_ _

W--..

-pr-82

&a- ,am --Pr75

-r

_ 4--o gp69/71

_oi__p-.-pr65

__

p)30_

p151EI -.-p122rE pl15[E]

pl5-p12[EI

A B C D E F

Chlase- ' 11hr ) 20 20 20 20 20 c 20

H J K M N 0

0 2 7 20 0 2 7 20

FIG. 1. Normalprocessing of gag and env products of WT R-MuLV (WT-248). Subconfluent monolayers

ofNIH/3T3 cellsinfectedwith WTR-MuL V were pulse-labeled for 30minwith 100

,LCi

ofa'4C-aminoacid mixture (A-G) or with 75liCiof

L-[PS]methionine

(H-O),andradioactivity was chased for 0, 2, 7, and 20 h as indicated. The pulse-chase experiments wereperformedat 31°C(H-K), 37°C (A-G), or 39°C(L-O).After

lysis ofthe cellsasdescribed in the text, virus-specific polypeptides were analyzed by sodium dodecyl

sulfate-polyacrylamide gelelectrophoresisandscintillation autoradiography after immunoprecipitation with: anti-R-MuLVserum(A, B,H-O);anti-gp69/71serum(C);anti-pl5(E),pl2(E) serum (D); anti-p30 serum(E); anti-p15serum(F);oranti-BSAserum(G). Bands marked with * and*are discussed in the text.

theirsynthesis does not seem to be affectedin

anyofthemutants wehaveinvestigated. After

a chase period of20 h,

radioimmunoprecipita-tion analysis of labeled cultures (i.e., cells and

medium)withanti-R-MuLVserumshowedthat

the gag and env precursor polypeptides had

disappeared

and that the viral structural

poly-peptides gp69/71, p30,

p15(E),

andp12(E)were

formed(Fig.

1B).

These viral structural

polypep-tideswereidentifiedwithmonospecificantisera

(Fig.

1C-F).

Inacontrol

experiment

noneof the

polypeptides

could be

precipitated

with

anti-BSA serum

(Fig.

1G).

It should be noted that

thegag

products

p12andplOarenot

recognized

bytheanti-R-MuLVserumused.

Asacontrolfor the

analysis

of thetsmutants,

Fig.

l(H-O)

showsthe results ofa

pulse-chase

experimentat31°C

(Fig. 1H-K)

and

39°C

(Fig.

1L-O) with

NIH/3T3

cells infected with WT

R-MuLV

(WT-248).

Cells were

pulse-labeled

with L-[35S]methionine for 30 min, and,

there-after,

radioactivity

was chased for 0, 2, 7, and

20 h.

Radioimmunoprecipitation analysis

was

performed

with anti-R-MuLV serum. It

ap-pearedthatatbothtemperaturesthe precursor

polypeptide

env-pr82, as well asgag-pr75 and

gag-pr65, was cleaved into virus-specific

struc-tural polypeptides (Fig. 1H-O). It should be

noted thatp15does notcontainmethionine (7)

andconsequentlyisnotdetected whencellsare

labeledwithL-[35S]methionine.

Localization

of theblockageinthe

cleav-ageof the gag

gene-related

precursor

poly-peptides

of ts25 and ts26. The

cleavage

of

thegaggene-related precursor

polypeptides

of

ts25 and ts26 was shown to be blocked atthe

nonpermissive temperature (2, 19). To localize

the

blockage

in the

cleavage scheme,

the

syn-thesisandprocessingofvirus-specific precursor

polypeptides was examined in NIH/3T3 cells

infectedat31°C withts25andts26.Pulse-chase

experiments were performed at 31 and 39°C,

andthe

virus-specific polypeptides

were

immu-noprecipitated with anti-R-MuLV serum. The results obtained with ts25 arepresented inFig.

2. At31°C no differences in the synthesisand

processing of the precursorpolypeptidesofts25

and WT-248 could be observed (cf. Fig. 2A-D

and Fig. 1H-K). In accordance with the work

of Stephenson et al. (19), Fig. 2(E-H) shows

that the processing of the gag gene precursor

polypeptideswasimpairedatthe

nonpermissive

VOL. 25,1978 555

I-anit-- -

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.500.49.444.77.308.2]

(4)

pt-82_, pr-75

gl.)69/71

-pr 65*

a

a*~~~~b

O. - ama

4o

~~~~~~~.-. w4__~ _

p30

--p15[E I---

-121E2I1 E

A B (. D E F G H

Chasse' iTA("0 20 ( 2 20 2 4

FIG. 2. Thermosensitiveprocessing ofgagproductsofts25. Subconfluent monolayers of NIH/3T3 cells

infectedat31°Cwithts25werepulse-labeled for30min with 75,uCiof L-[35S]methionine, andradioactivity was chasedforvarious times asindicated (A-P). Pulse-chase experiments wereperformed at 31°C(A-D,

O-P)or39°C(E-H).Insome casesthe temperaturewasshifted forachaseat31°C (I-K)or39°C(L-N), as

described in the text. Thus, "chase in h2l/4" indicates a2-h chase at39°C followed by a 0.25-h chaseat

31°C,etc.Virus-specific polypeptideswereanalyzedasdescribed in thelegendtoFig.1.Immunoprecipitation

with: anti-R-MuLVserum(A-N);anti-BSAserum(O-P); reference V9-R-MuLV labeled with

L-[35S]methio-nine(Q).

temperature; gag-pr65 of ts25 is accumulated

and no p30isformed. On the other

hand,

like

WTgag-pr75, thegag-pr75 ofts25isconverted

into gag-pr65. When the temperature was

shiftedto31°C2hafter

pulse-labeling

the cells

at 39°C (Fig.

21-K),

the

proteolytic

cleavage

was resumed and p30was produced. When, in

another experiment, the temperature was

shifted from 31°C (during the pulse) to 39°C

(duringthechase), theproteolytic cleavage was blocked (Fig. 2L-N).

The results of parallel experiments with NIH/3T3 cells infected with ts26 (not shown) weresimilartothose observed with ts25; again,

at39°Cgag-pr65wasaccumulated, but the

mu-tationwasslightly leaky:cellsinfected with ts26

producedsmallamountsofp30attherestrictive

temperature(Fig. 3G-H).

A more detailed analysis of the blockage in

the proteolytic cleavage of the gag precursor

polypeptidesofts25andts26 wasobtained after

radioimmunoprecipitation analysis with

mono-specific antisera

(Fig.

3). Cells infectedwith ts25

orts26werelabeledfor30minwitha

"'C-amino

acid mixture at 31 or

39°C,

and radioactivity

waschasedfor20h. Figure3showsthatatthe

nonpermissive

temperaturegag-pr65 was

accu-mulatedincells infectedwith ts25

(Fig. 3M-O)

orts26(Fig. 3G-I);furthermore, the

production

of the

gag-related

structural

polypeptides

p30

and

p15

was inhibited. In addition to the

im-pairedprocessingof the gagprecursor

polypep-tides of ts25and ts26,ablockagein the

produc-tion of an env

gene-related

structural

poly-peptidewasalsoobservedattherestrictive

tem-perature.

Radioimmunoprecipitation

analysis

with anti-R-MuLVserum (Fig.2H and N; Fig.

3G and M), anti-gp69/71 serum (Fig. 3K and

Q), and

anti-p15(E),p12(E)

serum (Fig. 3J and

P) revealed that production of

p12(E)

was in-hibited. Production of the structural envelope

polypeptides gp69/71 and

p15(E),

however,

seemedtobe normal (Fig.2F-H andFig. 3G, J,

K, M, P,andQ).Itshould be noted that

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.500.67.454.76.349.2]

(5)

I

gp69/71-

d

pr65 _

I1.

a

_.-_-*

.l"i

p15(E) _ _

p15 a

p12(E)

I:

t-O

lb

7-

-_

^o

_im- --- ---P15(FE)

_-

_--A B C D E F _G _H _J _K _L _M _N

O p Q

FIG. 3. Leakythernosensitive processing ofgagproducts ofts26; thermosensitive productionof the env

productp12(E)in additiontothethermosensitiveprocessing ofgagproducts ofts25andts26.NIH/3T3 cells

infected at31°Cwith ts26 (A-L) orts25(M-Q) werepulse-labeled for30min with 100,uCi ofa 14C-amino

acidmixture,andradioactivitywaschasedfor20 h.Pulse-chaseexperimentswereperfornedat31°C (A-F)

or39°C(G-Q). Virus-specific polypeptideswereanalyzedasdescribed in thelegendtoFig.1. Immunoprecip-itation with: anti-R-MuLV serum (A, G, M); anti-p30 serum (B, H, N); anti-p15 serum (C, I, 0);

anti-p15(E),pI2(E)serum(D, J, P); anti-gp69/71serum(E, K, Q); anti-BSAserum(F, L).

times pl5(E) seemed to be composed of two components(Fig. 1, 2, and 3). Bothpolypeptides

were immunoprecipitated with anti-R-MuLV serum(Fig. 1, 2, and 3A, G, and M), anti-gp69/71 serum(Fig. 3E, K, and Q), andanti-p15(E),p12(E) serum (Fig. 3D, J, and P). [Incidentally, the

precipitation of p15(E) and p12(E) with anti-gp69/71 serum is presumably due to the pres-enceofacomplex betweengp69/71, p15(E), and

p12(E)asdiscussedbyvanZaaneetal. (24) and

Leamnsonetal. (11).]

Synthesis ofvirus-specific polypeptides

in NIH/3T3 cells infected with tsl7, ts28,

andts29. Twoearlytemperature-sensitive

mu-tants ofR-MuLV, tsl7andts29,wereshownto

bedefective inone or morefunctionsbefore the

synthesisofgroup-specific antigens (15). To

ex-amine the possibility that these mutants also

hadadefect in theproteolytic cleavageoftheir precursor polypeptides, the synthesis of virus-specific polypeptideswasstudiedat31and39°C, after infection at the permissive temperature.

Cells were labeledwith L-[35S]methionine, and

radioimmunoprecipitation was performed with

anti-R-MuLVserum.Theresultsobtained with

ts17 and ts29 are presented in Fig. 4 and 5,

respectively. In bothcases,gag-pr75 was

proc-essedtogag-pr65atthenonpermissive

temper-ature.However,proteolytic cleavagewasblocked

beyond gag-pr65, since no p30 was formed.

When the temperature was shifted from 39 to

31°C, cleavage was resumed, as could be

con-cluded from theproduction of p30. Furthermore, the inhibitionoftheproductionofp12(E)atthe

restrictive temperature, asfound incultures

in-fectedwithts25andts26,wasalsoobserved.On

the otherhand, the latemutant ts28, similarly analyzedinparallel experiments,wasnot defec-tive fortheprocessing of itsprecursor

polypep-tides. The relevant pulse-chase experimentsat 31and39°CareshowninFig. 6.

DISCUSSION

Some temperature-sensitive mutants of

R-MuLVarepartiallycharacterized (15, 16, 18, 19, 23,27). Amongthemarethe mutantsts17, ts25,

ts26, ts28, and ts29, which were used in this

study.

At39°C,someof thesemutantsaredefective in either early replication steps (class I

repre-sentedby tsl7andts29)orpostintegrationsteps (classIIrepresented by ts25andts26, andclass

'71

pr65

VOL. 25,1978 557

E-0 t

ago

ob.ft .

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.500.45.436.69.310.2]

(6)

."! 8-), _a

p6t75

-!!)69

J71

:.- (I5z 0

" .

_NW *N -0

maegm-

a*MOW

ow-.

_so

Ammww ---__ _w _4m- op

r)30

;5(L,([- _

;,12(ir ;:

rF L aj :A'\^ I'

FIG. 4. Thermosensitiveprocessing of gag products of tsl7and concomitantfailure to produce the env productp12(E).NIH/3T3cellsinfectedat31°C with tsl7werepulse-labeledwith75,iCi ofL-[35S]methionine

for 30 min, and radioactivity waschasedfor various times asindicated (A-M). Pulse-chase experiments wereperformed at 39°C (A-D) or31°C (H-M). The temperature was shifted from 39 to 31°C (E-G), as

described in thetext(seealsolegend toFig. 2). Virus-specificpolypeptideswere analyzedasdescribed in thelegendtoFig.1.Immunoprecipitationwith: anti-R-MuLVserum(A-K);anti-BSAserum(L-M);reference

V9-R-MuLV labeled withL-[35S]methionine (N).

IIIrepresented by ts28) (15). ts25andts26 are

postintegration defectivemutantswhose defect

is known to involve the cleavage of the gag

gene-related

precursor

polypeptides

(2, 19).

Mu-tantts29 possessesathermolabilereverse

tran-scriptase (23) and ts28 has an unidentified ts

defectat alate step in itsreplication (15, 27).

In this study cells were infected with these

mutants atthepermissive temperature (31°C),

and subsequently thesynthesis and processing

ofvirus-specificprecursorpolypeptideswere

ex-amined at the permissive and nonpermissive

temperatures. At 31°C no differences in the processing of the gag and env gene-related

pre-cursor polypeptides were observed in cells in-fected with either WT R-MuLV or oneof the

ts mutants (Fig. 1, 2, 4, 5, and 6). However, at

39°C,ablockagein theprocessingofagag

gene-related precursor polypeptide of the mutants

tsl7, ts25, ts26, and ts29 was observed (Fig. 2,

3, 4,and 5). When the temperature wasshifted

down to

31°C

after

pulse-labeling

the cells at

39°C, processing ofthegag precursor

polypep-tidesto the internal structural virion

polypep-tides p30 (Fig. 2, 3, 4, and 5) and p15 (Fig. 3)

was observed. The impaired processing of the gag precursor polypeptides at the restrictive temperature was localized beyond gag-pr65, since gag-pr75 disappearedbut gag-pr65

accu-mulated andp30wasnotproduced (Fig. 2, 3, 4, and 5). In all analyses, impaired production of

p12and plOescaped detection, since these

virus-specific polypeptideswere notrecognized by the

anti-R-MuLV serum. However, presumably

noneof the gaggene-relatedstructural polypep-tides isproduced at39°Csince all four subgene

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.500.72.459.69.382.2]

(7)

pr82 _ pr75_

gFp69/71 pr65

II 01- i

.*.. lo M a 4*

k._.

~~~~~~~~~~~mub-1

~ ~ ~ ~ ~ ~ ~~omkl 4

wql qwp w w qW

Pl5CE)

--p12(E}

Ch-ase in hr: A₀ B₁ C₂ ₂₀D E₀ ₁F G₂ ₂₀H _{21/4 21/2}J ₂₁K L

2.20

FIG. 5. Thermosensitiveprocessing of gag products of ts29 and concomitant failure to produce the env gene-related productp12(E). NIH/3T3 cellsinfectedat31°C withts29 werepulse-labeled for30minwith75

yCiof

L-[3S]methionine,

andradioactivity waschasedfor various timesas indicated(A-L). Pulse-chase

experiments wereperformed at31°C (A-D) or39°C (E-H). The temperature wasshifted from 39to 31°C

(I-L), as described in the text (see also legend to Fig. 2). Virus-specific polypeptides were analyzed as

describedin thelegendtoFig. 1.Immunoprecipitationwith: anti-R-MuLVserum(A-L).

products, p15, p12,p30, and plO,arepresent in

the smallergag precursor

polypeptide

gag-pr65

(van Zaane,

thesis).

This conclusion does not

exclude the

possibility

that

eventually

gag-pr65

isdegraded andmay give rise to anincomplete

setofgagsubgene products (cf. Barbacid etal.

[2]). The impaired processingof the gag

precur-sor polypeptide of ts25 and ts26 is consistent

with the observationsofStephensonetal. (19).

Thepresenceof thesamedefectinthemutants

ts17 and ts29 is

quite

unexpected, since both

werecharacterizedasearlymutants(class I).In

the case of ts29 the early replication defect is associated withathermolabilereverse

transcrip-tase (23).Furtherevidence forasecond

replica-tion defect in ts29 is obtained from an electron

microscopic study by Yegeretal. (27).

Upon ashift to the nonpermissive tempera-ture after infection atthe permissive tempera-ture, these authors found an accumulation of

immature virions at the cell surface. The

im-maturevirionswereinan

early

stage ofbudding,

suggestinga

replication

defectat an

early

stage

of virion

assembly.

Whereas cultures infected

with ts17, ts25, ts26, and ts29revealed an

im-paired

processing

of the gag precursor

polypep-tides at the

nonpermissive

temperature,

produc-tion of theenv

gene-related polypeptide p12(E)

wasalso inhibited (Fig. 2, 3, 4, and5). It isnot

yet clear whether or not there is a relation

between these defects.For

instance,

the defect

could be

primarily

invirion

assemblage, and,

as

aconsequence, thecleavageof different

precur-sorpolypeptidescould be blocked. As discussed

above, two of the mutants of this

phenotype

(ts17 andts29)possessanadditional

early

defect onthe basis of whichtheywerelistedasclass I mutants. Again, it is not known whether they

are double (or triple) mutants or single

pleio-tropicmutants.

25,1978 559

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.500.101.384.74.372.2]

(8)

.%*-i:W%m qa.*r- " *

-~~~~~~~~~~~~m._.

VISA,

pI5cE)---p12(

E)

-A B C D E F G H

Chase in hr: ₁ ₂ ₂₀ _22/4 _21/221 _2,200

21 2, f ,2

FIG. 6. Normalprocessing ofgagandenvproductsofts28.NIH/3T3cellsinfectedat31°Cwith ts28were

pulse-labeled for30minwith 75 jCiofL-[35S]methionine,andradioactivitywaschasedforvarious timesas

indicated(A-L).Pulse-chaseexperimentswereperformedat39°C (A-D)or31°C(I-L).Thetemperaturewas

shifted from39to31°C (E-H),asdescribed in the text.Virus-specific polypeptideswereanalyzedasdescribed

in thelegendtoFig.1 (seealsolegendtoFig.2). Immunoprecipitationwith: anti-R-MuLVserum(A-L).

Finally,the structuralpolypeptidesof ts28are

produced normally at the restrictive

tempera-ture(Fig. 6). However,thedistortedmorphology of the virions formed at 390C (27) suggests a

defectinalatestageof theassembly.

ACKNOWLEDGMENTS

We thank J. R. Stephensonand S. A. Aaronson for the clonal stockofwild-typeR-MuLV and the ts R-MuLV

mu-tants, andwethankM. Strand and J. T.Augustfor the anti-gp69/71 andanti-p15sera.

These investigationswere supported by the Foundation

for Medical Research FUNGO,which is subsidized by the NetherlandsOrganizationforAdvancement of PureResearch (Z.W.O.).

LITERATURE CITED

1. Arcement, L. J., W. L. Karshin, R. B. Naso, G. Jamjoom,and R. B.Arlinghaus. 1976. Biosynthesis ofRauscher leukemiaviralproteins: presence ofp30 andenvelope p15sequencesinprecursorpolypeptides.

Virology 69:763-774.

2. Barbacid, M.,J. R.Stephenson,and S. A.Aaronson. 1976. Gag-gene of mammalian type-C RNA tumour

viruses. Nature(London) 262:554-559.

3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids inpolyacrylamidegels. Eur. J. Biochem. 46:83-88.

4. Chopra, H. C., and G. P. Shibley. 1967. Infection ofan

establishedmousecell line with cell-free Rauscher

leu-kemia virus.I.Cytological studywith the electron

mi-croscope.J. Natl.Cancer Inst. 39:241-256.

5.Duesberg, P. H.,and W. S. Robinson. 1966. Nucleic

acids andproteinsisolated from the Rauschermouse

leukemia virus (MLV). Proc. Natl. Acad. Sci. U.S.A. 55:219-227.

6.Friis, R. R., K. Toyoshima, and P. K. Vogt. 1971.

Conditionallethal mutants of aviansarcomaviruses. I.

Physiologyof ts75 and ts149.Virology43:375-389.

7. Ikeda, H.,W.Hardy,Jr.,E.Tress,and E. Fleissner.

1975.Chromatographic separationandantigenic

anal-ysis of proteins of theoncornaviruses. V. Identification

ofanewmurineviralprotein, p15(E).J. Virol. 16:53-61.

8. Jamjoom,G.A.,R. B.Naso,and R. B.Arlinghaus. 1977. Further characterizationof intracellularprecursor polyproteins of Rauscher leukemia virus. Virology 78:11-34.

9. Kawai, S., and H. Hanafusa. 1972. Plaqueassay for

somestrains of avian leukosis virus.Virology48: 126-135.

10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assemblyof the head ofbacteriophageT4.

Nature(London)227:680-685.

11. Leamnson,R.H.,M. H. M.Shandler,and M. S.

Hal-pern.1977. A structuralprotein complexinMoloney leukemiavirus.Virology76:437-439.

12. Naso, R. B., L.J. Arcement,W. L.Karshin, G. A.

Jamjoom, and R. B. Arlinghaus. 1976. A fucose-deficientprecursortoRauscherleukemia virusgp69/71. Proc. Natl. Acad. Sci. U.S.A. 73:2326-2330.

13. Scolnick,E.M.,J. R.Stephenson,and S. A.

Aaron-Pr

82I

pr

75

gP9/7

1 0

Pr65

0

J K

1 2

L

20

MWAO.Immmm.-.... ,WI; .110mmo.. .:...

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.500.90.439.71.350.2]

(9)

son. 1972.Isolationoftemperature-sensitive mutants

of murinesarcomavirus. J. Virol.10:653-657.

14. Shapiro, S. Z., and J. T. August. 1976. Proteolytic cleavage events in oncornavirus protein synthesis. Biochim.Biophys.Acta 458:375-396.

15. Stephenson, J. R., and S. A. Aaronson. 1973. Charac-terizationoftemperature-sensitive mutantsofmurine leukemia virus.Virology 54:53-59.

16. Stephenson, J. R., and S. A. Aaronson.1974. Temper-ature-sensitive mutantsof murineleukemia virus: III. Mutants defective inhelper functions forsarcomavirus fixation.Virology 58:294-297.

17. Stephenson,J.R.,R. K.Reynolds, and S. A.

Aaron-son. 1972. Isolation oftemperature-sensitive mutants

ofmurineleukemia virus.Virology48:749-756. 18. Stephenson, J. R., S. R. Tronick, and S. A. Aaronson.

1974.Temperature-sensitivemutantsofmurine leuke-mia virus. IV. Furtherphysiological characterization and evidence for genetic recombination. J. Virol. 14:918-923.

19. Stephenson,J.R., S. R. Tronick, and S. A. Aaronson.

1975. Murineleukemia virusmutantswith temperature-sensitive defects inprecursorpolypeptide cleavage. Cell 6:543-548.

20. Strand, M.,and J.T.August.1976.Structural proteins of ribonucleic acid tumor viruses. J. Biol. Chem. 251:559-564.

21.Todaro, G., and H. Green. 1963. Quantitative studies

onthegrowth ofmouse embryo cells inculture and theirdevelopment into established lines. J. Cell. Biol. 17:299-313.

22.Toyoshima, K., and P. K. Vogt. 1969. Temperature-sensitivemutantsofanaviansarcomavirus.Virology

39:930-931.

23.Tronick, S. R., J.R. Stephenson, L.M.Verma, and S. A.Aaronson. 1975. Thermolabilereverse

transcrip-taseofamammalianleukemia virusmutant tempera-ture-sensitivein itsreplication andsarcomavirushelper functions. J. Virol. 16:1476-1482.

24.vanZaane, D., M. J. A. Dekker-Michielsen,andH. P. J. Bloemers.1976.Virus-specificprecursor polypep-tides in cells infected with Rauschermurineleukemia virus:synthesis, identificationandprocessing. Virology 75:113-129.

25.Wong, P. K. Y., and J. A. McCarter. 1973. Genetic studies of temperature-sensitive mutants ofMoloney murineleukemia virus. Virology 53:319-326. 26. Wyke, J.A.1973.The selectiveisolation of

temperature-sensitive mutants of Rous sarcoma virus. Virology 52:587-590.

27. Yeger, H., V.I.Kalnins, and J. R. Stephenson. 1976.

Electron microscopy ofmammalian type-C RNA

vi-ruses: useof conditional lethalmutants instudies of virion maturation andassembly. Virology 74:459-469.

561