Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

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Vol.40,No.1 JOURNALOF VIROLOGY, Oct.1981,p.45-55

0022-538X/81/100045-11$02.00/0

Fv-1

Host Restriction

of Friend Leukemia Virus: Analysis of

Unintegrated

Proviral DNA

JEFFREY CHINSKY' AND RUY SOEIRO'-*

Departments ofMedicine2andCellBiology',AlbertEinsteinCollege ofMedicine,Bronx, New York 10461 Received 6 March 1981/Accepted 15 June 1981

The murine gene Fv-1 predominantly controls the outcome of infection by

murineecotropic retroviruses. The inhibition of virus replicationby theFv-1gene

product has been determined to be at an early stage in virusreplication.

Mecha-nistically, its effectappears tobe on the accumulation of unintegrated proviral

DNA oritsintegration or both. We investigated the synthesis of unintegrated

proviral DNA, usingseveral clones of B-, N-,orNB-tropic Friend murineleukemia

virus. Our results indicate that the accumulation of B-tropic proviral DNA in

NIH cellsmaybeinhibited ateither the level of linear (form III) orcovalently

closed circular DNA (forn I), dependingupon the degree of restriction of the

clone of virus used. We confirmed that there isaneffect of theFv-1gene on the

accumulation of form I DNA of eitherB- or N-tropic Friend murine leukemia

virus. However, the decrease in infectiouscenterseffected by the Fv-1 genedid

notcorrelatequantitatively with the effectonform Iproviral DNA produced by

N-tropic Friend murine leukemia-virus in nonpermissive cells. Lastly, we

dem-onstrated in nonpermissively infected NIHcells thatarapidly migrating doublet

of viral DNA is formed.

ThemurinegeneFv-1 isanautosomal

domi-nantwhichexerts amajor controloverthe

out-comeof infection of micebymostmurine

retro-viruses(3, 14; forareviewseereferences6 and

12). This gene, whose phenotype canbe

dem-onstrated in tissue culture, confers resistanceto

thereplication of retroviruses. Thegenehastwo

codominant alleles, Fv-ln and Fv-1

b.

Fv-1n

homozygousanimalsarepermissive for N-tropic,

but resistant to B-tropic, murine leukemia

vi-ruses(MuLV's). Fv-lb1b animalsarepermissive

for B-tropic, but nonpermissive for N-tropic,

viruses. Most murine virusesmay beclassified

as either N- or B-tropic, according tothe host

range they exhibit. However, some

laboratory-derived ecotropic viruses exhibit NB-tropism,

whichis definedasthe abilitytoreplicate well

incells ofanyFv-1 type.

Thedegreeof host resistanceconferred by the

Fv-1geneis relative (3);thatis,a50- to

10,000-fold decrease in infectious centers is observed

whenagivenvirus infectsapermissiveversus a restrictive host.The degree of restriction, which

dependsupon the exact virus andhostcellused,

may showeither single- or two-hit kinetics on

thenonpermissivecelltype (1, 7, 15, 19).

The replication of virus in a restrictive as

comparedwith apermissivehost wasstudied for

bothearlyandlateeventsin the virus cycle. It

was shown in our laboratory, as well as

else-where, thatearlysteps,suchasadsorption,

pen-etration, and initiation ofproviralDNA

synthe-sis,are unimpaired in the restrictive host (5, 8,

11,23).However,biological studies indicate that

the Fv-1 restriction effectoccursduring theearly

phase of the viralreplication cycle (2, 24).

Fur-thermore,ourunpublishedstudies indicate that

if an infectious center is established by a

re-stricted virus, there is no restriction of the

expression of either viralproteinsorRNA. Our

conclusion, therefore, is that the Fv-1gene

prod-uct acts at anearlyphase in viralreplicationto

decrease the efficiency ofestablishingan

infec-tiouscenter.

Weand others have shown that the

integra-tion ofproviralDNA afterexogenousinfection

issuppressedduring Fv-1 host restriction(8, 23).

Thestudies ofYangetal. (26)andofJolicoeur

and Rassart (9), suggestedthatthe

Fv-1-medi-ated inhibitionofproviralDNAintegrationmay

be due to an inhibition of the

production

or

accumulationorboth of form I circular

proviral

DNA, thepresumedprecursortotheintegrated provirus.

We studiedthe earlyforms ofnonintegrated

proviral DNAproduced during acute infection

byclonesof either B-tropic orN-tropicFriend

MuLV (F-MuLV). Ourresults indicatethat,

de-pending upon the clone of B-tropic F-MuLV

used, restriction in NIH cells (Fv-1

f/f)

may be

45

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observed at either oftwosteps ofproviralDNA

accumulation. That is, highly restricted clones failed to accumulate linear proviral DNA, whereas less restrictedvirusshowedadecrease only in form I circular DNA. Secondly,

N-tro?ic

F-MuLV infection of BALB/c cells (Fv-1

16)

manifested a decrease in the level of circular DNA accumulated. However, the decrease in the level of circular DNA found in the restrictive infection did not correlate quantitatively with the decrease in the number of infectious centers formed.

(The data in this paper are from a thesis to be submitted inpartial fulfillment of the require-ments for the Ph.D. degreein the SueGolding

Division of Medical Sciences, Albert Einstein College ofMedicine, Yeshiva University.)

MATERIALS AND METHODS Cells.NIH, BALB/c,and XC cellswereobtained from Janet Hartley and Wallace Rowe. They were

maintained inmonolayer culture withJoklikmodified Eagle medium containing 10% fetal calf serum and nonessential amino acids. DBA/2cells were the gen-erous gift of Robert Bassin and weremaintained in monolayer culture with Dulbecco modifiedEagle

me-diumcontaining10%ofetal calfserum.

Viruses.N-tropicandB-tropic F-MuLVwere ob-tained from Frank Lilly ashomogenates ofspleens

from infected NIH andBALB/c animals,respectively.

The viruscomplex containingboth thespleen

focus-forming virus, as well as the replicative competent helperF-MuLV, waspassed in tissue cultureathigh

dilutioninFv-1permissivemousefibroblastcell lines. Progeny viruswassubsequentlypassed in uninfected fibroblastcellstoeliminate the spleenfocus-forming

virus component. The N and B typelymphatic leu-kemia virus components (F-MuLV) were then cloned twicebylimitingdilution (using XC assay[18]),and thecloned viruswaspassagedtohightiter andfrozen inaliquots.

Virus infection. Cloned virus passaged to high

titer was usedtoinfect permissivesubconfluent mouse fibroblasts. Virusaliquotswerequicklythawed, passed through Millex HA 0.20-,um filter units to remove virus clumps, and titers were determined by direct XC plaque assay.Similarly,processed aliquots were used forexperiments. Confluent mouse fibroblastcelllines weretrypsinized and seeded at adensity of1 x 106 cellsper 100-mmdish or 3 x 106 to 5x 106cellsper T150 flask (Falcon Plastics, Oxnard,Calif.)inmedium containingPolybrene at5

jig/ml.

At 9to 12 h later, the cellswere infectedwith appropriate amounts of virusin mediumcontaining Polybrene (5iLg/ml).At thistime,asample dishwastrypsinized, andthe cells

were counted to ensure anaccurate multiplicity of infection(MOI) determination.

Isolation of viral DNA.Atappropriatetimes after infection withvirus,cellsweretrypsinized,rinsed with serum containing medium, centrifuged into apellet,

andsuspendedin10mMTris-hydrochloride (pH 7.5)-10mM EDTAat <2.0 x 106cellsper ml.Cellswere

heatedat65°C (personal communication,AnnSkalka)

for 15 min, then lysed in 1.0% sodium dodecyl sulfate at65°C for 20 min. NaCl was added to 1 M (4), and thesampleswerekept at4°C overnight. Samples were

centrifugedat12,000rpm in aSorvallSS 34 rotor, and the supematantswereextracted withphenol and chlo-roform-isoamyl alcohol (24:1) and precipitated with 2 volumes of ethanol at -20°C.

Agarose gelelectrophoresisandblot hybridi-zation. DNA samples wereseparated byhorizontal electrophoresis in 0.8% agarose slabgelssubmerged2

to 3mmunder therunningbuffer(0.089MTris-0.082 Mboric acid-0.002 M EDTA). The gel was stained with 1,ug of ethidium bromide per ml in 0.5 M am-monium acetatefor Polaroid photography under UV illumination, and the DNA wastransferred to nitro-cellulose by the Southern transfer procedure (22). Pretreatment with0.15M HClwas used forpartial depurinationof DNAtoenhance the transfer of the larger fragments from the gel (25). Viral DNA trans-ferred to nitrocellulose paper was detected by molec-ularhybridization to 32P-labeled F-MuLV DNA

pre-paredby nick translation (17) of F-MuLV DNA cloned into the plasmid vector pBR322, a generous gift of A.

Oliff (13). The specific activities routinely obtained

were 0.5x 108to 2.0 x108 cpm/yg of DNA. Hybridi-zationof the nitrocellulose-boundviral DNA to the

[32P]DNAprobewasperformedin 10% dextran sulfate (25), 5xSSC (lx SSC=0.15MNaCl,0.015Msodium

citrate),50%formamide,0.01Mphosphate buffer (pH 7.0), and100jigof salmon sperm DNA per ml at 42 to

44°Cfor 12to 16 h.This wasfollowed by extensive

sequential washingsat650Cin3xSSC,lxSSC, 0.4x

SSC, and 0.2x SSCcontaining0.2%sodiumdodecyl sulfate. Filterswereairdried andexposedat-700Cto

KodakX-Omat Rfilm, usingaPickerMAX-1

inten-sifyingscreen.

Quantitationof viral DNA. The band intensities of theautoradiogramsweremeasured with a double-beamrecording microdensitometer (MKIII C; Joyce, Loebl and Co., Ltd., England). Several

autoradi-ographic exposures ofeach hybridizedblotwere ob-tainedsothatasingle wedge settingonthe microden-sitometer could be usedtocompareadjacentlanes for bands representing both forms I and III DNA. The

backgroundexhibitedonthemicrodensitometry

trac-ing was thencarefullyconsidered, and the peak area representing either form III orform I wascutfrom thetracingandweighedon ananalyticalbalance (Met-tler).Itwasdemonstratedthat,withinawell-defined range,the microdensitometer tracing method gavea

linear responsetodifferentamountsofhybridizedviral DNA, similar to the standardizationprocedures

de-scribed by Keshet et al. (10). Therefore, only those peak weights obtained from asingle autoradiogram whichfellwithinthisdefined rangewerecompared.

Restriction endonucleases. Restriction enzymes were obtained from Bethesda ResearchLaboratories,

Gaithersburg,Md. Twotofive units ofenzyme perjig ofDNA wasused with the reaction conditions pro-vided by the supplier. The extent of reaction was

monitored with either lambda DNA or adenovirus

type2DNA.

RESULTS

Quantitation of nonintegrated proviral

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Fv-1 HOST RESTRICTION OF F-MuLV 47 DNA from acutely infectedcells. Acute

in-fection by retroviruses initially results in the

formation of free nonintegrated proviral DNA

andsubsequently in theintegration of thisDNA

into the host genome. Previous studies have

demonstrated two predominant forms of free

DNA in the infected cells: a double-stranded

linear DNA (forn III), which gives rise to a

covalently closed circular supercoiled DNA

(form I), which in turn is presumed to be the

precursor of the integrated provirus (21).

Be-cause relatively few copies of viral DNA are

synthesized percell,ourinitial studieswere

di-rectedatoptimizingtherecoveryof thesemajor

nonintegrated DNA forms.Avery poorrecovery

of closed circular proviral DNA was obtained

when the Hirt extractionprocedure (4)was

ap-pliedto 5 x 107permissivelyinfectedcells

grow-ing in roller bottles. Our suspicions were

that

this lowyield of circular DNA could be dueto

extraction artifacts. Accordingly, we modified

both the extractionprocedure andtheinfected

cell number. The modification of the Hirt

pro-cedure involveslysingandtreating the infected

cells at 65°C before salt precipitation of

chro-mosomal DNA.Using this method,wewereable

toisolatesignificantamountsof both form I and

form Ill DNA from as few as 1 x 106 cells.

However, scaling up the procedure to process

larger numbers of cells grown inroller bottles

still resulted inapoorrecoveryofform I DNA.

We attribute these results eithertomechanical

entrapment ornuclease degradation of

uninte-gratedproviral DNAdue to agreatercell

con-centrationduring extraction.

With this new meihod, and using only

be-tween1 x106and5 x106cells,weexamined the

proviral DNAaccumulated inseveral cell lines

considered either permissive or restrictive in

termsof theirFv-1genotype.Ourresults, shown

in Fig. 1A, indicated that distinctly different

amounts of nonintegrated proviral DNA are formedindifferent celltypes evenif the cellsare

consideredequallypermissiveatthe Fv-1 gene.

UsinganNB-tropicF-MuLV,avirus whose host

rangeis notlimited bythe Fv-1 gene, we

com-pared the proviral DNA accumulated in NIH

(Fv-1f'/),

BALB/c (FV-l

b/b),

and DBA/2

(Fv-IJn/f)

cells.Extrachromosomal DNA obtained by

the modified Hirt extractionwasseparated by

electrophoresis inagarose, transferred to

nitro-cellulose (22), and hybridized to a

[32P]DNA

probeprepared by nicktranslationofcloned

F-MuLVDNA(13).It is evident thatthe recovery

ofboth form I and form III from each of the

three cell lines was different. Recovery was

greatest fromNIH (lane 2), least from DBA/2

(lane 3), and intermediate from BALB/c (lane

1) cells. A study comparing N-tropic virus in

A

2 3

123

w:p1q

Form

II-FormI(- Cs

B

2 3

-14 2

--iO __ m mFo

- 6.1 -- 4.5

[image:3.496.247.439.52.210.2]

-U:.

FIG. 1. Autoradiograms offilterblothybridization analysis ofHirt DNAextractsfromvirally infected cells. Subconfluentmousefibroblastcelllines(5.0x

106cells)wereinfected with either B-tropic F-MuLV orNB-tropicF-MuLV(MOI of1.0)inthepresenceof

Polybrene (5pg/ml) for 16 h. After infection, cells

wereextracted bythemodifiedHirtprocedure (see

Materials andMethods), and the Hirt supernatant DNAwasloadedonto0.8%agarosegels. Electropho-resiswasfor 20 hat50 V. Eachlanecontainsthe DNAobtainedfromthe entireextractof5x10'cells.

Afterelectrophoresis, the DNAwastransferredtoa nitrocellulose filterandhybridizedto a nick-trans-lated [32P]DNA (specific activity, 0.5 to 1.0 X 108

cpm/pg)probecontainingF-MuL Vsequences. Auto-radiography wasperformedwith X-Omatfilm and

intensifierscreensat-70°C. (A) NB-tropicF-MuLV

infection ofBALB/ccells(lane 1),NIHcells(lane 2),

and DBA/2cells(lane3). (B)B-tropic

F-MuLVinfec-tionofBALB/ccells(lane1),NIHcells(lane 2),and

DBA/2cells(lane 3). The bandsof hybridizedDNA

areindicatedatthe sidesofeachautoradiogramand

representformIcircular andform III linear viral

DNA. The numbers between theautoradiograms

rep-resentthe locationsand sizes (inkilobasepairs) of BamHI-digested adenovirus type2 DNAfragments migratingin the samegel. The broad bands atthe

topofthegelsrepresentresidual hostcellDNAthat wasnotcompletelyeliminatedbytheHirtprocedure.

NIH versus DBA/2 cells gave similar results (datanotshown). The basisfor thesedifferences

maybeatthe levelofsynthesis,degradation,or extractability of proviralDNA from the individ-ualcell lines. The relative platingefficiency of each cell line also differsand must be controlled to ensure identical cell numbers, both at the time of infection and at the time of harvest.

These possibilities have not all been explored. We concludedthatthe differencesintotal

recov-ery between cell lineswould complicate an at-tempttocompare the effectof the Fv-1 alleles in these different cell lines and that this fact mustbeconsidered inanysuchanalysis.

Inadditiontovisualanalysis of the

autoradi-ogram,wecomparedthe amounts of form I and form III DNA by assaying the labeled probe })FormI VOL. 40,1981

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48 CHINSKY AND SOEIRO

bound tothe nitrocellulose filter. Inourhands,

the totalamountofradioactivity that could be

directly ascribed to the DNA bands was very

low,usually of the order of50 to 200cpmabove

abackgroundof approximately50cpmfoundin

theinterband regions. We consideredthat these

small differences of radioactivity wouldnot

per-mitus tomakeanaccuratecomparison between

different cells.

Toquantitate thepresenceofproviral DNA,

microdensitometrytracing of the autoradiogram

presentedan alternative toscintillation

count-ing. By analyzing the weight of theareaunder

the curveof the microdensitometry tracing,we

measured the total amount of annealed

radio-active DNA and minimized the background.

Furthermore,weelectedtocomparetheratioof

formsIand III DNA inthosecells which differed

inaccumulationor recovery or both ofproviral

DNA. Inthisway, ananalysis ofratios could be

made with amethodthat wouldnormalize

re-sults, regardless of the total proviral DNA

re-covered. In Fig. 1A, for example, the ratios of

form IIItoform I DNA for the threeNBtropic

F-MuLVinfectedcellsareverysimilar; forNIH

it is1.34, for DBA/2 it is1.39,and for BALB/c

it is 2.12. We feel that, within the limits of

resolution of the technique, these numbersare

not significantly different. Comparison of the

ratiosof the proviral forms thereforepermits the

determination of the effect of different Fv-1

al-leles onthesynthesis oraccumulation orboth

of these proviral DNAforms by asingle virus

type.

B-tropic F-MuLV infection of Fv-_jln

cells. Thedegreeofrestriction of growth of

B-tropic F-MuLV in NIH Swiss fibroblastcelllines

can vary up to 100-fold depending upon the

stock of B-tropic virus examined. We cloned

stocks of B-tropic F-MuLV that are restricted

either 10-2or10-4whenanalyzed by the direct

XCplaqueassay onNIHversusBALB/ccells.

The synthesis of unintegrated viralDNA after

infection of various cell lines by the

10-2

re-stricted B-tropic F-MuLV

(B-2)

was examined

by the Southern blot hybridization technique.

The resultsareshown in Fig. 1B. In both

per-missiveBALB/ccells(Fv-1b/b)and

nonpermis-siveNIH cells

(Fv-1'1)

(lanes 1 and2,

respec-tively), nearly equivalentamounts of linear

pro-viral DNA (form III) werefound. However,in

the nonpermissive cell, essentially no form I

DNAcould bedetected(lane 2). By comparison

with DNA size standards, weestimatethe size of thelinearproviralDNAinbothcelltypes to be about 8.8 to 9.1 kilobase pairs (kb). Using

CsCl-ethidium bromide density gradients, we

demonstrated for the permissive cell that the

bands migrating between the 6.1- and 4.5-kb

markersrepresent the two closed circular

pro-viralDNAspecies(formI) known toaccumulate

inretrovirus-infected cells (20, 27). By analogy

withpublished data, the slower migrating of the

twospeciesof formIshould containtwocopies

of the long terminal repeat sequence (LTR),

whereas the faster band should contain onlya

single copy (20, 27). This clone ofB-tropic

F-MuLV thusappearednottoberestricted in its

production of form III DNA, but accumulated

markedly diminished amounts of both form I

DNA species. In contrast, in another Fv-1

1'-type cell (DBA/2), the accumulation of both

form III and form Iwasdiminiishedrelative to

the NIH cell (lanes 3 and 1, respectively) as

would bepredictedfromourearlierresults(Fig.

1A). InthecaseofDBA/2cells,however, both

species of formIDNAareevident(Fig. 1B,lane

3).

The ratio of linearDNA tothe combinedtwo

species of closed circular DNAwasobtainedby

means of comparison of the areas under the

curves describedbymicrodensitometry. In the

case ofB virus infection ofBALB/c cells, the

formn

III/form Iratio equaled 0.76, whereas B

virusinfection of DBA/2 cells resulted inaratio

of 1.38. Noratio could be determined for NIH

cells because no measurable form I was

accu-mulated (Fig. 1B, lane2). By direct XCassay,

this clone ofB-tropic virus wasrestricted

100-to 500-fold on NIH cells, but only 50-fold on

DBA/2 cells. In agreement with published

re-sultsfrom otherlaboratories,Fv-1 host

restric-tion of this B-tropic F-MuLV in NIH cells is

associated withadrastic inhibitionof the

accu-mulationofformI (9, 26). However, in the less

restrictive DBA/2 cells,clear-cut accumulation

ofclosed circular DNAwasfound,andat aratio

tolinear formsonlyslightlydifferent from that

found in thepermissiveinfection. Forreference,

on this autoradiogram, a nonrestricted

NB-tropic virus infection ofDBA/2cells resulted in

aratio of1.67 (Fig. 1A). These resultssuggest

thatthe decreased accumulation of formIDNA

found inB-tropicinfection of restrictiveDBA/2

cells (ratio, 1.38)relativetothepermissive

infec-tionofBALB/c cells(ratio, 0.76)maynot

com-pletelyexplainthe50-folddecrease in infectious

centersobservedin theXCassay.

ThesynthesisofproviralDNA in NIH Swiss

cells was further examined with a clone of

B-tropicF-MuLVthat exhibits10,000-fold

restric-tion (B-4) whenexamined bydirect XC assay.

Theresultsareshown inFig.2.Linearproviral

DNAspecies(form III)weredrasticallyreduced

in thenonpermissive NIHcells(Fig. 2A,lane3;

Fig.2B, lane7)comparedto

permissive

BALB/

ccells (Fig. 2A, lane 2; Fig. 2B, lane 6) in two

different experiments withtwo different

input

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VOL. 40, 1981

A

12 3

B

[image:5.496.43.235.63.197.2]

1 2 3 4 5 6 7

FIG. 2. Autoradiograms offilter blot hybridization analysis of cells infected with highly restricted B-tropic F-MuLV. Subconfluent mouse fibroblast cell lines (1.5 x 106 to 2x 106 cells) were infected with either B- orN-tropic F-MuLVfor 24 h. Hirt

super-natantDNA was subjected to electrophoresis in0.8% agarosefor (A) 11.5 h at 50 V or (B) 16 h at 50 V. DNA, transferred to nitrocellulose, was hybridized to

32P-labeled MuLV DNA. (A) Lane 1, N-tropic

F-MuLVinfectionof NIH cells (MOI of 0.4); lane2,

B-tropic F-MuLV infection of BALB/c cells (MOI of 0.4);lane 3, B-tropic F-MuL V infection of NIH cells

(MOI of0.4). (B) Lanes 1 to 5, N-tropic F-MuLV infection of: NIH cells (MOI of 1.0), DBA/2 cells

(MOIof 1.0), BALB/c cells (MOI of 1.0), BALB/c cells(MOIof 2.0), BALB/c cells (MOI of 5.0), respec-tively; lane 6, B-tropic F-MuL V infection ofBALB/c cells(MOIof 1.0); lane 7, B-tropic F-MuL V infection of NIH cells(MOI of 1.0).

multiplicities. Othersetsof permissiveand

non-permissive infectionsarepresentedinthis figure

forcomparisonoftheamountsof formIII DNA

synthesized; thesewill be discussedlater. This

result, demonstrating theabsenceofformIand

reducedamountsofformIII,isclearly different

fromthat whichwasobserved for the

100-fold-restricted B virus(B-2),whichexhibited little to

nodecreaseinlinearDNA(formIII).

Independ-entclonesofB-tropic viruscanthereforeexhibit

different types of restriction of their proviral

DNAinNIHSwiss cells. Thishighly restricted

viral clone (B-4) did not appear to be able to

accumulate linear proviral DNA to the same

extent as the less restricted clone (B-2). These

datamayexplain the different degreesof

restric-tionexhibitedbythese twoB-tropic clones.

The effect of the multiplicity of infection

(MOI)onthe synthesis ofproviral DNA in

Fv-1permissivecellscomparedwith nonpermissive

cells was examined. B-tropic F-MuLV which

exhibits 102restriction in NIH cells (B-2)was

used to infectNIHand BALB/c cellsatMOIs

rangingfrom 0.25 to 1.0. Theresultsareshown

inFig.3. Asexpected,thetotalamountof

pro-viral DNA increased relative to the MOI. At each MOI, the amount of form III DNA

ob-Fv-1 HOST RESTRICTION OF F-MuLV 49

served in thenonpermissive cellswasequivalent

to that observed inthe permissive cells.

How-ever,regardless of the MOI, theamountof form

I DNA in the nonpermissivecells was greatly

reduced.Itisunclear whether the lack of

detec-tion of form I in thenonpermissive hostat an

MOI of0.25(lane 1)is duesimplytoinsufficient

numbers ofinfected cells usedinthisanalysisor

whether this form is completely absent atthis

MOI.

Two rapidly migrating bands of DNA are clearlyobserved at thehigherMOI in the

non-permissivecells(lanes3and4).Thesebandsare

seen to migrate more rapidly than the fonn I

DNAs found in the permissive BALB/c cells

(lanes5 to8). Because these bands in the

non-permissivecellsarepresentatlowconcentration,

wehave thus far beenunabletodemonstrate,as

wehave inpermissive infections, thattheyare,

in fact, form I DNA either by CsCl-ethidium

bromide density gradient orby restriction

en-zyme criteria (datanot shown). In the

nonper-missivecell, these presumed form I bands were

present at aratio of1:10tothe amountofform III present. If theproviralDNAsynthesizedin the nonpermissive cell had a lower molecular

1 2 3 4 5 6 7 8

-Formm

-}Form I

FIG. 3. Autoradiogram of filterblothybridization analysis ofeffect ofMOIbyB-tropicF-MuLV. Sub-confluent NIH Swiss (lanes1 to4) andBALB/c cells

(lanes5 to8)wereinfectedwithB-tropicF-MuL Vat

variousMOIsfor16h andsubjectedtomodifiedHirt extraction asdescribed in thelegendtoFig. 1. Hirt supernatantDNAwassubjectedtoelectrophoresisin 0.8% agaroseat 60Vfor16h.Afterelectrophoresis, DNAwastransferred bythe Southern blotprocedure

to nitrocellulose and hybridized to 32P-labeled F-MuLV DNA.Autoradiographywasperformedasin Fig. 1. Lanes 1 to 4: B-tropic F-MuLVinfection of NIH Swiss cellsatMOIsof0.25(lane 1),0.50(lane 2),

1.0(lane 3), and2.0(lane 4). Lanes5 to 8:B-tropic F-MuLV infection ofBALBIc cells at MOIs of0.25

(lane 5), 0.50 (lane6), 1.0(lane 7), and2.0(lane 8).

The hybridized bands of viral DNA representing

formIIIlinear andformIcircularDNA are

indi-catedonthe sidesof the autoradiogram. knothi[L

and ud

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weight than thatin thepermissivecell,

circular-ization would result in a form I DNA which

would migratemorerapidly than itspermissive

equivalent. Asmall difference in the size of the

linear proviral DNA (form III) synthesized in

thenonpermissive cell wouldnotbe detected by

directvisual examination ofourgels because of

thelimitedresolvingpowerof 0.8%agarosegels

for DNA species thataregreaterthan8.0kb in

size. We therefore digested the proviral DNA

obtained fromnonpermissiveversuspermissive

cells with the restriction endonuclease EcoRI.

This enzyme cuts the viral genome only once,

producingtwobands of5.7and3.4 kb

(unpub-lished data and reference 13). Ifadifference in

the sizeof the linear DNA from the

nonpermis-sive cellswerepresent, itmight be detectedas

an altered migration ofone ofthe viral bands

resulting from EcoRI digestion. The results of

thisanalysisareshown inFig.4. Thepattern of

digestion obtained from proviralDNAextracts

from nonpermissive cells was similar to that

obtained fromproviralDNAextracted from the

permissive cells. Preliminary studies using

HindIII,anenzymewhichappearsto cuttwice,

confirmed these results. Therefore, it does not

appearthat the basis for the restriction in the

nonpermissive cells isamajordeletionor

incom-plete synthesis of the form III DNA

accumu-lated in these cells. The basis for the altered

migration pattem of the doublet DNA

(pre-sumed circular species) is not yet understood

and isunderinvestigation.

Additionally, we tested whether extensive

methylation of form III in different host cells

could be associated withdifferential

accumula-tion of circular DNA. The results (data not

shown) ofHpall digestionoflinear DNA

accu-mulated in eitherapermissiveor a

nonpermis-sive host reveal noresistant fragments of

pro-viral material. Since resistance tothis enzyme

correlates with methylation of the internal

cy-tosine in the HpaII recognition sequence (16),

these resultssuggestthat extensivemethylation

of proviral DNA doesnot occur in either cell

type,butthey donotexclude limitedsite-specific

methylbasemodification.

Insummary, independent clones ofB-tropic

virus exhibited wide variation in the restriction

byNIHSwisscells whenexaminedbythe direct

XCplaque assay. Thedegreeofrestriction

ap-pears to correlate not only with the ability to

accumulate normal amounts of proviral DNA butalso with thetypes of DNA made.B-tropic F-MuLV thatexhibitsahigh degree of

restric-tion in NIHcells

(B-4)

appearedtoaccumulate

severely reducedlevels oflinear,andessentially

no circular, forms of proviral DNA. Viruses

which are lessrestricted (B-2)were ableto

ac-64

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_Irni * -.34

FIG. 4. EcoRI restriction enzyme analysis offormn IIIDNA.Subconfluent ceU lines (5 x 106 cells) were

infectedwith B-tropic F-MuLV(MOI of1.0) in the presenceofPolybrene (5pg/mi)for 23 h and subjected

tomodifiedHirt extraction. Viral DNA samples were treated withendonuclease EcoRI (5U/pgofDNA) at 38°C for 3 h and then subjected to electrophoresis on 0.8% agarose to 60 V for 17 h. After electrophoresis, DNAwas transferred to nitrocellulose and hybrid-izedto32P-labeledF-MuLV DNA.Autoradiography wasperformed as in Fig. 1. Undigested DNA from B-tropic F-MuLV infection ofBALB/c cells (lane 1) and NIH cells (lane 2) was run in parallel with

digestedDNAfromBALB/c cells (lane 3) and NIH cells(lane 4). The numbers at the side of the autora-diogram represent the molecular size (in kilobase pairs) estimated from restriction fragmentsof ade-novirustype2DNA and lambda DNA that were run in parallel. In lane 4, the band migrating at 9.0 kb represents endogenous cellular sequences found in genomic DNAofuninfectedaswell as infected cells. Inthis gel, these sequences resultfrom the contami-nating chromosomal DNA that is found in all Hirt

extracts. In lane 3, the bands migrating to 9.0 kb represent these same cellular sequences as well as form III (DNA) derivedfrom formIafter a single cut

byendonuclease EcoRI.

cumulate normal amounts oflinearDNA, but

accumulatedmarkedlyreducedlevels of

abnor-mally migrating presumptive circular DNA.

Theseresultssuggestthat,in NIHcells,

restric-tion ofB-tropic virusreplicationmay occur at

either the accumulation of linear or circular

DNA.Whetheronlyone orbothof theseeffects

may be correlated with the Fv-1phenotypeisat

thistime notresolved.

Furthermore, in DBA/2 cells, B-tropic

F-MuLVwasrestrictedto alesserdegreethanthat

observedwith NIHcells. However, the ratio of

the accumulation of circular forms relative to

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VOL. 40, 1981

linearappearsequivalenttothatobserved with

apermissive infection (NB-tropic virus) in those

same cells (Fig. 1). Therefore, the degree of

biological restriction observed in tissue culture assays with DBA/2 cells didnotcorrelate well withadecreased accumulation of form I proviral DNA.

N-tropic F-MuLV infection of BALB/c

cells: analysis of nonintegrated proviral

DNA. Several clones ofour stock N-tropic F-MuLVweretested and allconsistently exhibited between 100- and 500-fold restrictionon

nonper-missive BALB/c cells when examined by the direct XCplaqueassay.Tocompare thesite(s) of Fv-1 restriction, we examined the proviral DNAspecies of N-tropic F-MuLV accumulated inNIHversusBALB/c cells. The results repre-sentinfectionatarangeof MOI from 0.25to1.0

at16to 17 hpostinfection (Fig. 5). Visual eval-uation of theautoradiogram of the blot hybrid-ization suggests thatateach MOI, theamount

of formIIIspecies accumulatedwasslightly less in thenonpermissive, when compared with the permissive, infection.Inotherexperiments, the

amounts of form III were identical. However,

theamountofform I DNA in theBALB/c cell

1 2 3 4 5 6 7 8 9

[image:7.496.246.439.323.549.2]

Y

FIG. 5. Autoradiogram of filterblothybridization analysis of effect ofMOIby N-tropicF-MuLV.

Sub-confluentBALBIc(lanesIto4)andNIH Swisscells

(lanes5to8)wereinfectedwithN-tropicF-MuLVat

variousMOIsfor16 h andsubjectedtomodifiedHirt extractionasdescribed in thelegend for Fig.1.Hirt

supernatant DNAwassubjectedtoelectrophoresisin 0.8%agaroseat60 Vfor16h.Afterelectrophoresis,

DNAwastransferred bySouthern blotprocedureto

nitrocelluloseandhybridizedto32P-labeled F-MuLV DNA.AutoradiographywasperformedasinFig. 1. Lanes1to4:N-tropicF-MuLVinfection of BALB/c cellsatMOIsof0.25(lane1),0.50(lane2), 0.75(lane 3),and 1.0(ane4). Lanes5to8: N-tropicF-MuLV

infection ofNIHSwisscellsatMOIsof0.25(ane5),

0.50(lane 6), 0.75 (lane 7), and1.0(lane 8). Lane9

representsDNAobtainedfrom B-tropic F-MuLV

in-fection ofNIHSwiss cellsatanMOIof5.0and is

shownfor comparison oftheelectrophoretic

migra-tionof formI(Fig. 3).

Fv-1 HOST RESTRICTION OF F-MuLV 51

(lanes 1 to4) was reduced ateach MOI, when

compared with the permissive cell (lanes 5to8). Microdensitometry tracingsweretaken foreach

lane, and theareasunderthepeakforeach DNA

form were weighed for quantitation (Table 1). Two points are evident: (i) at each MOI the amountof form III inpermissiveversus

nonper-missive cellswasnearlyequivalent, and (ii) the amountsof form Ifound in the nonpermisive

cellwerereduced. The ratiosof form III to form Iindicate that onlyatwo-tothreefolddecrease

in the amount of circularspecies occurredinthe restrictive infection. Aseries of similar experi-ments, in which the proviralDNA formswere examinedatbetween 6and20 h afterinfection, all confirmed these findings. Within 6 h after

infection, we documented the accumulation of

linear DNA inexcessofcircularDNA,thatis,a high ratio of form III toform I. With time this

ratioapproachedthatseenat12 to 16 h(Table 1). In the nonpermissive infection, a parallel time-dependent changeinaccumulation of DNA

species occurred.But thesamerelative decrease

in accumulation of circular DNAseen at 16 h

TABLE 1. EffectofMOI onproviralDNA in

N-tropicF-MuL Vinfection

Hostcell MOI Form 11 Form1

Ratio

(A) NIHSwiss 0.25 0.016 0.015 1.07 0.50 0.031 0.030 1.03 0.75 0.040 0.050 0.80

1.00 0.057 0.0655 0.87 (B)BALB/c 0.25 0.0285 0.011 2.59

0.50 0.045 0.019 2.37 0.75 0.065 0.027 2.41 1.00 0.092 0.030 3.07

NIHSwiss 0.25 0.053 0.055 0.96 0.50 0.076 0.066 1.15 (C) BALB/c 0.50 0.049 0.021 2.30

0.75 0.059 0.022 2.68 1.00 0.061 0.023 2.65 NIHSwiss 0.50 0.034 0.024 1.42

0.75 0.051 0.032 1.59 1.00 0.074 0.045 1.64

(D)NIHSwiss 1.00 0.123 0.062 1.98 BALB/c 1.00 0.092 0.017 5.41 2.0 0.115 0.030 3.83

5.0 0.199 0.048 4.15 aNumbers indicate weightingramsofpeakareas

cutfrom themicrodensitometrytracingsof autoradi-ograms.All the measurements in each letteredgroup

weremadeusingasinglewedge settingonthe micro-densitometer. (A) and(B)representdifferent

autora-diographic exposurestothesamehybridized Southern

blot. (A) and (B):5 x 10'cells wereinfectedin the

presence ofPolybrene (5

,ug/ml)

for16h. The

auto-radiogramcanbeseeninFig.5.(C):2x 106cellswere

infected in the presence ofPolybrene (5 Lg/ml)for16

h.(D):1.5x 106 cellswereinfected in the presence of

Polybrene (5 Lg/ml)for24h. Theautoradiogramcan

beseeninFig.2B.

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[image:7.496.43.233.349.482.2]

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was observedateach timepoint in the nonper-missive infection. That is, although both the absoluteamountand thetypeofproviral DNA species accumulated in cells varied with time, therewas aconsistentfinding (indicated in the ratio ofform IIItoformI) ofadecreased

accu-mulation of only two- to fourfold of circular DNA inBALB/c cells infected with N-tropic F-MuLV relative to that seen in the permissive infection. These data are consistent with the decreased accumulation of forn I reported by other investigators (9, 26). However, they are

inconsistent with the degree of reduction of form Inotedby Jolicoeur and Rassart (9). Yangetal. (26) suggested a multiplicity-dependent effect ontheformation of form I DNA in nonpermis-sivecells.Our datasuggestthat thedecrease in form I DNA accumulation is independent of multiplicity, since the ratio of form IIItoform Iwasconstantoverawiderangeof viralinput.

The phenomenon of the Fv-1generestrictive

effecthas, however, been shown inmany labo-ratoriestofollow two-hitkinetics. That is, titra-tion of either infectious centers or ofprogeny

virus production in nonpermissive cells de-creases geometrically rather than linearly with virusdilution. Incontrast tothepermissive cell infections, these findings have been interpreted

to indicate that a nonpermissive cell must be infected by at least two infectious viruses to

establish an infectious center. By contrast, the permissivecellrequiresonlyasingle "hit" (15). However, single-hit restriction curves, appar-ently due to the Fv-1 host gene, have been

reported inmanylaboratories (reviewed by

Jol-icoeur,6).

OurN-tropic F-MuLVappearstoberestricted by single-hit kineticsasmeasuredby the direct XC assay. These kinetics may also be

demon-stratedbyprogenyyield. NIH andBALB/c cells

wereinfectedatanMOI ofeither 2.0or0.2. The

progenyyield from suchpermissivelyor

nonper-missively infected cells wasmeasured on NIH

cells at24 through 96 h postinfection. The

re-sults, shown in Table 2, were (i) at 24 h in

permissive cellstherewasthe expected 10-fold

difference in yield, comparing an MOI of0.2 with oneof2.0; (ii) atboth 24 and 48 h

postin-fection, there was a 100-fold decrease in yield,

comparingpermissivewithnonpermissive

infec-tion, and this result occurred at eitherMOI; (iii)

at72 and96h, therewasonlya10-fold restric-tion inyield,aresultpresumably due to

cell-to-cellspread,aphenomenon previously found by

us (Soeiro, unpublished data) and others (7).

Similar experimentsstudying infectiouscenters

producedby N-tropic F-MuLV infection of NIH versus BALB/c cells have also indicated such single-hit kinetics. Our results, interms of the ratio of form IIItoform I inanFv-1 restrictive infection, musttherefore be interpreted in the light ofapparentsingle-hit kinetics.

Toexamineproviral DNA formed undermore extremeconditions ofmultiplicity,wecompared theN-tropic F-MuLV infection of BALB/c ver-susNIHcellsatanMOI of 5.0. At thisMOI,at

least 96% of the cellswere infected bytwo or moreinfectiousparticles. It would be expected, accordingtothe two-hitmodel, thatatthisMOI restriction would be completely overcome and the patterns ofproviralDNAsynthesismightbe

identical. For comparison, we also infected a muchlarger numberofBALB/ccellsatanMOI of0.5,sothat theamountsoftotallinear DNA produced by N-tropic F-MuLV would be

ap-proximately equaltothat foundby infection of fewercellsatahigherMOI. Theresultsof this experiment depictedasmicrodensitometry

trac-ings from the autoradiogram are shown inFig.

6. Figure 6A represents the twocircular forms (form I) and the linear formIII ofpermissively infectedNIHcells.Figure6Brepresents similar tracings takenfromBALB/ccellsinfected atan MOI of 5.0. Figure 6C is the tracing obtained

fromagreaternumber ofBALB/ccellsinfected atan MOIof0.5. Theratio ofweights of form TABLE 2. Effect ofMOIonprogenyyield from permissive versus restrictive infection'

Progenyyield at hpostinfectionof:

MOI 24 48 72 96

N B N B N B N B

2.0 4 x 104 3x 102 1.2x 107 1.2x 105 0.9 x 106 1.3 x 105 1.4x 106 2 x 105

0.2 3x 103 NDb 8 x106 4 x 104 9 X 105 5 x 104 3 x 106 3 x 105 aSubconfluent mouse fibroblast cell

lines

(1.0 x

106

cells) were infected with N-tropic F-MuLV in the presenceof Polybrene (5,ug/ml)at an MOI of 2.0 or 0.2. After a 3.5-h adsorption period,cellswerewashed and given fresh medium. At thetimesindicated,the medium was replaced, and the titers of the progeny virus in the removed mediaweredetermined on permissive (NIH) cells by direct XC plaque assay. The results obtained from media removed from infected NIH Swiss cells (N) or BALB/ccells (B) are given as the number of macroscopic PFU permilliliter.

bND,Notdetected.

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A

B

C

DNA Forms: I m I m I m

Area Under: Curve (g)

Ratio m/I:

.147 .133

0.90

.064 .147 .051 .142

2.30 2.78

FIG. 6. Microdensitometrytracings offilterblothybridizationanalysesof infection by N-tropicF-MuLV.

SubconfluentceU lines wereinfectedfor24h in thepresenceof Polybrene (5

lg/ml)

andsubjected tomodified

Hirtextraction(seeMaterialsandMethods).Afterelectrophoresisthrough 0.8%agarosefor16h at 50V,the DNA wastransferred bythe Southern blotproceduretonitrocellulose andhybridized to 32P-labeledF-MuLV DNA.Autoradiographywasperformedasdescribed in thelegendtoFig. 1,and theexposed filmwasanalyzed

lanebylanewithadouble-beamrecordingmicrodensitometer.After carefulconsiderationof background,the peak areas were cut out and weighed; thus, the areas under the curves are given in gram units. (A) N-tropic

F-MuLVinfectionof 1.0 x106NIHSwiss cells,MOI of5.0. (B)N-tropic F-MuLVinfection of 1.0 x106BALBE

c cells, MOIof5.0. (C) N-tropic F-MuLVinfection of4.0 x 106 BALBIc cells, MOI of 0.5. Thepeaks representingformIDNA andformIIIDNA are indicated.

III toform I is included in the figure.We can

conclude from comparing both the totalamount

of linear DNA formed and the ratio of linearto

circular formsthat,even at a veryhighMOI,at

which restriction is expected to be overcome,

there is a two- to threefold reduction in the

accumulation ofform I in nonpermissive cells.

Furthermore,even at anMOI of0.5,by

compar-isonwith5.0,the ratio of lineartocircularforms

innonpermissive cells remains thesame.

Theseresultsconfirm that,in anonpermissive

infection, accumulation offormIis reduced. But

they also show that thedegree of reduction is

much less than that expected by the 100-fold

restriction ofviral progenyyield. Furthermore,

atMOIs of5.0and0.5,the ratiosof form III to

form I are essentiallyidentical, a result

incon-sistent with a two-hit restriction phenomenon.

We conclude that decreased accumulation of

formIdoesoccurin restrictive infectionwith

N-tropic F-MuLV but that thedecrease is

incon-sistent with the degree ofrestriction observed in

tissue cultureassays. In addition,with our virus

and cell combination, the ratio ofform III to

formI inBALB/c cells did not reflect a two-hit

effectonthe decrease ofcircularDNA.

DISCUSSION

Fv-1-mediated host restriction of

B-tropic

MuLV affects the accumulation of

noninte-gratedproviralDNAinFv-1 'i" cells. The results

previouslyreported byotherinvestigators (9, 26)

have shownthat, in NIHcells,there isablock

in theaccumulationof forn III DNA(and

there-fore no

form

I is found) after infection with

WN1802B. Other Fv-1'1'cells(DBA/2)infected

with thissamevirusproduceamountsof linear

DNA equivalent to that forned in permissive cells, but levels offorn I are reduced. It has been suggested that the differences observed

between these cell types could be due to the

presence of a second gene in NIH cells which

modifies the Fv-1 effect (9,26).

N-tropic

MuLV

(WN1802N) infection ofFv-1b/b cells resulted

also in theaccumulationofnormalamountsof

linearbut decreasedamountsofcircular DNA.

Since previous resultsfromthislab and others

(8, 23) haveindicated that Fv-1 restriction

oc-curs at or before proviral DNA integration, it

wasconcludedthat theblockinaccumulation of

circular proviralDNA is the primary effect of the Fv-1 gene.

VOL. 40, 1981

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Wehave demonstrated that individual clones

of B-tropic F-MuLV can exhibit different

de-grees of restriction byNIH cells. Cloned virus

stock exhibitingalow level of restriction (10-2)

byXCassay wereshowntoaccumulatenormal levels of form III, but reduced levels of form I,

DNA. However, a second clone, showing high

(10-4)restriction,wasunabletoaccumulateeven

normallevels of formIII DNAinthesecells.

ThesedatasuggestthatNIHcellsareableto

restrict B-tropic virus atseveral points in the

earlyproviralDNAphase of replication. These

effectscould be dueto asingle pleiotropicFv-l1

allele capable of interacting at different steps

during proviral DNA accumulation.

Alterna-tively,NIHcellsmay carry asecondgenewhich

modifies the Fv-l" allele. Our studies withtwo

clones ofonevirus thatappearblockedat

differ-ent steps leadingto integration do notresolve

thisquestion.They do, however, show that these

two clones are recognized differentially by the

samecell, suggesting thepossibilityofmorethan

asingle recognition feature in host cell

restric-tion. There might be two sites on the virus

recognized by a single Fv-1 gene product or,

altematively, two gene products. This

conclu-sion is underscored by the fact that both

B-tropic clonesarerestricted tothe same degree

(50-fold) in DBA/2 cells

(Fv-1'1).

Thissuggests

thatDBA/2 cellsmayonly be abletorecognize

asingle feature shared by both viruses. These

patternsofproviralDNAaccumulationmaybe duetothepresenceofasecondrestrictinggene

product(s) inNIH cells which isnotpresent in

DBA/2 cells or to an increased efficiency of a

single Fv-l"geneproductinthe NIHcells.

Our studies of the restriction ofN-tropic virus

inBALB/ccells have revealed adecreased

ac-cumulation of form I DNA in agreement with

studies from other laboratories. However, we

observed that the reduction of form Iwas only

of theorder oftwo- tothreefold when compared

with theamountofproviralDNAobtained from

permissively infected cells. Our results are

clearly different from the estimated 25- to

50-fold reduction observed by Jolicoeur and

co-workers (9) and do not show amultiplicity

de-pendence as observed by Yang et al. (26). A

possible reasonfor the differences observed,in

addition to modifications of technical

proce-dures, may reside in the virus stocks used in

thesestudies.The titrationpatternobservedby Yang and co-workers isclearlytwohit in

non-permissive cells, whereas our results

demon-stratedaone-hitrestriction curve, thatis,alack

ofmultiplicity effects on virus yield or on the

proviralDNApattern.

SeveralfeaturesofFv-1restriction have been

described, including "hitness," "refractoriness,"

J. VIROL.

"expression,"and"prozone" effects(15). These

arewell discussed by Jolicoeur (6), whose

con-clusion is that both refractoriness and two-hit

kineticsappear tobepartof the Fv-1geneeffect.

Our findings are that decreases in the

accu-mulationof form I (two-tothreefold)are

incon-sistentwith the degree of restriction (100-fold)

and thatthe decreaseinform I accumulationis

notmultiplicity dependent (Fig. 6).These data

suggestthat forourcells and virus the decrease

informImaybe associated withrefractoriness,

thatis, anon-multiplicity-dependent

Fv-1-me-diatedrestriction effect. (The word

"refractori-ness" is usedtoimplyadecreased efficiency of

formation of infectiouscenters in nonpermissive

hostcells.)

The data of Yangetal., which showatwo-hit

effectonthe accumulation of form IDNA,

sug-gestthatadecrease incircular DNA is the site

ofFv-1 restriction. Our data, which didnotshow

such a multiplicity-dependent effect on form I

accumulation,suggestthat Fv-1 restrictionmay

notbe due simply to effects on circular DNA

accumulation. An alternative model for the

site(s) of Fv-1 gene effect is suggested by our

data.If theefficiency of integration varies geo-metricallywith theconcentration ofform I, then small decreases in accumulation of form I might

result in large differences in the establishment

of infectiouscenters.Therefore, in additiontoa

small decrease in form I accumulation, there

might bealarge effectontheintegrationprocess

itself. This second effect might be considered

secondary, but could result ina geometric

out-come ofalinear process. There is no evidence

against suchamodel thatwe are awareof.Also,

itmustbeconsidered that thebiological

signifi-canceof theaccumulatedproviral DNA species

found after the first few hours ofinfectionis yet

tobecompletely defined andmayrepresent an

easily measurable but dead-end feature of an

acuteinfection.

Lastly, we have attempted to assay

mecha-nisms whichmight resultindecreased

accumu-lation ofform I DNAintherestrictivecell.Our

data, which show no residual HpaII-resistant

proviral fragments,suggest thatextensive

meth-ylationof the DNA atcytosineresidues in either

a permissive or restrictive infection is not

in-volvedinthis effect. Also,no large deletionsin

the length of linear DNA could be found by

preliminary restriction analysis of the forn III

DNAaccumulatedinrestrictivecells.However,

these studies do notexcludethepossibilitythat

verysmall, especiallyterminal, fragments may

bemissing or base modified. The easily

recog-nizedalterationintheelectrophoretic migration

of putative form I in Fig. 3 and 5 is as yet

unexplained. It may represent a more highly

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VOL. 40, 1981

supercoiled structurethan isnormally

accumu-lated. Whether this structure is a result ofan interaction of proviral DNA with the Fv-1 gene

productmustbe determined. An understanding of this result may define mechanistically how the Fv-1 gene effect is mediated. Finally, the mechanism(s) for the decreased accumulation of form I hasnotbeen determined andmayresult from eitherchangesinsynthesisordegradation orboth of circlesfonned in therestricting cell.

ACKNOWLEDGMENTS

This workwassupported bygrantsMV-40from the Amer-ican CancerSociety, P3 OCA-13330 from the NationalCancer Institute, andtraininggrant5T32GM7288 fromthe National Institute of General Medical Sciences.

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16. Razin, A.,and A.Riggs. 1980. DNAmethylationand gene function.Science210:604-610.

17. Rigby, P. W. J., M. Dieckmann,C.Rhodes, and P. Berg. 1977.Labellingof DNAtohigh specificactivity

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19. Schuh, V., M.Blackstein, and A. Axelrad. 1976. In-herited resistance to N-andB-tropicmurine leukemia viruses in vitro: titration patterns in strains SIM and SIM.R congenic at the Fv-1 locus. J. Virol. 18:473-480. 20.Shank,P.RK, S.H.Hughes,H. J.Kung,J. E.Majors,

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21.Smotkin,D., A. M.Gianni,S.Rozenblatt,and R. A. Weinberg. 1975. Infectious viral DNA of murine leu-kemia virus. Proc. Natl. Acad. Sci. U.S.A. 72:4910-4915. 22. Southern, E. M. 1975.Detection ofspecificsequences among DNAfragmentsseparated bygel

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