A single point mutation in the envelope gene is responsible for replication and XC fusion deficiency of the endogenous ecotropic C3H/He murine leukemia virus and for its repair in culture.

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0022-538X/88/030932-12$02.00/0

CopyrightC) 1988,American Society for Microbiology

A

Single

Point Mutation in the Envelope Gene

Is

Responsible

for

Replication and

XC

Fusion Deficiency of

the

Endogenous

Ecotropic

C3H/He Murine Leukemia Virus and for Its Repair in Culture

GUNAMANI SITHANANDAM ANDULF R. RAPP*

Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701 Received25June 1987/Accepted 2 November 1987

The molecular basis has been determined fordifferences in infectivity and XC phenotype of endogenous ecotropic murine leukemia virus of the low-leukemia mouse strainC3H/He,its relative in thehigh-leukemia mouse strain AKR, and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/1OT1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformedC3H/10T1/2cells(MCA5)areXC positive and infectious. XC-negative viruses can be converted to XC-positive viruses by being grown in certain transformed cell lines. We have cloned the endogenous ecotropic provirus ofC3H/Hefrom MCA5 cells, which is XC negative and replication deficient,as well as two XC-positive C3H proviruses derived by in vitro conversion. Fragment exchange between the XC-negative molecular clone pllO and the XC-positive AKR virus clone p623 revealed that thedefect in pllO lies3'of the

Sail

site located in the pol region. Nucleotide sequencing established that the C3H pllO provirus wasintegrated within theR region ofanendogenous VL30 long terminal repeat (LTR)in reverse orientation and that thevirusdifferedfrom the infectiousAKRp623 provirus byapoint mutation, substituting Lys for Arg atthepotential precursor cleavage site forgp7Oandpl5E.Invitro-convertedXC-positive C3H proviralclones 3211 and4211are identicaltoXC-negative C3Hp110,except thattheyhaveArgatthis site and the normal cleavage site is thus regenerated inthese clones. The XC-negative C3H pllO was blocked in processing of Pr85env, whereas clones 3211 and 4211 had normal cleavage of the env precursor into gp7O. Both the XC-negative C3H provirus and the in vitro-converted XC-positive C3H proviruses had a single copy ofa 99-base-pairenhancer elementin theLTR, whereastwocopies of this sequencearepresentintheAKRproviral

LTR. Substitution of Arg for Lys at the envelope precursor processing site of C3H pllO by site-directed mutagenesis is sufficient by itself to convert the virus to the XC-positive replication-competent phenotype. Thus,wehave established thatasingle point mutationat theprocessing site of the envelope precursor protein Pr85 is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic murine leukemia virusfromC3H/HeandAKRmice and that the basis for in vitro conversion isamutationatthissite. Inbredmouse strains differ in their incidence of leukemia

and the number and inducibility of endogenous ecotropic

murine leukemia virus (MuLV)proviruses(1, 15, 26, 30, 47, 53). Leukemia-negative strains lack

endcogenous

ecotropic

MuLV, whereas low- and high-leukemic strains contain at least one copy of this provirus (30, 47). Initial studies on virus inducibility with halogenated pyrimidines (32)

indi-cated that viruses from low-leukemic strains were more

difficulttoinducethanvirusesfromhigh-leukemic strains (1,

32, 53, 54). These findings were interpreted to indicate

differences in cellular control ofexpression of endogenous ecotropic virus(21, 35,47,48)ordifferences intheir biolog-ical activities (1, 41, 53). Specifically, ecotropic virus recov-eredfrom induction of cellsfromhigh-leukemic strains such as AKR formed large XC plaques, whereas virus from the

low-leukemic BALB/c mouse cells formed small (1) or no (41, 45) plaques.

Inthe courseof studiesexamining the role of endogenous

C-type viruses in chemical carcinogenesis, we have ob-served that untransformed or transformed cells from low-leukemic C3H/He (C3H/1OT1/2 Cl 8) and BALB/c (BALB 3T3) mice produced XC-negative,replication-deficientvirus

early (phaseI)afteriododeoxyuridine(IdUrd) treatment (41, 44, 45). XC-positive virus did emerge late after IdUrd

*

Corresponding

author.

induction ofchemically transformed C3H/1OT1/2 Cl 8 cells (phase III),presumably owingtogenetic changesthatwere selectedduring serialreinfection(44, 45).The lackof

infec-tivity and XC fusion activity of endogenous C3H/He and BALB/c ecotropic virus in phase I after induction was

interpretedtoreflectthedefectivenatureoftheendogenous proviruses, and their conversion to large-XC-plaque-in-ducing, infectious viruses similar to the AKR

ecotropic

provirus (41) was thought to result from mutation during

serial reinfection (41, 45). Moreover, the genotype ofthe

endogenous ecotropic provirus appeared to be a

major

determinant of leukemia

incidence,

since inoculation of NFS/N micewithXC-positiveAKRand C3H virus,butnot withXC-negative endogenousecotropicC3Hvirus, resulted

in the development of leukemia (41). The existence of

XC-negative ecotropic BALB/c virus in the company of excess XC-positiveviruswasalsodescribedbyothers(18);

however, since this virus stock had been maintained in

chronically infected NIH 3T3 cells for some time before

analysis, no conclusion regarding the properties of the

endogenous BALB/cproviruscould be drawn.

Thepossible existence of cellularregulatory geneswhich control virusexpressioninBALB/cand otherlow-leukemic mice has since been furtherexplored (21, 34, 35).To date,

the nature of potential cellular control genes

determining

ecotropic

virus inducibility in BALB/c mice remains un-932

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MuLV ENVELOPE GLYCOPROTEIN PROCESSING MUTANT 933

clear,

whereas structural defects

affecting

viral infectivity

havebeen observed (22). Nevertheless, ithas been

demon-strated that in other systems,nonviralfactors suchasDNA

methylation

(7, 13, 17)andchromatinstructureofintegration sites affect the

expression

ofexogenousC-type virus (6, 13, 17, 25). Moreover, transregulation of C-type viral RNA

expression by

activation of

protein

kinase C has been

demonstrated for the VL30

long

terminalrepeat(LTR) (46).

Toestablishthemolecular basisforthe lackofinfectivity of

endogenous ecotropic

virusfrom C3H/Heand theinvitro

conversion to an

infectious, XC-positive form,

we deter-mined thestructures of both viruses.

MATERIALS AND METHODS

Cells. All cell lines were maintained in

Eagle

minimum

essential medium with10% heat-inactivated fetal calfserum.

C3H/MCA5,

a

methylcholanthrene-transformed

C3H/1OT1/2

Cl 8 cell

line,

and the NIH R+Cl3 cell line

(NIH

cells

infected with

XC-positive

converted C3HR+C13

virus)

were

used.The

origin

andcharacteristics of these cell lineshave

beendescribed

previously

(41, 44).

Molecular cloning.

High-molecular-weight

DNA from

MCA5

and NIHR+Cl3celllineswasextracted and

digested

with the restriction enzyme EcoRI to

completion

as

recom-mended

by

themanufacturer

(New England BioLabs,

Inc.).

The

digested

DNAs were

separated

ina 10to40% sucrose

gradient,

and MCA5DNAfractions

containing

21-kilobase-pairs (kb) fragments

were

pooled,

dialyzed,

and

precipitated.

Similarly, samples containing

9- to 11-kb

fragments

from

NIH R+Cl3

gradients

were

pooled

and

precipitated

(33).

The 21-kb EcoRI

fragment

was

ligated

to

gradient-purified

EcoRI-digested

Charon 35 arms

(29).

Since BamHIcleaves the stuffer

fragment internally,

Charon 35 DNAwasdouble

digested

withEcoRIand

BamHI,

and the smaller

fragments

of stuffer were

easily separated

from vector arms in the

gradient.

NIH R+C13 EcoRI

fragments

were

ligated

to A

WesB EcoRIarms

(28)

and

packaged

invitro

(9),

and thetwo

libraries were screened

by using

the Benton and Davis

method

(3)

witha

32P-labeled p400 eco-specific

env

fragment

(5).

Positive clones were

plaque purified

and subcloned in

pBR322.

Gel

electrophoresis

and hybridization analysis. DNA

sam-ples digested

with restriction endonucleases were

electro-phoresed

in 0.8to0.9%agarose

gels

withTris-acetate buffer

(pH 7.8),

and the DNA was transferred to nitrocellulose

paper

by

the

procedure

of Southern

(51).

The filters were

prehybridized

at

60°C

foratleast2to3 handthen

hybridized

overnight

at

60°C

in the

hybridization

buffer

containing

0.5 x 106 to 1 x 106

cpm

of

32P-labeled

probe per ml. After

hybridization

the filters were washed twice with lx SSC

(0.15

M NaCl

plus

0.015 M sodium

citrate)-0.1%

sodium

dodecyl

sulfate

(SDS)

at

65°C

and then

subjected

to three

30-minwashes at650Cwith0.lx SSC-0.1% SDS. Air-dried

filters were

exposed

to Kodak X-ray film at -70°C with

intensifying

screens.

Invitroconstruction of chimericviralgenomes. AKRp623

contains the entire AKR MuLVgenome cloned inpBR322at theHindlllandEcoRIsites.HindIllandEcoRIsites inp623

are located in 5' and 3'

flanking

cellular sequences,

respec-tively (31).

C3HpllOcontains the C3H endogenous

ecotro-pic proviral

genome with 12-kb cellular

flanking

sequences cloned into the EcoRI site of

pBR322.

Both p623 and pllO

have a

single

Sall restriction site within the ecotropic

provirus

in the pol

region

and the otherSall site within the

pBR

sequences.

Fragments

3' of the Sall site in the pol

region were switched between the two plasmids. AKR p623 and C3HpllO DNAs were digested with Sail, thefragments

were separated in a preparative gel, and the desired frag-mentswere isolated andligated with T4 DNA ligase (at14WC for 16h) and used to transform EscherichiacoliHB101 cells. Colonies were then screenedby the method of Grunstein and

Hogness (14), and the DNA from positive clones was mo-lecularly analyzed by using appropriate restriction enzymes to determine the orientation of theexchangedfragments.

DNAsequencing. DNA wassequenced by the dideoxynu-cleotide chain termination method (2, 50) on fragments inserted in pUC18, pUC19, and single-stranded M13mpl8 andM13mpl9 vectors. Commercially available primers were used for priming, and primer extensions were carried out with the large fragment of E. coli. DNA polymerase I

(Klenow)and[35S]ATP (Amersham Corp.)wereused as the sourceof labeled triphosphate.

Site-directed mutagenesis. Theprocedureused for oligonu-cleotide site-directed mutagenesis was essentially that de-scribed by Zoller and Smith (57). A 520-base-pair (bp)

KpnI-to-XbaI fragmentwas cloned into the M13 vectorand was used as a template for site-directed mutagenesis. Two

oligonucleotide primers were used, a 26mer synthetic

ol-igonucleotide, 5'-GCCAAATATAAAAGAGAACCCGTC TC-3', and the universal sequencing primer (New England BioLabs). Preliminary tests were conducted by using the

mutagenic oligonuclotide as a sequencing primer in a stan-dard dideoxynucleotide sequencing proceduretodetermine the optimum conditions of primer-to-template ratio. Both

primers were simultaneously annealed to single-stranded

template DNA at 60°C for 5 min, extended with DNA

polymerase I (large fragment), and ligated at 15°C for 7 h. Diluted DNA wasthen used totransformE.coliJM103 cells. Theplaques were screened for mutants byfilter

hybridiza-tion with 5'-end-labeled mutagenic oligonucleotide as a

probe,andfilterswereprehybridizedat65°C for1 h and then

hybridized at 37°C overnightunderconditions of low strin-gency inwhich bothmutantandwild-typeDNAhybridized with oligonucleotide. The filters were then washed three times with 6x SSC at various temperatures. At 37°C both mutantandwild-typeDNAs weredetected,whereas at62°C only afewhybridizing plaques remained, which were puta-tive mutants. Strongly hybridizing plaques were purified, single-strandedDNA wasprepared,andportionswere spot-ted onto nitrocellulose and hybridized with the labeled

mutagenic oligonucleotide. Filters were again washed at

differenttemperaturesranging from37 to65°C.The

hybrid-izing

signals of wild-typeclones werelost aftera47°Cwash. Incontrast, hybridizing signalsfromputative mutantphage

clones were stable even atveryhightemperatures.

Transfection of NIH 3T3 cells withproviral clones.

Trans-fection ofNIH 3T3cellswithproviralclones wasperformed by a modification ofthe method described by Graham and van der Eb (12). Linearized plasmid DNA was mixed with

pSV2neoDNA(ratio, 10:1) and carrier salmon sperm DNA to a DNA concentration of 20

pug/ml

and suspended in

transfectionbuffer to afinal concentration of0.13 MCaCl2.

At 2dayslaterG418 was added to the medium at 400 ,ug/ml. Clones resistantto G418were selected and expanded.

Viralassays. Production of infectious virus particles was monitoredby XC andreversetranscriptaseassays. The XC test was aslight modification of thatdeveloped byRowe et al.(49). Target SC-1cellswereseededat105 cells per 60-mm dish. At 24 h later the cells were treated with Polybrene

(Sigma;

20

,ug/ml)

for 1 hand then infected with 0.2 ml of virussuspension.At2 hafterinfection the virus suspension

VOL.62, 1988

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wasremoved and 5 ml of medium was added. At 4 days after

infectionthecells were lethallyirradiatedwith aUV

germi-cidallamp andwereoverlaid with 106ratXC cells. Depend-ingupon the confluency ofthe XC monolayer, the test was

terminated and the cells were fixed with methanol and

stainedwith Giemsa. Infectious-center assays were used as asensitiveassay to detectsingle-virus-producing cells.

neo-positive cells from transfections (test cells) were seeded at various concentrations and allowed to form small islands

duringgrowth for 2 days and then were lethally irradiated withUVandoverlaidwith XC cells. Plaques were scored 2 to3days later. Cocultivation assays with virus-free mouse

cells were used to determine the original phenotype of neomycin-positive clones in terms oftheir XC fusion

activ-ity. This test also determines whether there was a

conver-sion from the XC-negative to the XC-positive virus on

growth in different cells. neo-positive clones were mixed with uninfected SC-1 cells at various ratios (5:95, 10:90,

40:60, and 80:20), grown to confluency, and then lethally irradiatedand overlaid with XCindicatorcells. For reverse

transcriptaseassays, culturefluidsatearlyconfluency were

collected, cleared of cells and cell debris, and assayed for

reverse transcriptase activity essentially by the method

previously described (55).

Immunoprecipitation. Transfected cells were starved in leucine-free medium for2hbefore40,Ci of

[3H]leucine

was

added. The cells werelabeled for 30 min andchased for0

and30min and 1, 2, 3, and4h. Labeled cellswere lysed in lysis buffer (0.05 M Tris [pH 7.2], 0.15 M NaCl, 0.5% Nonidet P-40,0.5% desoxycholate,

0.5%

SDS), and 1ml of the clarified lysates was incubated overnight with 10

RI

of Raucher

gp7O

antibody and100

RI

of 10%protein A-agarose inlysis buffer at 4°C with gentle shaking.

Immunoprecipi-tates were washed five times with lysis buffer, and pellets

weredissolvedin 50,ulofsample buffer,heatedfor2min at

100°C, and run on an SDS-10% polyacrylamide gel. After electrophoresis for 16 h at 35 V, the gel was soaked in

destainsolution for30 minand then foranadditional30min

in Amplify (Amersham Corp.). Thegel wasthen driedand

exposedto SB-5 film(Kodak). RESULTS

Cloning of the endogenous C3H ecotropic provirus from MCA5 cells. High-molecular-weight DNA from the MCA5

cell line was digested with various restriction enzymes, electrophoresed through agarose gels, and analyzed by

Southern blothybridization (51). UsinganecotropicMuLV env-specific probe, p400 (5), we detected a single 21-kb EcoRI fragment (Fig. 1A, lane 1). Since this enzyme does

notnormally cleave within thegenomes ofecotropic

provi-ruses(39), the 21-kb Eco-RI fragment contains the prototyp-ical 8.9-kb provirus and approximately 12 kb of flanking mousecellular DNA. Sucrosegradient-purified21-kbEcoRI

fragmentswerecloned intogradient-purified lambdaCharon

35EcoRI arms,and theresulting phage

library

wasscreened withthe envprobe p400(5).Afterscreeningapproximately5 x

105

recombinant phages, we identified two positive plaques (clones C1109 and ClllO). Both clones had a

com-plete proviral genome and identical flanking cellular se-quences. The results of Southern blot analysis of cloned

DNA confirmedthat the correct provirus was cloned(Fig.

1B). The 21-kb EcoRI insert ofClllO was subcloned into pBR322 and designated p110. To characterize the proviral

sequenceandtoidentify the unique cellularsequenceinthe

clonedDNA, we performed restriction enzymeanalysis of

the 21-kb fragment. The restriction mapping of the 8.9-kb provirus and approximately 2.1 kb of 5' and 2.5 kb of 3' flankingcellular DNA isshown inFig. 1C.

A

0 m

o a _ c

M us x n. T e

23.5 -_

9.59--o4

6.64-I4

4.45- i

2.29n 1.95 _

B

ccc

8 i (n . 0

w x

._.

Ki

FIG. 1. Comparison of thegenomic andcloned C3Hecotropic provirusby Southern blotanalysis. (A) MCA5genomic DNA; (B) C3H pllODNA. DNAs weredigestedwith the indicated enzymes, electrophoresed, transferredto nitrocellulose, andhybridizedtoa nick-translated 400-bp env-specific ecotropic probe. 32P-labeled HindIII-digested lambda DNA was used as marker. Blots were exposed for 6 h (panel B) to 4 days (panel A). (C) Restriction endonuclease map of C3H p110. Restriction enzyme sites: B, BamHI; Bs, BstEII;Cl, ClaI;H,Hindlll; Hp, HpaI; K, KpnI; P, PstI;Pv,PvuI; PvII, PvuII;Ss, SstI; S,Sall; Sm, SmaI; X,XbaI; Xh,XhoI.Theprovirus isintegratedwithin the RregionofaVL30 LTR inreverseorientation.

C VL30

r n 11 X.11 PB1 tt XhKSm

X s 5s Us R Uus as

gag I pol

a H K B XS Xh

Hp S. PullSs

pIO

env

CC Hp 8 K

11 I

Sm PW11 S Sm as

1Kb

VL30 r--l

I PSMKSs XK P H K

I ---I

T

---T-Pvli Ss

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[image:3.612.59.529.421.723.2]

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MuLV ENVELOPE GLYCOPROTEIN PROCESSING MUTANT 935 Biological activity of the C3H ecotropic proviral clonep110.

Totestthe biological activity of the proviral clonep110,NIH

3T3 cellswerecotransfected with pllO andpSV2neo,which

carries thegenefor neomycin resistance, ata10:1ratio (52).

Transfections with AKR p623 and pSV2neowerealso

per-formed as a positive control. Transfected cells were first selected for their ability to grow in the presence of the

neomycin analog G418, and individual colonieswereisolated

and assayed for XC fusion activity by theinfectious-center

assay at their first confluency. These conditions minimize the chance for secondary rounds of virus spread and

con-version in NIH 3T3 cells. Supernatant culture fluid was

tested forreleasedreversetranscriptase-containing particles

(55) and was also tested in XC assays (49). A total often neo-positive cloneswereassayed for each transfection. With

thecontrol p623 DNA, 10 of 10 G418-resistant clones were

positive forreverse transcriptase,indicating thepresenceof released viral particles. In thisway,producer cell lineswere

readily isolated. Incontrast,cellstransfectedwith C3H pllO DNA did not form fusions with XC cells, and release of

reverse transcriptase-positive particles was barely

detect-able(Fig. 2).

In vitro restriction fragment exchange and localization of thedefect in C311 p110.The fact that the XC-negative C3H

clone pllO and the XC-positive AKR infectious clone p623 share severalidentical restriction endonuclease sites allowed

ustomakeconstructs by switching fragments 3'tothe Sall site in thepolregion (Fig. 2). Chimeric plasmids were then

screened by colony hybridization (14), and plasmid DNA

waslinearized and transfected along with pSV2neoataratio

of10:1 onto NIH 3T3 cells. neo-positive single-cell clones

were tested for their reverse transcriptase activity and for

p623

p110

UDR

t

the presence of infectious virions as described previously

(49, 55). Switching the fragment 3' of the Sall site in p623 with that inpllO restored the biological activity of the pllO virus and generated infectious virus withreverse

transcrip-taseactivity (p335 in Fig. 2). Incontrast, 10 of 10 NIH 3T3 cells transfected with chimericplasmid p474 which has the 5' upstream Sall fragment from p623 and the 3' downstream SalI fragment from pllO didnotrescue the XC phenotype,

andparticle productionwasbarely detectable by thereverse

transcriptase assay. These results demonstrated that the

C3Hproviral clonepllOhadnodefectupstreamof theSalI site in thepolregion and that the defectmusttherefore lie somewhere downstream of this restriction site.

In vitro conversion of XC-negative to XC-positive pheno-type. We observed previously that poorly infectious, XC-negative ecotropic virus induced from C3H/1OT1/2 and BALB 3T3 cells convert to replication-competent, XC-positive viruswhengrown in certain transformed cell lines suchasSC-1, MCA5, and NIH 3T3 (41, 44). Totestwhether pllO could be converted, virus-negative target cells (SC-1)

were mixed in various ratios with neo-positive pllO-trans-fected clones and grown in the presence of Polybrene.

Different ratios ofvirus-positivetovirus-negative cellswere

used to allow forone or several cycles of infection in the

negative cells. With ratios of 5:95 and 10:90 of virus-positive pllO-transfected NIH 3T3 cells to virus-negative SC-1 cells, conversion from the XC-negative to the XC-positive phenotype was observed on continued coculture. No conversion was observed at a mixing ratio of 60:40or

80:20, in accordance with our previous observation with

biologically cloned viruses (44). These mixing ratios donot allow multiple rounds of infection in theconvertercells.

RU3

Supernatant Reverse Transcriptase Activity

Construct (103cpmof[3H]TMP) XC Phenotype

p623 119.8

p110

p335

p474

+

8.9

188.8 +

[image:4.612.145.473.405.660.2]

8.2

FIG. 2. Infectivity of endogenous ecotropic proviral clones AKR p623, C3Hp110,andconstructsp335 and p474. Schematic

representa-tions ofthe proviral clones p623, p110, and the constructs p335 and p474 are shown. First-confluency supernatant culture fluid from

transfected celllineswasharvested 24 h after mediumchange. Mediumwasfreed of cells and cell debris by centrifugation and assayedas

previously described (45) forreversetranscriptase activity. Results areexpressed as103counts perminute of3[H]TMP incorporated ina

30-min incubation periodpermilliliter ofsupernatant; 105cpmrepresents1.2 pmol of TMP incorporated. Reverse transcriptase activities in thefigure arefor clones whichweretested bypulse-chase experiments. The XC phenotype wastested by the infectious-centerassay(45),

aswellasby the ability of thesupernatant toinduce syncytium formation in Rous sarcomavirus-transformedratXC cells.

gag

I

pol env

Sa gp7O

PIE

Sal 9P0 Pl5E

VL30 Sal VL30

TR LTR""2 z" "'z' "

VOL.62, 1988

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Ca)

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tm

0E

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sup 5.6

-awfl

40

cCT TGA

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PvP

K P R +Ct3 3211 , I

P Ss Sm

A 4 C13 4211

TACCITGA

RI

RI R+C13 7-1111

Ss

ga I

S HK a . 33

IL ~ I I I I I I

Ba I

II I

Hp So Pulls Sm

I env TCA CCTT

CC Hp BK X P K

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c4I

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R+C131013

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Kb

FIG. 3. Comparative Southern blot analysis of proviruses in NIHR+Cl3cellular DNA and C3Hecotropic provirusesisolated form NIH

R+Cl3 cells. (A) NIH R+C13 genomic DNA. (B) NIH R+C13 ecotropic proviral clones C13211, C14211, C17111, andC11013. DNAs were

digested with the indicatedenzymes,electrophoresed, transferredtonitrocellulose,andhybridizedtoanick-translated400-bp env-specific ecotropic probe. Blotswereexposed for 6 h (panel B)to4days (panel A). (C) Restriction endonucleasemapof C3H R+C13proviralclones

C13211, C14211, C17111, and C11013...Internal deletion. Cleavage sitesare denoted asfollows: B, BamHI; Bs,BstEII; Cl, ClaI; H,

Hindlll;Hp, HpaI;K, KpnI; P, PstI; Pv, PvuI; PvII, PvuII; Ss, SstI;S,SalI;Sm, SmaI;X, XbaI; Xh, XhoI; RI,EcoRI. The4-bpcellular

directrepeatattheintegrationsite is shown for C13211and C14211.

Molecular cloningof in vitro-converted replication-compe-tent C3H virus from NIH R+C13 cells. To compare the genetic structure of in vitro-converted XC-positive C3H

viruswith thatof the parentalXC-negative form,wechosea

biologicallycloned isolate for molecular analysis. R+C13 is

anXC-positive, replication-competent variantgeneratedby

growth ofIdUrd-induced XC-negative C3H/1OT1/2 virus in C3H/MCA5 cells. Thevirus wasclonedbyisolation from a

large XC plaque that developed in cultures infected at limiting dilution, and agar stabs from these plaques were

transferredtouninfectedNIH 3T3 cellcultures(40).

South-ern blot analysis with the eco-specific env probe (5) of

EcoRI-restricted NIH R+C13 genomic DNA detected a

stronghybridizing region of 9to10kb(Fig. 3A). Hybridiza-tion of PstI digested DNA with p400 probe detected two

bands of 8.2 and 6.8kb.ComparativeSouthern blotanalysis ofequal amounts ofPstI-digested MCA5 DNA and NIH R+C13 DNA indicated thepresence ofthree 8.2-kb

ecotro-picproviruses integrated in C3HR+C13-infected NIHcells (datanotshown).The 6.8-kbPstI fragmentwaspresumably

notderived fromendogenousxenotropicviruswhich carries

aninternal PstI site,sinceithybridizedwithaneco-specific env probe. Gradient-purified 8- to 11-kb EcoRI fragments

werecloned into XWES B(28)EcoRIarms,and theresulting phageswerescreenedwithp400. Fourpositive plaqueswere

identified after5 x 10i phagewerescreened(clones C13211, C14211, C17111, and C11013). To analyze the structure of ecotropic proviruses cloned form this cellline, wedigested cloned DNAs with the restriction enzymes PstI and KpnI, which should give internal viral fragments of defined size

A

Kpnl CD)

0-2.29 1:955

4.4

-#..m.

C

H RI I

11.2-

40 40,40

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MuLV ENVELOPE GLYCOPROTEIN PROCESSING MUTANT

when hybridized with eco-specific probe (Fig. 3B). C13211, C14211, and C11013 showed hybridizing fragments of 8.2 kb with PstI and 4.4 kb with KpnI. Incontrast, the hybridizing fragments from C17111 were 6.8 kb for PstI and 5.1 kb for

KpnI,indicatinganinternaldeletion ofa ca. 1.9-kb fragment

which includes one of the internal KpnI sites. C13211 and

C14211 had identical restriction maps within the provirus,

buttheir flankingsequences weredifferent. Nomajor struc-turaldifference could be detected for C11013 onthebasis of

Southern blots. However, restriction analysis indicated lack of the HindIlI site at nucleotide 2668 (nucleotides were

numbered by using AKR, p623 sequenceof Herr [16]), and

this clone had eitherapoint mutationor aminor deletion in

that region (Fig. 3C). All four clones were subcloned in

pBR322.

Infectious properties ofprovirus clones from NIH R+C13 cells. NIH 3T3 cells were cotransfected with linearized

plasmid DNA of C13211, C14211, C17111, or C11013 and

pSV2neo, and G418 (52)-resistant clonesweretested for XC

fusion activity by the infectious center assay. Supernatant

culture fluid from these cells was tested for the release of

reverse transcriptase-containing particles andwasalso used

for the XCassay. Cellstransfected with C13211 and C14211 DNAwere XC positive and released reverse

transcriptase-containing particles, whereas cells transfected with C17111 and C11013werenegative for XC fusion activity and released no reverse transcriptase-containing particles into the

super-natant medium (Table 1). For comparison, results are also

shown for cells transfected withendogenous C3H viral DNA

p110,testedattheir firstconfluency(phase I) and again after five subculture generations (phase III), aswellaswith AKR

p623 viral DNA. C3HpllOclonesreleasedbarely detectable levels of reverse transcriptase-containing particles early

after transfection, butwere highly positive afterpassage in

culture, atwhich time theywerestill XC negative.

Sequence analysis of XC-negative C3H pllO and in vitro-convertedXC-positive C3H R+C13 proviral clones: molecular basis forthe XCphenotype. Toidentify the defect in the C3H proviral clone p110,the clonewas sequenced from the Sall

siteatnucleotide 3720tothe3' end of theprovirusandwas

compared with the XC-positive infectious AKR provirus p623sequencepublished by Herr (16). pllO fragmentswere

subcloned in pUC18, pUC19, and M13mpl8 and M13mpl9 vectors and were sequenced by dideoxy chain termination

(2, 50). Of the 4,660 bp sequenced, there were 21 base

substitutions: 10wereinthe3'pol region, 8wereintheenv

region, and 3 were in the LTR region. Of these, only 6

changes ledtoamino acidsubstitutions(2inthe3'pol region

and4in theenvregion), correspondingto99.5% amino acid homology between AKR p623 and C3H p110. The most interesting amino acid substitution occurred atthe proteo-lytic cleavage site of Pr85, wherea 1-bp change of GtoAat position 7191 substituted lysine for arginine (Fig. 4). A similar 1-bp change has been observed ina BALB/c

endog-enous ecotropic provirus clone (22), presumably because germlineinfection occurredpriorto divergence of BALB/c and C3H/He mice from a common ancestor. Nucleotide

sequenceanalysis of the converted C3H R+Cl3 virus clones

showed that all four coded for arginine at this potential cleavage site. Comparison ofa ca.1,500-bpsequenceof C3H R+C13 clones C13211 andC14211 shows thatthey differ from thatof C3HpllO attheproteolytic cleavage site by coding for Argatposition 7191 (Fig. 4). No othersequencechanges

between C3H R+Cl3 clones C13211 and C14211 relative to C3H pllO were detected in this 1,500-bp segment of the envelopegene sequence(Fig. 4). However, it is possible that

the XC-positive, replication-competent variant clones have additional changes in the remainder of the envelope gene, for example, at either or both of the other two positions outside

this segment in which the AKR env sequence differs from thatofC3HpllO(Fig.4). Wedidnot explore this possibility further, but instead concentrated on evaluating the effect of the Lys-to-Arg mutation on the XC and replication pheno-type, because of its likely consequences for envelope pre-cursorprocessing.

Comparison of LTR sequence and integrationsitesof endog-enous C3H XC-negative and converted XC-positive provi-ruses. Analysis of flanking sequences of the C3H

XC-negative clone pllO and the in vitro-converted XC-positive

C3H R+C13 clones showed that the pllOprovirus is inte-grated within the R region of a VL30 LTR in reverse

orientation (Fig. 5) (23, 24). A similar integration site has

also been observed for the BALB/c endogenous provirus

(20, 22). Flanking sequence analysis of C13211 and C14211 show that they are different from each other and are not integrated within the VL30 LTR, but within other unique sequences.Figure5BshowstheflankingsequenceofC14211

with the integration site, and Fig. 3C schematically shows the 4-bp direct repeat of cellular DNA at theintegrationsite

for C13211. The LTR from both endogenous (p110) and

converted (C13211 and C14211) C3H MuLV had a single

enhancer element in the U3 region oftheir LTRs (Fig. 4), whereas in the AKR provirus there are two copies ofthe enhancer sequences (16). In addition, the pllO LTR se-quence shows three nucleotide differences (two in the U3

region andoneintheUSregion) relativetop623,all of which

are identical to those changes observed for the BALB/c ecotropic viral LTR (22). Apart from sharing these three

nucleotide changes, proviral clones from NIH R+Cl3 cells differ from the LTR sequence ofpllO at onenucleotide at

position7889. Sinceall theecotropic provirusesclonedfrom

NIHR+Cl3cells sharethis 1-bpchange,they were

presum-ablyderived from asingle converted virus.

Site-directed mutation of C3Hp110.Todetermine whether the difference observed in infectivity between the C3H

XC-negativeand invitro-convertedC3H XC-positive clones isduesolelyto thepoint mutationattheproteolytic cleavage site or to otherfactors such as integration site orthe 1-bp change observed in the LTR, we performed a Lys-to-Arg

substitution in C3H pllO by site-directed mutagenesis. A

[image:6.612.317.559.585.678.2]

520-bp KpnI-XbaI fragment which includes the gp7O/pl5E junction of the env region of pllO was subcloned into M13mpl9and used as atemplatefor site-directed mutagen-esis(seeMaterialsandMethods). Sequence analysis of these

TABLE 1. Infectivity of C3H endogenous ecotropic proviralclonesa

Supernatant

XC

Clone RTactivityb phenotype

R+Cl3 3211 129.2 +

R+C134211 162.5 +

R+C137111 1.4

R+C13 1013 1.8

pllOphaseI 8.9

pllOphaseIII 125.1

AKRp623 191.9 +

aCulture fluids fromNIH 3T3-transfected cellsat their first confluency

wereharvested24hafter mediumchange.Infectivitywasassayed byreverse

transcriptaseandXCassay asdescribedin thelegendtoFig.2.

b Reversetranscriptase (RT) activityisexpressedas103countsper minute of[3H]TMPincorporatedpermilliliter ofsupernatantculturefluid.

VOL.62, 1988 ₉₃₇

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ulmalns,.al vrklsLvsevvelrlntvI Iavt^r^"rb^^Ii I^rt I.t_t__;IL-a^ S_"_-A

C3H GTcG _ 3839

T

LouhrgM-tValAlaAalsiAlaValLsurhrLssAsal*GlYLYsfuThrMotGlyGlnPro. uValIllL*uAiProHislaValGrluAlaiLuValLysGlnProProAs

C3H CCGTGGTAGCAGCCATTGCCGTcTACAAAGA 3959

AKR

ArgTrpLuSrAsnAM ArqMetThH GArValGlnPholYProValVAlaLouAsnProAlaThrLuL5 uProLuProGluGlu

C3H CGCTGCTACCAACGCCCGCATGACCCACTACCGCATGCTCCTAG^iCACTGACCGAGTTCAGTTGACCAGTGGTGGCCCTAACCTGCCACCTTACTCCCTCTCCCGGAAGhM 4079

AKR

GlYAlaProHisAspCysL*uGluXleLouAlaGluThrHisGlvrThrAr ProAspL6uThrAspGlnProIlloProAspJuAspHis*ThrTrpTErThrAsDG1YSirS*rPh*LAu

C3H GGAGCCCCCCA7rGCTGACAGCCCATCCCAGACGCCGAGCCACTCACT AACC TG;ACArC5T0 4199

AKR

GlnGluGlyGlnArqLvsAlaGlvAlaAlaValThrThrGluThrGluValIllTrMl ArgAlaL*uProAlaGlyThrSerAlaGlnArgAlaGluL*uIleA1aLuThrGInAla

C3H _CCACCGCCATACACTC CC 4319

AKR A

L*uLYsMotlaGlu1LYLsAFrqIuAsnValTYrThrAspS*rArgTrAlaPheAlaThrAlaHisIlHisGl1yGuIleTyrAr&Ar rFG1yL*uL*uThrS*rG1uG1YAr

C3H TXAT 4439

AKR G

GluIleLysAsnLysSrImI1L*u/laLouLouLysAlaL*uPhoLFuPrOLvsAr L*uS.rSlleZl.HisCysLouGlyHisGnLsGlvAs *rAlaGluAlaArqg1n

C3H GAMTCAAAACAGACCGAGAC CAAAGACTCAGTATAATT C AGC C 4559

AM

ArqLouAl AspGlnAlaAiaArgGluAI&lall*TysThrPcroProAspThrSorThrL uL*uIl-&lUAsSSrThrProTyrThrProAlaT

rPhqoHisTyrThirGluThrAsp

C3H CGCACAGACCCAGCGGCCCGC;GAGGCTGCCTTACTACGCCT 4679

AKR

L.uLysLysLauArqGluL.uGlyAlsThrTyrMrAnGlnS-rLysGiyTyrrpalPhoGlnGl LysProValM*tProA- uAsS*rLeuHisAr C3H CTATAACAAACCAGAGAGCTTCvGGCCACCTATAACTGAGTTTACTCCAGCCGGGTGGTGCCCG heVaPTA GTTAA LCATCAC 4799 AKR

Lu~ThrHis'LuGlyTYrGiDnLYsM tLysAla*)uLuAspArgGlyGluS*rProTyrTyrM tLouAsnArgUDLyeThrLuGlnTyrValJilaAs S-r YsThirValCysAla C3H CTChCCCACCTCGG,TACCACACTCCTTGAICCCTCCGgXCCCGCCTAACCCCGGACAACCCTCCAATATGTGA CCTGCjCoGTshTGCC 4919

AK

G1nValAsnAlaS*rLys sIlGlyAlaGlyValArgValArqGlyHisArqProG r isTrGluIl1Ph*hrGluValLysProGlvLsuTvrG1yTyrLysTY

C3H CACGTACTGCCCGCCTGCATG:TAGGGGATCGATCNAACAGT3CACGCAGCTGTA AT.TAC 5039 AKR

L.uLeuValPh-7ValAspThrPhSruaPh*ProThryhslTh lValSrsL LruLouGluGlullPheProArPhOGl tPro

C3H CTC CCTCCAAC 5159

AKR

GlnValLsuGlySerAsDpsnGlyProAlaPheThrSerGlnValSerGlnSerValAlaAspL;uL.uGl IleAspTr LsLI*uHisCysAlaTyrArgProGlnSFrSrGlyGln

C3H CAGGTATTGGGATCTGAACGGGCCTGCCTTCACCTCCCAGGTAAGTCAGTCGGTGGCCGAACCCAGAGTCTCAG 5279

VlGluArqM

tAsnArFThrIlsLysGluThrLouThrLysLeuThrLeuAlaAlaGlyThrArgAspTrpValLeuL*uLeuProLeuAlaLeuTyrArgAlaArFAsnThrProGly

C3H C539CATCA9CTCTAACTAA9TTACGCTTGCAGCTGGCAC GTACTCCTACTCCCCTTAGCTCTCTACCGAGCCCGGAACACTCCGGGC 5399

AKR T

ProHisGlYLeuThrProTYrGluIleL.uTyrGlyAlaProProProLeuValAsnPheHisAspProAspMetSerGluILuThrAsnSerProSerL.uGlnAlaHisL*uGlnAla

C3H CCCCATGGATTGACTCCGTATGAAATCTTGTACGGGGCGCCCCCGCCCCTTGTCAACTTCCATGACCCCGACATGTCAGAATTACTAATAGCCCATCTCTCCAAGCTCACTTACAGGCC 5519

AKR C T

LouGlnThrValGlnArqGlull*TrpLysProLouAlaGluAlaTyrArq As uAspG1nProVa1I1ProFHisProPheArFIlGlvAs S*rValTr ValAr r His

C3H CTCCA5ACGG73GCAGGAATTGGCCACTGGCCGAGGCCTACCGGTGATACCACACCCCTTC9;lG RCCGTr3MWTGCGC98CAC 5639

Al~

In

A~

A

G1nThrLysAsnLeuG1uProArFTr LYsGl ProTyrThrValLsuLsuThF 'rOThrAlaeuLysValAs GlyIl*SrAlaTr IlHisAlaAlaHisValLysAlaAla

C3H CAGACCAAAAACTTAGAACCTCGTGGAG ACCCTACACCGTCCTACTGACCACCCCACCGCTCTCAAGGTAGACGGCATCTCTGCAGGATACACGCCGCCCACGTCGCG 5759

AKR.

LtGluSG1 rThrThrL.uS.rLysProPh.LysAsnGlnValAsnProTrpGlYProLsuI1*ValLsuLouIl-LsuGlyGlyVaiAsnProVa

ThrThrProProIlLysProSrTrpAr ValGlnArqsorGl AsnProLsuLysIllArgLvuThrArqGlyAlaProEnd

C3H ACCACACCCCCGATAAAACATCATGACAACTCTCAAAACCC7'TAAAAALTCAGGTTAACCCGTGGGGCCCCCTAATTGTCCTTCTGATTCTCGGAGGGGTCAACCCG 5879

AlR G

1LuGlyAsnSFrProHisGlnValPheAsnL.uThrTrPGluValThrAsnGlYAspArgGluThrValTrpAlaIleThrGlyAsnHisProLuTrpThrTrpTrpProAspLou C3H 6119ACAGCCCCCACCAGGTTTTCCTCACCTGGGAGTGACTATGGAGACCGAGAAACGGTGTGGGCAATACCGGCAATCACCCTCTGACTTCGTGACCA 5999 AKA

roAsprouCsA AltAouAaLuisF GlyProSoT

rGlyuGlPhuTyrCrgMaProPhGiyCyrProProPrGlyArorCsCy

sSYrG

cy

rS.rASrThrProG

C3H CSSAACTCTTTTTGCTCACGGGCCGTCCTTTCGCCTAGAATATCGGGCTCC-Trrl7lTCCTCCTCCCCCGGGOCCCCCCTECTETTCAGGAAGtGCGACTCCACGCCAG 6119 AKR

lyCysS-rArgAspCvsGluGluProLouThrSrTyrThrProArqCysAsnThrAl aTrpAsnArgLouLysLoeuSrLysValThr}iisAlaHisA.n G1 lyhTyrValCysP

C3H GCTGCCAAATGGGGAGCCCCTGACTTCATATACTCCCCGGTGCAATACGGCCTGG;ACAGACTTAAGTTAT=AAAAGTG CACAAGC GG G CTATGTCTG;CC 623 9

AKl A A

roGlyPoHisArProAr TrAAgSCyGlyGlGyProluS-rPheTyrCvysAlaSorTr GiCsluThrThrGlyArgAlaesrTrEw rSSrTApT

C3H CCGGGCCACATCG CCC GCCTCG CCAGAATCCTTTGCCT CTGCGAAACCACAGGCCGAGCATC CCATCCTCGTCCTCGGACT 6359

AKR A

vrIlThrValSerAsnAsnLeuThrS-rAspGlnAlaThrProValCYsLysGlyAsnGluTrpCysAsnSerLouThrIleArqPheThrSerPheGlyLysGlnAlaThrSerTr V

C3H ACATCACAGTAAGCAACAATCTAACCTCAGA67CAGGCAA9CCCAGTATGAGTGGTGCAACTCCTTAACTATCCGGTTCACGAGCT97GAAAACAGGCCACCTC6479

AlR

alThrGlyHisTrDTrDG1 LouArgLo uTyrVal1SrGlyHi sAspProGlyLouIl1 PhoGlyIl1eArgL uLyslIl*ThrAspSe rGlyProArgVal1ProIl*GlyProAsnP roV

C3H TAAGC

W

1GCCTTCTT7GAAGCCGGTA GACGCTAATCGTCGGCCGTCATGGCACCG6599

AKR

alLeuSerAspArgArgProProSerArqProArgProThrArgSerProProProSerAsnSerThrProThrGluTh'rProLeuThrL.uProGluProProProAalyValGluA

C3H TCTGTCAGACCGACGACCACC7TCCCGGCCTAGACCCACCAGATCTCCCCCGCCTTCAAACTCCACCCCAACCGAGACACcccTcAcccTcCCCGAACCCCCGCCAGCGGGAGTCGAAA6719

AKR

6nArgLeuLsuAsnLeuValLysGlyAlaTyrGlnAlaLeuAsnLeuThrSerProAspLysThrGlnGluCysTrpLeuCysLeuValSerGlyProProTyrTyrGluGlyValAlaV

CM AC 6839

C3H ACCGATTGTTAAATCTAGTAAMGACCTACCAAGCCCTCAACCTAACCAGTCCTGATAAAACCCAAGAGTG;CTGG;TTATGCCTAGTATCGGGACCCCCATACTACGAGGGGGTTG CG 683

AKR C

R+CL3 - R+CL3

alLeuGlyThrTyrSrAsnHisThrSrAlaProAlaAsnCysS rValAlaSrGlnHisLysLouThrLuSrGluValThrGlyGlnGlyL*uCysI lGlyAlaVa1ProLYsT C3H TCCTAGGTACCTACTCCAACCATACTTCTGCCCCAGCTAACTGCTCTG9GGCCTCTCAACACAAATACCTGTCCGAAGTGACC oGGAcTCTGCATAGGCGGTcCCTALbA 6959

AM

[image:7.612.64.562.59.648.2]

R+CL3

FIG. 4. Envelope gene sequenceof C3Hp110. The sequence ofC3HpllO from theSall siteat

position

3719ofthepol

region

tothe virus/cellularjunction (top row)iscomparedwith thatofAKRp623provirus (16).C3HR+Cl3cloneswere

sequenced

from nucleotide6835 tothevirus/cellularjunction. Clones C13211andC14211 hadidenticalsequencesinthis region. Different aminoacids encodedatthepoint

mutations areboxed. - - - -,Absenceofone copyofthe99-bpenhancer element in the U3

region

of C3HpllOandC3HR+C13clones. clones showed(Fig. 6)that thecorrectA-to-Gtransversion from the

replicative

form of the mutant

phage.

The

BstEII-wasinsertedinto themutant.Noother

base-pair change

was to-XbaI

fragment

from a 1.8-kb BamHI-SstI subclone of observed in this region. C3H pllO was substituted

by

the

corresponding

fragment

For reconstruction of C3H p110-R7191, the

KpnI-XbaI

from the

mutagenized

subclone.

Subsequently,

the

BamHI-fragment containing the

gp7O/pl5E

junction was recovered SstI

fragment

was substituted into the HindIII-Sall

pBR

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C3H

R+CL3 C3H R+CL3 C3H

ARM

R+CL3

hrHisGlnValLeuCYsAsnThrThrGlnLysThrSerAspGlySerTyrTyrLAuAlaAlaProThrGlyThrThrTrpAlaCysSerThrGlyL*uThrProCysIleSerThrThrI

CCCATCCTCTACTAATACCACCCcTGc TATTTGGCCCTCCCACAGGAACTACCTGGGCTrOTAGTACTOGACTCACTCCCTGTATCTCAACCACCA 770 79 T

1e*L.uAsp euThrThrAls pTyrCysVa LUValGluL*uTrpProArqValThrTyrHisSrProSrTyrValTyrHisGGlnPh*lGuAr Ar A1 sr 1uProV

TACTA7GACCTCACCACCGATTACTGTGTCCTGGTCGAiCMCCAAGGGTGACCCATCCCCCACCAATTAAAGACGAGCCATwIAACCCG 7199

alS*rL.uThrL.uAlaLeuLuLJulGl 1 L.uThrM.tGlyGlyIlOAlaAlaGlyValGlyrGlThrThrA1aLeuVa1A1aThrGlnGlnPh.GlnGlnLu'±lnAlaA1aM

TCTCACTAACTCTGGCCCTACTATTGCTCACTATGGGCGCCCTAGGGCCACTCAGCAGTTCCAACAiCTCCAGGCTGC-ACCG

*tHi sAsDAsoL uLy5GluValGluLysS-rIlThrAsnL*uGluLysS rLeuThrS-rLouS-_GluVal1Val1LouGlnAsnArArqglyLuAs2LeuLeuPheLeuLysG1uG

C3H TGCACGA GAtCT A AGTCCATCA aAATTCCCTCCGTCCGAAGTAGTGTTACAGAATCGTAGAGGCCTAGATCTACTATCCTMGAGG

AICR

R+CL3

lvcflvLAuCvsAiaAjaauLvsG1uG1uCv3CVSPh.TyrA5pHihThrGyLUValArqASSrM@tAlaLysL4uArqGluArgL.uSerGlnArqGlnLysLeuPheGluS

C3H

T?GGTACGGACATGGCCAAAC7TAGAGAAAGAGTCAGAGACAAAAGCTCTrGAPAT

AKR

R+CL3

C3H AMm R+CL3 C3H R+CL3 C3H AM R+CL3 C3H

AM

R+CL3 C3H R+CL3 C3N R+CL3 C3H R+CL3

C3H

R+CL3

*grOlGniYTr hiGuGlL*uPhoAJnLYsS*rProTrDPhbThrThrLeuI

_{CTGTITAATAXGTCCC}

lSeSrThrIlul*MtGlyProL*uI lolIl*L*uLouLosul 1*L*uLouPhoGlyProCYsIl eL

C CCTGTATCCACCATCATGGGTCCCCTGATAATCCTCTTGTTAAT

TTACrCTTTGGGCCTATIC

*uAsnAr L[uVaIGlnPheIl*LvsAspArgIllSrValValGlnAlaLuValL4uThrGlnGlnTyrHisGlnu sThrIl spCy sLVsS.rArgGluEnd

TCAATCGCCTCGTCCAGTTTATCAMGACAGGATTTCGGTAGTGCAGGCCCTGGTTCTGACAACAATTCATCAACACAT TTGTAACACGTGAATAAAAGATTT

I-) U3

TA,TCAGTTACAGAAAGAGGGGGAT*AAACCCCTTC ATAAGGCTTAGCCAGCTAAC TGCAGTAACGCCATCCTGCAAGGCATGGGAAAATACCAGAGCTGATG1TTCTCAGAAAAA

T 0

A

CA8;AACAAGGAAGTA

CCGGGACTACGGGCCAAACAGGATATCTGTGGTCAAGCACTAGGGCCCCGGCCCAGGGCCAAGAACAGATGGTCCCCAGAAA---CAGAG

TAGCTAAAACAACAACAGTTTCAAGA

AGOCTOGAAAGTACCGGGACTAGGGCCAACAGGATATCTGGWTCAAGCACTAGGGCCCCGGCCCAGGGCCAAGAACAGAATGGTCCCCAGAAA

LACCCAACT rCCCCC AGTACsCGATCTACCCA TCTT TCTCTCGC TCTTC TGTACCCGC GCTTATTGCTGCCCCTAT

- ) R 82651-> US

AAAGGGTAAACCCCACACTCCGCGCGCCAGTCCTCCGATAGACTGAGTCGCCCGGGTACCCGT'GTATCCAATAAAGCC --- CTGBTTGCATCGAATCGTGGTCTCGCTGATCC'TT G

GGGAGGGTCTCCTCCAGAGTGATTGACTGCCCAGCl iWGGGGTCTrrCA

FIG. 4-Continued.

7319

7439

7559

7679

7799

7919

8034

8060

810

25

74

subclone. Finally, the 5' part of the viral genome with cellular flank was ligated to this construct to generate the provirus C3H pllO-R7191.

InfectivityassayofC3H

pllO-R7191.

DNA from the muta-genized clone C3HpllO-R7191wastransfectedontoNIH 3T3 cells with pSV2neo as described above. Ten neo-positive

colonies were selected, and the infectivity of these clones was determined by release of reverse

transcriptase-con-taining particles and assay of their XC fusion ability. In contrast to cells transfected with C3H pllO DNA, cells transfected with the mutagenized C3H

pllO-R7191

DNA released reverse transcriptase-containing particles and

scoredpositiveinthe XC assay(Table 2).

Processing of Pr85to gp7O and pl5E. During the biosyn-thesis of the murine leukemia virus envgene products, the

precursorpolypeptide Pr85 isproteolyticallycleavedtogp70 andpl5E. It has been shown that the cleavage recognition

sequences forthe envgeneproducts lieatthe Arg(Lys)-X-Lys-Arg for a number of retroviruses, as well as for the

hemagglutinin glycoprotein of avian and human influenza viruses(11, 38, 56). Eucaryotic endopeptidases with speci-ficity forLys-Arg have been identified in theGolgi compart-ment(27). To examine whether the point mutationobserved inC3HpllOfunctionally blocks the cleavage of Pr85togp7O andpl5E,wecompared therateofprocessing of Pr85env in NIH3T3 cells transfected withdifferentproviral DNAs. The cells were pulse-labeled with [3H]leucine for 30 min and

chased for0 and 30min and 1, 2, 3, and 4 h. The labeled proteinswere immunoprecipitated with ananti-gp70 antise-rumand separated byelectrophoresis inan

SDS-polyacryl-amide gel. In cells transfected with AKR p623 DNA, the

precursor protein Pr85 was present at high levels after a

30-min pulse and cleavage was observed after a 1-h chase (Fig. 7A). Similarly,cellstransfectedwith the DNA of the in

vitro-converted infectious C3HR+Cl3 virus (clone C14211) or with the AKR p623-C3H pllO construct 335, which containthe 5' halfof C3HpllO and the 3'half ofAKRp623,

showed cleavage ofPr85 (Fig. 7B). Alternatively, in cells

transfected with C3HpllO(phaseIII) there was no cleavage

ofPr85.Instead, aftera1-hchaseahigher-molecular-weight formmigratingas aPrlOO wasobserved whichwasstablefor

theremainder ofthechaseperiod(Fig.7A). Incontrast,Pr85 wascleavedinto

gp70

with identicalkinetics to those of the AKR virus control in cells transfected with DNA of the

mutagenized clone C3H

p110-R7191

(Fig.

7C).

DISCUSSION

Wehave previously described, on the basis of work with

biologically cloned viruses, that the endogenous ecotropic

virus in at least two low-leukemic mouse strains, C3H/He

and BALB/c, is XC negative and replication defective. XC-negative, replication deficient virus could, however, be

convertedto aninfectious XC-positive form whengrownin certain transformed mouse cells in culture. The change in phenotype involved mutation and selection as judged from

conversionexperimentsatlimitingdilutions (41, 44, 45) and under conditions which restricted successive rounds of infection (41). The converted virus was comparable with virus fromhigh-leukemic AKR mice inits ability to spread and form large XC plaques. The nonpathogenicity of the

XC-negativevirus insusceptible NFS/N mice,incontrast to the similarity of the converted, XC-positive infectious

C3H/Hevirus and ecotropic AKRvirus(41) in induction of VOL.62, 1988

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A

TGGATCTCTGAGTTTGAGGCCAGTCCATCTACACAGTGAGTTCTAGGACATTCAGGGCTACACAGAGAAA 70

cellular flank VL30 US

CCTTGTCTTGTAAAACAAATCAAAACAAAAAATTACCCTGAAAGACCCTCGAGGGGAGACCCTCACTCAG 140

VL30 R

ACACTCAAGTCCCGGGACAGCCGCGTACCCAATGAAGACACTGAGACCATACATAAAATGTAGAAGGCAA 210

GATTTAATAAGGCAGCAACATGAAAGC1I- C3H pllO provirus I-gIAGACCAGAGCTC 257

VL30 R

TGGGGTCGAAATAAAGCAGCAACATGGAAGCACACAGAGCTCTGGGGTCGAAACTTCATACACCTTAGCA 327

CAGGGTAGAGGAGTCTCGACGGTCAGCCAGAATTTTTCACAGGCTTATATAGTAAAACTCAAAGGGGGAG

VL30 U3

AACTGGGCAGGGAAAGTACAAGTTTACATCACTAGGGAGTTCTGCCAAGGGACAAGGGGTTCTGCCAAAG

GATTCTACGTAACTAAGGAGTCATGTCCTATCAAGGAACCTACGTAACTAAGGAGTACCTGGTTCATTTT GAGGTTGTTCCAGGAGGCCTTTATCTCA

397

A5,7

TABLE 2. Comparisonof infectivity of C3Hp110-R7191

andR+Cl34211provirusesa

Plasmid Supematant XC

RTactivity' phenotype

p110 10.1

-p110R7191 111.4 +

R+Cl3 4211 183.7 +

a NIH 3T3 cells weretransfected with plasmid C3Hp110-R719,and

R+C13

537 4211. Culturefluid from transfected NIH 3T3 cells was tested for reverse

565 transcriptase and XC fusion activity as described in the legend to Fig. 2.

b Reverse transcriptase (RT) activity is expressed as103counts per minute

of[3H]TMPincorporated per milliliter of supernatant culture fluid. B

TCTTGCCTCTGTTAAATACAGCCCCAAAACTATCTGGATGGGACTTCATACTGAAATTACCATGTCTACC 70

TAAACAAAAAACTTAGCCTGGTAGGATCTCCTACCATCACCACTCCCAAATATCTTTATCTCCTTCCTTA 210

GTATCATCCTGACAAGGTGGCTCACAGCACAGCTACTTTTGTTCCTTTAGATTCGGGAAGCCACACATAT 280

AATTCATACTCCATCCTTATTTTTTTGAGAAATTACATATTTTTGAACCCTGGTTTTAGAGTCCTTTCCA 350

GAGCCTGAAGGTCCCAGCATT|A---4211 PROVIRUS--- ATTGCTGAG 388

ACATAGCACACCTTTGTGTTTCTGATTATCATGGAATTCATTGGCATTAACAG 441

FIG. 5. Flanking cellular sequences of XC-negative and XC-positive C3H proviruses. (A) Flanking cellular sequence ofC3H p110. C3H pllO is integrated within a VL30 LTR in reverse orientation. The directrepeatsequences that occur at the junction of the VL30LTRand the ecotropicprovirus are boxed. The U3, R, andU5 regions of VL30 LTR are indicated. (B) Flanking cellular sequence of the C3H R+C13 4211 provirus. The direct cellular repeat attheintegration site of the provirus is boxed.

leukemia, led us to conclude that the infectivity of the

endogenous ecotropic virus was a major determinant of

leukemia incidence. The properties ofthe molecular clones

of endogenous and in vitro-converted ecotropic C3H/He

viruses presented here provide definitive evidence for our

previous conclusions and furthermore precisely locate the

lesion in theendogenous C3H/He virus.

The sourcefor molecular cloning of endogenous ecotropic

C3H MuLV consisted of C3H/MCA5 cells, a chemically

transformed derivativeofC3H/1OT1/2Cl 8 cells, from which

XC-positive virus could be obtained late (phase III) after

induction withIdUrd (41, 44, 45). The fact that the proviral DNA from these cells encoded a XC-negative replication

deficient virus for which these cells were homozygous

(unpublisheddata) established that chemical transformation

A

had not mutated the proviral DNA to yield the infectious virus form, confirming ourbiological observations (41, 45). The nature of the defect in infectivity and XC fusion activity of endogenous C3H/He virus is a point mutation at

the precursor cleavage site of Pr85 as judged by sequence

comparisonand DNAfragment exchange with the ecotropic AKRprovirus. An identical mutation has been observed in theendogenous BALB/c ecotropic virus (22). In contrast to our findings, the biological activity determinations with

cloned BALB/c ecotropic viral DNA were interpreted to

indicate levels of infectivity comparable to those of AKR virus clone p623 (22). However, the assay conditions used were different from ours in that they allowed for multiple

cycles of infection and thus selection (22). Moreover, the target cells were of the converter category (see below), according to ourexperience, and the time of reverse

tran-A

AKR 623

O' 30 1hr 2hr 3hr 4hr

C3H p110 Phase III

O' 30'1hr2hr3hr4hr Pr100_

gp7l _ r

B Construct 335 O' 30'1 hr2hr3hr4hr

R+C134211

0' 30'1hr 2hr 3 hr4hr

Pr851

4000V

***

004

am

gp70

-,-*

B

A T G C A T G C

-.NO_

A. C3HpI1O 5' TTT GAA AGA CGA GCC AAA TATAAAAA GAA CCC 3' Phe Glu Arg Arg Ala Lys Tyr LysILysiGIu Pro B. R7191 5' TTT GAA AGA CGA GCC AAA TAT AAAIAGAIGAA CCC 3

Phe Glu Arg Arg Ala Lys Tyr Lys ArgJGlu Pro

FIG. 6. Site-directed mutagenesis of C3H p110 (Lys-7191) to C3H p11O-R7191 (Arg-7191). The M13 dideoxynucleotide sequence ofp110 and R7191 clones in the region of mutation is shown. The position of the A-to-G transition is indicated by an arrow. Amino acids encoded by the region shown in the sequenceare indicated below. TheLys-to-Argtransition is boxed.

C

C3H p11OR7191

O' 30'lhr2hr3hr4hr

Pr85

-@w-gp70"O

FIG. 7. Pulse-chase analysis of the env gene product Pr85 ex-pressedfrom cells transfectedwith (A) AKR p623 andC3H pllO phase III, (B) construct 335 and C3HR+Cl3 4211, and (C) C3H pllO-R7191.Transfected cells were labeledfor30min with 40,Ciof

[3H]leucine,chasedfor0 and 30 min and 1, 2, 3, and 4 h, and then lysed. Lysates were immunoprecipitated withanti-gp7Oantiserum. Immunoprecipitates were analyzed on SDS-10% polyacrylamide, and the gels were exposed for 3 weeks. Sizes of proteins were determined from theirmigration relative to a 14C-labeled marker protein.

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scriptase assay waslate aftertransfection, presumably

cor-responding to what we have termed phase II/III of virus spread (44,45).

Thesequenceofthecleavagesite intheXC-negativevirus is Arg-Ala-Lys-Tyr-Lys-L-Glu-Pro and Arg-Ala-Lys-Tyr-Lys-Arg-Glu-Pro in the AKR virus (the Lys-to-Arg

substi-tution is underlined).ThisArg(Lys)-X-Lys-Argcleavage site has been conserved in a number of other retroviruses, including humanimmunodeficiency virusand some

myxovi-ruses (11, 38, 56). The Arg(Lys)-X-Arg-Arg sequence at the

cleavage site ispresent in human T-cell lymphotropic virus type I,bovine leukemia virus, and feline leukemia virus (56). Fromexperiments with surface-labeled IdUrd-induced C3H/ 1OT1/2 Cl 8 phase III cells, wehadprevious evidence fora

defect in envelope glycoprotein processing (N. Famulari, and U. Rapp, unpublished data). Pulse-chase experiments withcellstransfected with C3H/HeproviralDNAclonepllO

confirmtheseearly observations,aspresentedin thisreport,

since proteolytic cleavage of Pr85 into gp7O and pl5E is

blocked (Fig. 7) and leads to the accumulation ofa

unpro-cessed precursor. Processing of precursor polyprotein to

functional gene products plays a fundamental role in the

infectivity of viralparticles. Forexample, ithasbeen shown for avian influenza virus that the

proteolytic cleavage

of hemagglutinin glycoproteinto HAl andHA2is

required

for full infectivity but not for full assembly (4). We have

observed, by electron-microscopic examination of virus produced by

C3H/1OT1/2

Cl 8 cells acutely infected with C3H/10T1/2 phase III virus, that the budding particles

ap-peared bald relativetoecotropic AKRvirus, indicative ofa lackof mature envelope glycoprotein (unpublished data).

Conversion of

XC-negative

to

XC-positive

infectious virus in cultureby growthintransformedmousecells restores the Pr85 cleavage site by a point mutation (A-to-G transition, resulting in a Lys-to-Arg substitution) as demonstrated for

the molecular clones C13211 and C14211 derived from C3H R+Cl3 grown in NIH 3T3 cells. Recombination with

non-ecotropic

endogenous

envelope

sequences as a source for

the amino acid substitution is

virtually

ruled out, since no

additional sequence alterations relative to clone pllO were

observed in this

region.

Both C3H R+C13 clones and the

infectious AKR ecotropic virus have an identical sequence atthecleavage

site, indicating

that otheramino acid

substi-tutions that could be achieved by

single-point

mutation of this region may not be

compatible

with Pr85 cleavage. We have examined the consequence of this mutation for

enve-lope glycoprotein precursor processing by pulse-chase

ex-periments andfound it to restorecleavage.

Why does conversionoccur

preferentially

incertain

trans-formed cells? One

possibility

isthattransformedcells which behave as converter cells haveanincreased mutation rate.

However, several lines of evidenceargue

against

this.

First,

the endogenous C3H/He virus in chemically transformed C3H/MCA5 cells was not mutated, as shown above. Simi-larly, infection of converter cells under conditions that

minimize subsequent virus spread (as achieved by using

high-percentage

virus donor cells and

by

the absence of Polybrene in the cocultivation) eliminate conversion.

Fi-nally, long-termpassage ofC3H/10T1/2 Cl 8 phase III cells foratleast 18months didnotyield convertedvirus(40, 41, 44) as might be expected iftheir potentially lower rate of mutation during DNAsynthesis was the limiting factor for conversion. Therefore, we conclude that mutation occurs

duringreversetranscription. Transformed cells tendtogrow virus to ahighertiter than normal cellsdo,and this would be

expected to facilitate subsequent rounds of

infection,

even

for

poorly

infectious

particles.

Once a

highly

infectious

mutantemerged, it would

quickly

be

amplified by selection,

provided that a sufficient reservoir of virus-free cells was available. The basis forbetter virus

growth

by

transformed

cells is not known and may include differences in DNA

methylation activityand enhancerutilization.

The LTR ofthe

ecotropic

C3H/He

provirus

has

only

a

single

enhancer

element,

raising

the

possibility

thatsomeof

the differences in

infectivity

relative to

ecotropic

AKR MuLV

might

be LTRmediated.

However,

virus

production

levels,

as determined

by

reverse

transcriptase

assays, are

only

<5-fold lower in chronic

producer

cultures of C3H/10T1/2 Cl 8

phase

III than in AKR

phase

IIIcell lines

(41, 44),

indicating comparable

transcription

ratesfrom the

proviral

LTRs. A

single-nucleotide change

has been

ob-served at

position

7889 between the

proviral

LTRs ofthe

endogenous

and converted

large

XC

plaque-derived

C3H virus clone

R+C13, suggesting

that LTRmutationsmay also

contribute to the increased

infectivity

of this converted virus. However, these mutations were not necessary for conversion oftheXC

phenotype,

as shown inthisreport

by

DNA

fragment exchange

between

endogenous

C3H/Heand AKR

proviral

DNA.

The step in the

replication cycle

at which

XC-negative

ecotropic

C3H/He virus is blocked in acute infections is at thelevel of virusrelease

(41).

Thus,

the

specific infectivity,

as measured

by p30

antigen production

in

acutely

infected

cells,

was

comparable

between

XC-negative

C3H/He,

XC-positive

converted

C3H/He,

and

XC-positive

AKR viruses

(44), whereas an assayfor release ofreverse

transcriptase-containing particles by

infected cells showed a 100- to

1,000-fold

lower

infectivity

for the

XC-negative

virus

(41,

44). The

initially

nonproductive

infection

eventually

be-comes

productive

oncontinued subculture of infected non-converter

cells,

without anydetectable

change

in the stable

properties

ofthe

produced

virus. Themechanism

underlying

this processisunknown. One

possibility

isviral gene

ampli-ficationvia

repeated

cycles

of infectionas aresultof

initially

incomplete

levels ofinterference. C3H/10T1/2Cl8

phase

III cells

chronically producing

XC-negative

C3H/He virushave

glycoprotein

ontheir

surfaces,

areGlX cell surface

antigen

positive,

and are

completely

resistant to

superinfection by

XC-positive

virus

(P.

0. Donnell and U. R.

Rapp,

unpub-lished

data).

In contrast, cells

acutely

infected with

XC-negative

C3H/He virusexpress

p30,

but

only

very low levels

of

gp70,

are GlX

antigen negative,

and are

only

partially

resistantto

superinfection.

The lackofGlX

antigen

may be due to the low level of

envelope

glycoprotein expressed

at the cell surface or may be a consequence ofthe structural

mutation

(19).

The

inability

of

XC-negative

C3H/He virus to

rapidly

establish a

productive

infection is

presumably

the basis for

its lack of XCfusion

activity.

From

experiments

inwhichrat

XC cell infection was facilitated

(use

of

Polybrene)

or

blocked

(addition

of

virus-neutralizing antibody)

after

over-lay

of

XC-positive

virus

producer

cultures,

it appears that suchaninfection isa

requirement

for effective fusion

(U.

R.

Rapp,unpublished data). These

findings

do not

explain

the basis for the lack ofXC fusion

activity

of other hostrange classes of

XC-negative

type C

retroviruses,

nor do

they

establish that all

XC-negative ecotropic

murine type C viruses must be defective atthe same site.

Clearly,

thecell surface

receptor-binding specificity

of viral

gp70

for the

ecotropic

viralreceptor

(8, 43)

isa

requirement.

The

ecotro-pic

viral receptor is a

potential

fusion receptor, whereas other

xenotropic

or mink cell

focus-forming

viralreceptors VOL.62, 1988

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http://jvi.asm.org/

(11)

are not.Thus, introduction of a different receptorspecificity by recombination between ecotropic and endogenous am-photropic minkcellfocus-forming-class murine type C virus (43) destroys XC fusion activity of the parental ecotropic

virus. Our experiments establish that cell surface

presenta-tion of ecotropic Pr85 with an intactreceptor-binding siteis not sufficientto induce XC plaque formation. We ascribe the

basisfor this inabilitytothe associatedreplicationdeficiency

of the virus, which is not effective in establishing a rapid productiveinfection of XC cells.

Multiple mechanisms contributetothe controlofleukemia

in low-leukemic mouse strains. In BALB/c mice, virus

spread is suppressed by the restricting allele of a host resistancegene, Fv-J (36, 37), and, additionally,the

endog-enous ecotropic virus has a structural defect (22). For C3H/He mice it appears that the structural defect of the

endogenous virus is by itself sufficient to contain virus spread, since this strain carries apermissive Fv-J allelefor

its endogenous ecotropic virus (36, 37). With the molecular clones described in this report, we are now inapositionto test the impact of a single proviral point mutation on leukemia incidence in C3H/He mice. Mutantviruses,

gener-ated by site-directed mutagenesis, have nowbeenprepared and are being tested for their ability to increase leukemia

incidence in C3H/He mice.

After submission of this manuscript, inhibition of

enve-lope precursor processing by an Arg-to-Lys substitution in AKR MuLV was reported (10).

ACKNOWLEDGMENTS

WeacknowledgePatriciaLloyd and Robert Nalewaikfor excel-lent technical assistance. Wethank Steve Oroszlan for providing gp7O antibodyand JamesIhleand John Cleveland for criticalreading of themanuscript.

G. S. is a Ph.D. student in thegraduateprogram of Geneticsat George WashingtonUniversity, Washington,D.C.

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