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0022-538X/83/050538-09$02.00/0

Copyright © 1983, American Society for Microbiology

Deletion

Mutants

of Moloney Murine Leukemia Virus Which

Lack

Glycosylated

gag

Protein Are Replication

Competent

PAMELASCHWARTZBERG, JOHN COLICELLI,ANDSTEPHEN P.GOFF*

Departmentof Biochemistry, Columbia University, College of Physicians and Surgeons, Cancer Research

Center,

New York,New York

10032

Received 28 December 1982/Accepted23February 1983

Aseries of deletion mutations localizednearthe 5' end of the Moloney murine

leukemia virusgenome was generated by site-specific mutagenesis of cloned viral

DNA. Themutants recoveredfrom such deleted DNAsfailed to synthesize the

normal glycosylatedgagprotein gPr80Wag.Twoof the mutants made no detectable

protein, and a third mutant, containing a66-base pair deletion, synthesized an

altered gag protein which was not glycosylated. All the mutants made normal

amounts of the internal PrO5gag protein. The viruses were XC positive and

replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These

results indicate that the additional peptides ofthe glycosylated gag protein are

encoded near the 5' end, that the glycosylated and internal gag proteins are

synthesized independently, andthatthe glycosylated gagprotein isnot required

during the normal replication cycle. In addition, the region deleted in these

mutants apparently encodesno cis-acting functionneeded for replication. Thus,

all essentialsequences, including those for packaging viralRNA, must lie outside

thisarea.

The structure of the Moloney murine

leuke-miavirus(M-MuLV)genome is known ingreat

detail; indeed, the complete nucleotidesequence

hasbeen determined (32). The detailed functions carriedoutby the

products

of thegag,

pol,

and

env genes, however, are largely unknown (5).

For

example,

although it is known that thegag

proteins are structural elements in the virion

particle,

the actual role of each mature

protein

(termed P15, P12, P30, and P10) is uncertain.We

have begunagenetic

analysis

of thegenomeof M-MuLVinanattempt todetermine the various functions of the

proteins

and regulatory

se-quences

required

in the viral life

cycle.

The first 620 nucleotides of the viral RNA

(extending

from the

capped

5' end totheAUG

codon utilized for the synthesis of the Pr65gag

protein) are required for several functions

im-portant tothereplication ofthevirus. Contained

inthisregionare(i)thebinding site for

tRNAPro,

whichprimesDNAsynthesis (26);(ii)thesplice

donorsite for the formation ofenvmRNA; (32;

J.

Champoux, personal

communication); (iii) signals for packaging of viral RNA into virion particles (30);and(iv)aregion ofunknownsize encoding N-terminal peptides ofthe glycosylat-ed gag protein termed gPr80gag (7). The region fromthe splice donor site(atnucleotide205) to

the start of

PrO5gag

(at nucleotide 621)

consti-tutes the largestregion ofthe virus with

uncer-tain function. Work on avian retrovirus mutants

(30)hasshown thatpartofthe

region

isessential for RNA

packaging,

but theextent of the

puta-tive recognition site is unknown. In the caseof M-MuLV, a large part of this RNA must also encode the

glycosylated

gag

protein

gPr80gag.

Thisprotein

contains

essentially all of the

pep-tides of Pr65gag and

approximately 4,000

to

10,000 daltons of additional

protein

at the N

terminus (7); additional amino acids may be utilized as a signalpeptide

directing

the

protein

to the endoplasmic reticulum fortransport and glycosylation. Mannose-rich core

oligosaccha-ride chainsareaddedtotheprotein,

probably

in

the P15 and P30regions (7), andarethen

modi-fied furtherbytheaddition of

complex

oligosac-charides. The

protein

is

transported

to the cell surface, and a portion may be shed from the

cells (6). The AUG sequence at which

protein

translation starts is unknown. The function of this protein is equally unclear,

although

it is known to constitute the GCSA

antigen

on the

surface of infectedcells;aroleinvirion

matura-tion has also beenproposed.

Many laboratorieshavegenerated mutants of leukemiaviruses which wereblockedatvarious

stages ofthe lifecycle (1, 9, 13, 20-22, 27, 31,

36, 40, 43); these mutantshavebeen

extremely

useful in definingthe variousfunctions carried

outby the virus. Theavailability of

fully

infec-tious DNA clones of the M-MuLV genome

makespossible theconstruction of defined

dele-538

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tion mutations at known sites within genes.

Thus, it should be possible to mutateany gene by modifying cloned DNA, to recover virus

fromcellstransfectedwith the altered DNA, and toobserve the effect ofthemutationon thelife

cycleofthe virus. To definemorepreciselythe

extentandpositionof thesequences neededfor

viral genomic packaging and for synthesis of

gPr809'9,

wehavecarriedoutsite-specific

muta-genesis ofacloned DNAcopy ofthe M-MuLV

RNAgenome. Surprisingly, several of the mu-tantDNAs gave riseto fully infectious replica-tion-competent virus,eventhough no

glycosyl-atedgag proteinwasmade.

MATERIALS AND METHODS

Cells and virus. NIH/3T3 fibroblastsweregrownin

Dulbecco modified Eagle medium (GIBCO

Labora-tories) supplementedwith10% calfserum(Flow

Labo-ratories,Inc.).M-MuLVreleased byNIH/3T3 clone4

wasthesourceofinfectious wild-type virus. This cell

linewasderivedas asingle-cell clone of NIH/3T3 cells

infected at lowmultiplicity (<0.05) with the clone 1 strain of M-MuLV(11). Viralinfectionsathigh

multi-plicity were carried out in the presence of 8 ,ug of

Polybrenepermlat37°Cfor 2h,and theinfectedcells

were then maintained in 1.6 jig ofPolybreneper ml

overnight.LymphocytecelllinesSWR/4 (34)and 70Z

(25) were grown in RPMI 1640 medium (GIBCO

Laboratories) supplementedwith10% fetal calfserum.

Cloned DNAs. Plasmid p8.2 contains a full-length permutedcopyof M-MuLVcircular viralDNA, joined

attheuniqueHindIll site in thecenterof thegenome

and containing only one copy of the long terminal

repeat(LTR).Theclonewaspreparedby subcloning

theHindIll viral DNAfragmentof lambda8.2(33)into

theHindIlI site ofplasmid pBR322 bystandard meth-ods.

PlasmidpZAPcontainsafull-lengthproviralcopyof M-MuLV plus approximately 8 kilobases offlanking rat sequencessubcloned from phage lambdainto the EcoRI site ofplasmid pBR322(15).PlasmidpSV2GPT

(gift from R. Mulligan) contains theEscherichia coli

gptgenelinkedtoasimian virus 40promoter(24).

DNAenzymology.Partial restrictionenzymedigests

ofplasmidDNAswerecarriedout at20°Cover arange

of time periods (5 min to 1 h). Full-length linear molecules were isolated by electrophoresis in 0.6% agarose (SigmaChemicalCo.) gelcastinTEA buffer

(50mMTris-hydrochloride, 1 mMEDTA,aceticacid

topH 8.0) containing1 ,ugof ethidiumbromideperml

and were purified bythe glass powder method (41).

This linear DNAwasdigestedwith Bal 31exonuclease

at25°Cfor 20sinbuffer(0.3MNaCl,6mMCaC12,6

mM MgCl2, 10 mMTris-hydrochloride [pH 8.0], 0.5

mM EDTA). The reaction was quenched with the

additionof EDTA toafinalconcentrationof20mM,

and the DNAwaspurified bysuccessive extractions

withphenoland ether andprecipitationwithethanol.

The DNAwascircularized with T4ligase (New

En-glandBioLabs)ataDNA concentration of1 Fg/mlin

buffer (50 mM Tris-hydrochloride [pH 7.5], 10 mM

MgCl2,10 mMdithiothreitol,1 mM ATP).

Bacterial cultures. E. coliHB101(4)wasgrownin L broth(10goftryptone[DifcoLaboratories]perliter,5

g of yeast extract [Difco] per liter, 5 g of NaCl per liter,

1 mM NaOH) and was transformed to ampicillin

resistance by the CaCl2 method (4). Selective plates and media contained 50 1tg of ampicillin per ml.

Plasmid DNA wasisolated from small cultures (2 ml)

by the rapid preparation of Holms and Quigley (16)

andfrom large cultures (200 ml) by equilibrium

centrif-ugation incesiumchloride-ethidium bromide (17).

DNAsequence analysis. Mutant DNAs (1 to 5 ,ug)

were cleaved with BstEII (New England BioLabs),

and the terminal 5' phosphate was removed by

treat-ment with calf intestinal phosphatase (Boehringer

Manheim Corp.) in buffer (10 mM Tris [pH 8.3], 10

mMMgCl2) for 1 hat37°C. The DNAwaslabeled with

polynucleotide kinase (Bethesda Research

Labora-tories) and [-y-, 32P]ATP (Amersham Corp.) and was

recleaved with Sacl (New England BioLabs). The

end-labeled DNA fragment containing the mutation

was isolated by electrophoresis on a10o

polyacryl-amide gel andsubjected tothechemical degradation

protocol of Maxam and Gilbert (23). Cleavage

prod-ucts weredisplayedon8%polyacrylamide-8 M urea

gels(34 by 14cm) andexposedtoX-ray film (Kodak

XR5, Eastman KodakCo.)withanintensifyingscreen

(Lighting Plus, Du Pont Co.)at -70°C.

Mammalian cellular transfections and

transforma-tions.Cloned mutated viral DNAswereexcised from

the pBR322vectorplasmid by cleavage with HindlIl

(Bethesda Research Laboratories) andwerereligated

at a concentration of 100 to 500 ,ug/ml to ensure

extensive concatemerization. Plasmid DNAs pZAP

andpSV2GPTwereusedascircular DNAs.NIH/3T3

cells were transfected with the DNAbythe calcium

phosphate precipitation method (14, 35) with salmon

sperm DNA as the carrier. Transfections for XC

plaque assayswereperformed by precipitating0.5 ,ug

of cloned DNA and6,ugofcarrier inavolumeof 0.5

ml andapplyingthemto6-cm dishescontaining

10'

to

1.5 x 10- cells per dish for4 to 6 h. At3 days after

transfection, cells were split 1:10, replated in 10-cm

dishes, and allowedtogrowtoconfluency.The

pres-ence of viable virus was determined by overlaying

withXC cells (29) after UV irradiation of the

mono-layer.

Cotransfections with plasmidpSV2GPTDNAwere

carriedoutessentially in the same fashion.Amixture

of1 ,ugof viral DNA,1 ,ugofpSV2GPT, and 12 ,ug of

carrierwasappliedto3 x 105to5 x 105cells ona

10-cmplate. At2 days after transfection, cells were fed

with XAT medium (15 mM hypoxanthine, 0.2 mM

aminopterin,5 mMthymidine, 250 mM xanthine, 150

mMglutamine, 5 mM glycine,25 mMmycophenolic

acid).Cellswererefed every 5days, and colonies were

cloned 3 weeks later. The presence ofreverse

tran-scriptase activity in the media was assayed by the

methodofGoffet al. (13).

Viralproteinanalysis.Cells (2 x 105per6-cm dish)

wereinfected with filtered virus harvested from clones

of transfectedmutants.After1day, cellswerepassed

1:10 into 10-cmdishes. Whenmonolayers were half

confluent,thecells werestarved for0.5 hin Dulbecco

modified Eagle medium minus methionine, and

[35S]methionine (200 ,uCi;AmershamCorp.)was

add-edfor15min.The cells werelysed,and theproteins

wereimmunoprecipitated with goat anti-M-MuLV

se-rumorrabbitanti-gagserum507(giftofD.Baltimore)

by standard procedures. Immunoprecipitated proteins

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were analyzed by sodium dodecyl

sulfate-polyacryl-amide gel electrophoresis and fluorography as de-scribedpreviously (18, 39).Theproteinsweretreated

withendo-p-N-acetylglucosaminidase(endoH;gift of

P.Robbins)asdescribed(38).

RESULTS

Construction of deletion mutants incloned

M-MuLV DNA. The DNA used as the parent

molecule for all the mutagenesis described in

this work was plasmid p8.2, an infectious

full-lengthcopyof the M-MuLVgenomeinplasmid

pBR322. This clonewasprepared by cleavage of

proviral circular DNAatthesingle HindIII site

before cloning; thus, the M-MuLV insert was

circularly permuted relative to the usual linear

form of the genome. A map of the insert is

shown in Fig. 1.

Mutantswereconstructedby deleting

particu-lar restriction endonuclease recognition sites

and part of the flanking DNA. To direct these

deletionstotheregion encoding the5'end of the

viral RNA, recognition sites for the enzymes

PstI and PvuIwereselectedastheinitialtargets.

These enzymes made cleavages in the area of

interest,although they also cleave elsewhere in

the M-MuLV genome and in the plasmid

pBR322 vector DNA. Plasmid p8.2 DNA was

partially digested with either PstIor PvuI, and

full-length linears (cleaved only once)were

iso-lated fromanagarosegel.This DNAwastreated

with Bal31 exonuclease to remove a fewbase

pairs from each end. It was next purified and

recircularizedbyligationatalowconcentration.

The modified DNA was used to transform E.

coliHB101toampicillin resistance, and 12

resis-tantcolonies werepicked and grown into small

culturesfor eachstartingenzyme.Plasmid DNA

isolated from each culture was analyzed by

agarose gel electrophoresis after cleavage with

PvuII and Sacl to determine the position and

approximate size of the deletion.

Approximately one-third to one-half of the

plasmids examined contained deletions in M-MuLV sequences. Two ofthe DNAs

mutagen-izedbycleavage with PstI contained deletions of

the entire 176-base pair(bp) PstI fragment

be-tween 1.0 and 1.15 map units and were not

examined further. A fewmutants had deletions

atthePstI and PvuI sites inplasmidpBR322,but

neverthelesstheymusthaveencoded functional

ampicillinaseenzymes.Thoseplasmids

contain-ing small deletions mappcontain-ingateither the PvuI or

PstIsitenearthe5' end of theM-MuLVgenome

wereamplified in E. coli HB101 and purifiedon

CsClgradients for further analysis.

Mapping and sequencing of mutants. Three

mutantscontaining small deletions in the sites of

interestwere selected forfurther study. Two of

these mutants, termed d1902 and dl904, were

prepared by cleavage with PvuI; mapping

showed that they had lost the PvuI site at

nucleotide position421andapproximately 75bp of flankingDNA. The third mutant,d11003,was

prepared from PstIlinears and had lost the PstI siteat position 565as wellas approximately 50 bpnearby.Inallthree cases,theremaining PvuI and PstI sites wereintact.

The nucleotide sequences ofthe mutated

re-gions were determined as described above. Briefly, each mutant DNA was cleaved with BstEII, the 5' ends were labeled with 32p, and

the DNAs were recutwith Sacl. Maxam-Gilbert

cleavage reactions (23) were performed on the

fragment containingthesiteof the deletion, and

thesequence wasdetermined byelectrophoresis

ofthe DNA fragments. The numbers and

posi-tions of the bases missing in each mutant are

shown in Fig. 1. Mutants d1902 and

d1904

con-tained deletions of 56 and 83 bp, respectively,

and mutant d11003 contained a 66-bp deletion

veryclose to the start ofPr65gag. The sequences

obtained in this region agree with the data of Shinnicket al.(32),exceptforonebase

substitu-tion(aG to Achangeatnucleotide 617)near the

AUGfor Pr659'9 (Fig. 1).

Biological activity. Plasmid p8.2is apermuted

cloneofM-MuLV containing onlyone LTR. To

study theinfectivity ofthe mutagenized clones,

eachDNA wascleaved withHindIlI and ligated

intopolymerstoreconstruct acompleteproviral

structure.DNAs wereappliedtosensitive NIH/

3T3 fibroblasts in acalcium phosphate

precipi-tate, and the cells were tested for release of

viable virusby overlay with XC cells. All three

deletion mutants, dl902,

d1904,

and dl1003, formed XC

syncytia

similar in appearance and number to those formed by the wild-type viral clone p8.2. Media from these plates contained

measurable reverse transcriptase activity (data

not shown).

Clonal NIH/3T3 cell lines producing virus wereisolated bycotransfection withtwoDNAs, namely plasmid pSV2GPT DNA carrying the

selectablemarkerof resistancetomycophenolic

acid, and each of the polymerized mutant

DNAs. Cells expressing the gpt gene were

se-lected in XAT medium(seeabove), and several

mycophenolic acid-resistant colonies were

cul-tured and tested forreversetranscriptase

activi-ty. Between one-third and all of the colonies

exposedto DNAsofthe mutants werepositive

inthis assay(Fig. 2). To test thetransmissibility

of theseapparentlyviableviruses, medium from

the reverse transcriptase-positive clones was

filtered and used to infect new NIH/3T3 cells.

Cellsinfected in thismanneralsoproducedvirus

asmeasuredbythe XC assay, with titers in the

range of

104

to

105

PFU/ml(data notshown).

Insummary, these viruses renderedNIH/3T3

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r J

VOL. 46, 1983

t0(I 00

_ 0

O 0 N N

go%3 N' = 0N

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O-0 O

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1 2 3 4 5 6 7

pZAP * @ * *

di902*

209*

di904 *

d11003 Control

FIG. 2. Reverse transcriptase assays of

supema-tant fluids from clones of cells transfected by various DNAs. Each viral DNA was oligomerized, mixed with

plasmid pSV2GPT DNA, and applied to NIH/3T3

cells. Individual clones resistant to mycophenolic acid

were isolated, and the supernatantfluid was assayed

for reverse transcriptase on an exogenous template (13). Results for several independent clones derived

fromeach transfection are shown.

cells XC positive upon transfection, caused

themtoreleasereverse transcriptase-containing virions, and weretransmissible tofibroblasts.

Growth of mutant viruses in lymphocytes. A

number of strains of murine leukemia

viruses,

including the Grossvirusand a BALB/c

endoge-nousstrain, replicate poorly inlymphocyte cell lines and alsoactas poorhelpersfor the stable

transmission of Abelson MuLV into such cells (28). To examine whether these deletions could

cause such a

phenotype,

two

lymphocyte

lines

wereinfected with wild-typeor mutantvirusat a

multiplicity ofapproximatelyone, and the virus

released by these cells was monitored over

several days.In both70Z,aB-cell leukemia cell

line, and SWR/4, an Abelson MuLV-induced

(pre-B-cell) leukemiacellline,the mutant virus-es replicated normally and were secreted into

the medium at least as wellas wild-type virus

(Fig. 3). Thus,theregiondeleted isnotessential

for replication in theselymphocytes.

Analysis

ofgag proteins. Cells infected with

themutantviruseswere

pulse-labeled

for 15 min

with [35S]methionine, and extracts of protein

wereprepared foranalysis. Viralproteinswere

immunoprecipitated with avariety ofsera, and

theimmune

complexes

were collected

by

bind-ing to Formalin-fixed Staphlococcus aureus.

Proteinswereexaminedbysodiumdodecyl

sul-fate-polyacrylamide gel electrophoresis

fol-lowedby fluorography (Fig. 4). Wild-typevirus

(in NIH/3T3 clone 4 cells) clearly directed the

synthesisoftwo

gag-specific

proteins,

the

inter-nalPr65Rar andtheglycosylated

gPr8g9a9

(which migrated

slightly

faster than the

envelope

pro-tein gPr8Oe"v). Cells containing mutants d1902

andd1904 showed

only

theinternal gag

protein

andnodetectablelarger

species

evenupon

long

overexposure ofthe fluorograph. The cells

in-fected with mutant

d11003, however,

synthe-sized PrO5gag and a

large

amount of a novel

protein migrating at

approximately 71,000

dal-tons,detectedby

gag-specific

sera

(Fig. 4).

The

difference inapparent molecular massbetween the wild-type gPr80gag and the

dl1003

mutant

IL E

IL

0

ccI3

0

a

SWR/4

70

Z

2 4 6 8 10 2 4 6 8 10

Days Post

Infection

FIG. 3. Growth curves of various M-MuLV isolates inlymphocyte cell lines SWR/4(A) and 70Z(B). The

indicated cell lines were infected with virus on day zero, and supernatantfluidswere collected on successive

days. PFU in thesesupernatantswere titrated by XC assay in NIH/3T3 cells.Mock-infected cultures released no

PFU. Virus isolates used were clone 4 virus(0), d1902 virus (0), d1904 virus (O), and d11003 virus (A).

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VIABLE DELETIONS OF M-MuLV 543

N N

O

0

° O

a7) CY 0) _

_ _ _ _

tl-' rl--

ll-0 0 0

Z U Z 0 Z )

g

Pr

80e9-o

Pr6

5Q9_-

Pr 7190

4

Pr

65

9g9

4

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 4. Analysis of viralgagproteins synthesized in infected NIH/3T3 cells. Cell populationsinfected with

various M-MuLVpreparations werelabeled with [35S]methionine, and thelabeled proteinswereanalyzed by

immunoprecipitation,sodium dodecyl sulfate gel electrophoresis, and fluorography. Lanes1through4:Proteins

fromcells infected withwild-typeM-MuLV(clone4cells)wereprecipitatedwithnormalgoatserum(lane 1),

rabbitanti-gagserum507 (lane 2),goatanti-Moloneyserum(lane3),andgoatanti-RauscherPr60ga serum(lane

4).Thepositionsof thewild-type proteins Pr65gag andgPr80ga areindicated. Lanes5through12:Cells infected

withmutantsd/902(twoisolates), dl904, and d11003,asindicated,wereanalyzedwithnormalgoatserum(N)or rabbitanti-gagserum507 (507). The positions ofproteins Pr65gag and Pr7lgagareindicated.

protein was approximately 9,000 daltons and

wastoo greattobeaccounted for by the 66-bp

deletion. One possible explanation for this

dis-crepancy is that the deleted protein may also

lack thecarbohydrate foundonnormalgPr8099a.

To determine whether thenovel protein

con-tained carbohydrate, the immunoprecipitated

proteins were digested with endo H, which

cleaves mannose-richcarbohydrate sidechains,

and the treated proteinwas analyzed as before

(Fig. 5). Wild-typegPr8099a wasreduced in size

by endo H treatment, migrating as a protein of

approximately 73,000 daltons in these gels. The

mutant d11003 protein was completely

un-changed by endo Htreatment(Fig. 5). Thus,the

mutant protein appeared to lack the normal

glycosylation foundonthewild-type large

mem-brane-bound gagprotein.

Alltheseprotein phenotypeswerecompletely

stable for many cell generations (data not

shown). Thus, reversionorrecombination with

endogenous viruses did notinterfere with these

assays.

DISCUSSION

The site-specific mutations described in this

work were constructed by Bal 31 exonuclease

treatment of cloned viral DNA linearized by

restriction enzymes. This method provided a

rapid and reliable means for introducing

muta-tions at selected sites throughout the viral

genome: approximately 80%oofplasmids

isolat-ed after the mutagenesis contained deletions.

From suchtreatmentofclonep8.2 DNA,

linear-ized with PstI and PvuI, we obtained three

mutantscontaining small deletionsin theregion

between the5' LTR and theinitiation AUG for

the major gag and gag-polprecursor proteins.

Several larger deletions which were not viable

werealso obtained (datanotshown).

When these threeDNAswereappliedtoNIH/

3T3 cells in a calcium phosphate precipitate,

viable virus wasproduced, asdemonstrated by

XCplaqueassayandbythepresenceofreverse

transcriptase activity.When NIH/3T3 cellswere

cotransfected with viral DNAs and plasmid

0

:

.L

to-01

O-

2 (r

Zo

M Z 0O

VOL.46, 1983

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544 AND GOFF

Wild Type di1003

Endo H M

g PrB Pr4O"n

j- kP Pr659og

FIG. 5. Analysis of viralgagproteinssynthesized

bymutantdl1003 virus. Immunoprecipitates from cells

infected with wild-type M-MuLV or mutant d11003

were prepared with rabbit anti-gag serum 507. The

immunoprecipitates were solubilized, and portions

were mock treated or treated with endo H before

analysis by sodium dodecyl sulfategelelectrophoresis

and fluorography. Lane 1, wild-type M-MuLV

pro-teins without endo H; lane 2, wild-type M-MuLV

proteins with endoH treatment; lane 3, dl1003

pro-teins without endo H; lane 4, dl1003 proteins with

endoH; lane 5, marker proteinsproduced by

immuno-precipitation ofproteinsinclone 4 cellswithgoat

anti-M-MuLV serum.

pSV2GPT,amajority of the clones surviving in

XAT medium produced reverse transcriptase

activity in the supernatant medium, indicating

the presence of released viral particles. In this

way,producer cell lineswerereadily isolated. It

should be noted, however, that the percentage

of clones cotransfectedmaybeanoverestimate,

since we cannotrule outthepossibility of virus

spreadontheoriginal transfection plate. Each of

these clones produced fully transmissible virus,

which could be filtered and passedtonewNIH/

3T3 cellsto render them XCpositive.

Murine leukemia virusescontainanunusually

long leader sequence(620 bpin thecase of

M-MuLV) between the 5'end of the viral RNA and

the initiation codon for thePr658agand

Pr2009'9-PO/

precursors (32). We have shown that

dele-tions within an approximately 200-bp region

apparently do notaffect viral functions inNIH/

3T3 fibroblasts. Thus, we conclude that the

deleted regions containnocis-acting sequences

required for viral replication. R. Mann has

re-cently isolatedoneM-MuLV mutantcontaining

a 350-bp deletion in this region (personal

com-munication); thismutantisdefective and unable

to transmit stably its own genome to infected

cells. Because our mutantsare not impaired in

replicationfunctions,wecanfurthernarrowthe

window in which these control sequences are

located toa region close to the 5' end;

accord-ingly,we caneliminatemost ofthe region close

togag.

Itisparticularly strikingthat mutantd11003is

viable. This deletion eliminates sequences upto

19bp away from the initiation codon forPrO5gag

without affecting protein translation ofthis gene. Apparently, elimination of these sequences

causes no adverse effect on ribosomal binding

for Pr65gag and

Pr200gag-po

protein translation;

the signals for recognition of this AUG by the

ribosome, if they exist at all, must lie very close

tothe gene.

Analysis of gPr8OIga

proteip.

The gPr80gag

proteincouldbe readilydetected in cells

infect-edwith wild-type

virus,

but none of thisprotein

couldbe detected in cells infected withmutants

dl902 and d1904. Longexposures of the

fluoro-grams showed that the protein was present at

less than 5% of wild-type levels. The gels also

showed that the serum wasveryspecificfor gag

proteins and did not precipitate the closely

mi-grating gPr8oe"v protein. These results suggest

thatgPr80gag is notnecessary for replication or

transmission of M-MuLV in NIH/3T3

fibro-blasts. Furthermore, because these mutants

eliminate

gPr80gag,

they confirm that this region contains sequences necessary for the synthesis of thisprotein.

Immunoprecipitation of gag proteins from

mutant dllO03 revealed a protein

migrating

at

approximately 71,000 daltons; we interpreted this protein to be a shortened

gPr809'9.

The

observed shift inmobilitywastoo great,

howev-er, to be accounted for by a 22-amino-acid

deletion. Since

gPr80gag

is a glycosylated

pro-tein, the large shift in mobility of the mutant

protein might result fromthe combination ofa

lackof carbohydrate side chains andashortened polypeptide. When the mutant dl1003 protein

was treated with the enzyme endo H, which selectively removes the mannose-rich side chains, nochange in mobility wasdetected. In

contrast, wild-typegPr809a9was reduced in

ap-parentsizebyendoHtreatment to

approximate-ly 73,000 daltons, onapproximate-ly slightapproximate-ly larger than the

mutant protein. The difference in the apparent

size ofthedeglycosylated wild-typeproteinand thedl1003 mutantproteinis small: about 2,000

daltons,

very close to the difference predicted from a 66-bp DNA deletion. Thisgood

agree-ment suggests that the polypeptide moiety of

both proteins begins and ends at identical

se-quences and that the only difference is the

internally

deleted region of dl1003.

Previous studies (7)

tentatively

map the sites

of

glycosylation

in

wild-type

gPr80g'g

to the region shared with the

major

gag

proteins

P30

andP15,far downstreamfromthe

position

of the

deletion in d11003. This mutation must, there-fore,eliminate

glycosylation

of sitesata

consid-erable distance. We consider two

possibilities

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.490.80.219.74.221.2]
(8)

+

f

10) to leave a larger translation product. The

A

gP_

_g

r8o0°

d11003

protein, despite

its

deletion,

would be

R US 1_ Pr65°° largerthanwild-type

gPr80g'g

ifaleader

peptide

TY" Ii -- of 30 amino acids were not removed. Thus,

B Pr71 g

gPr809ag

may resemble the case of ovalbumin,

_P15

P12

P6

p9P0

5 whichcontainsaninternal

signal

sequence

need-dl U ed for membrane transport and which does not

undergo

cleavage

ofaleader

peptide

(19).

C P P P0_

Pr659°

Structure of the gene for

gPr8O'g.

The

dele-dIO2SU5 v tion mutantsdescribed inthispaper

help

define

dl

W4the

region

which encodes the

glycosylated

gag

,base

protein of M-MuLV. The extra amino acids

[image:8.490.50.242.58.202.2]

° 500 1000 1500 found in

gPr80gag

and notin

Pr65gag

havebeen

FIG. 6. Proposed structure of RNA genomes localized by peptide analysis (7) to the amino

(lines) and protein products (hatched boxes) of various terminal segment of the longer protein. The

M-MuLVisolates. (A) Two gag proteins,gPrgosasand

simplest interpretation

of the structure of

Pr65gag,

are synthesized by wild-type M-MuLV. The gPr80gag isthat translation is initiatedatasite in

structure of the RNA template for the synthesis of the leader RNA andcontinues in phase into the

gPr8.g2g

is unknown. Carbohydrates are added to sequence of the regular Pr65gag(Fig. 6). Three

gPr80gag at portions corresponding to P15 and P30 in AUG initiator methionine codons are found in

the maturecleavage products of

Pr659a8.

(B) Proteins the published sequence (32) upstream of the

synthsizeby mtantdllO0 arePr7ga adP68. gag

synthesized bymutantd11003are

Pr7.

a andp5 . AUG for the Pr65 ,butnone of these codons

The66-bp deletion in thisgenome causes ashortening is in thesametranslational

reading

frame asthe

ofthelargergagprotein withoutaffectingPr658a8 The gag

deletion also abolishes the addition of the carbohy- Pr65 . It is possible that proper translational

drate chainsusually found at the downstream sites.(C) initiation or RNA

splicing

sites have not been

Only Pr65gag is synthesized by mutants

d1902

and identified. A less

likely possibility

is that this

dl904; production of the larger protein is completely sequence may be translated starting from a

abolished by the deletions. valine GTG codon like that found in procaryotic

proteins

orfroman

entirely

different codon.

Possible protein function. Our work

provides

which could accountfor theeffect of this muta- thefirst

example

ofmutantsin the

glycosylated

tion. One

possibility

isthat thedeletion in

dl1003

membrane-bound gag

protein.

Previous efforts alters the structure of this

protein

in a manner to isolate such mutants

by

immunoselection of

which decreases the

accessibility

ofthe

glycosy-

infected cells with

anti-gag

sera and

comple-lation sites. On the average,

only

one-third of ment

lysis

have

only

resulted in isolation of the classic

tripeptide

sites

(-Asn-X-Thr/Ser)

are cellularmutants defective in membrane

protein

found

glycosylated

in membrane-bound and se-

processing

(8, 12).

The

production

of viable

creted

proteins,

and thus it has been

proposed

virus

by

someof these cell lines

despite

the lack

(37) that the addition of

carbohydrate

side chains ofa

glycosylated

gag

protein

on theircell sur-isdependentonthe

tertiary

structure surround- facesis in agreement with our

findings.

ing the

glycosylation

site.Adeletion of22amino Theconstruction of viable deletionmutantsof acids may well

change

the conformation of M-MuLV which lack

gPr80gag provides

conclu-gPr80gag

so as toeliminate

glycosylation.

Alter- sive evidencethatthis

protein

isnot

required

for

natively,

the deletion in

dl1003

may interfere

replication

and transmission in NIH/3T3 fibro-with a

signal

sequence which

normally

directs blasts or in

lymphocytes.

No known function this

protein

to themembrane. The mutantpro- has yet been associated with the

glycosylated

tein may not be translated on the

rough

endo- gag

protein,

but the

protein

may beinvolvedin

plasmic reticulum,

and

according

to current somelittle-understood function of

leukemogene-modelsfor the

synthesis

of membrane

proteins,

sis. The

availability

ofmutants in

gPr80gag

will

it would not be glycosylated or

transported

to facilitate a critical evaluation of

possible

roles

the cellmembrane. Weare

currently testing

the for this

protein

in the transformation of

lym-localization of the

protein by

surface

protein

phoid cells in vivo.

iodination with

lactoperoxidase

and

by

surface immunofluorescent stains.

If the

dl1003 protein

haslost part ofa

signal

sequence

normally

tagging

it as a membrane

protein, it must have one unusual property.

Normally, short leader

peptides

of about 30

amino acids are cleaved from membrane

pro-teins

during secretion,

and mutations in this

leader block

glycosylation

and

cleavage (2, 3,

ACKNOWLEDGMENTS

This work was supported by a grant from the National CancerInstitute(5R01CA-30488)andbyanawardfromthe Hirschl Foundation.

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J. VIROL.

on November 10, 2019 by guest

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Figure

FIG. 2.fromforcells.tant(13).plasmidwereDNAs. Reverse transcriptase assays of supema- fluids from clones of cells transfected by various Each viral DNA was oligomerized, mixed with pSV2GPT DNA, and applied to NIH/3T3 Individual clones resistant to mycophenolic acid isolated, and the supernatant fluid was assayed reverse transcriptase on an exogenous template Results for several independent clones derived each transfection are shown.
FIG. 4.from4).immunoprecipitation,rabbitvariouswithrabbit Analysis of viral gag proteins synthesized in infected NIH/3T3 cells
FIG. 5.byinfectedteinsendowereimmunoprecipitatesandanalysiswereprecipitationproteinsteinsM-MuLV Analysis of viral gag proteins synthesized mutant dl1003 virus
FIG. 6.(lines) Proposed and protein products

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