Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex.

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Further

Characterization of the Friend Murine Leukemia

Virus Reverse

Transcriptase-RNase

H

Complex

KARIN MOELLING'

Institut fur Virologie, Bereich Humanmedizin, Justus Liebig-Universitdt 6300, Giessen,Germany

Receivedforpublication25November 1975

The purified reverse transcriptase-RNase H complex from Friend murine

leukemiavirus consistsofasinglepolypeptideof 84,000 molecularweight,which

after mild protease treatment in vitroor after intentional

degradation

during

the purificationprocedure allows the generation ofseveral additional

polypep-tides. Degradation destroys theRNA-dependentDNApolymerase activitywith

native RNA templates and reduces RNase H butdoes not affect response to

synthetic template primers such as poly(rA)oligo(dT). The properties of the

intact murine enzyme consisting of a single polypeptide of84,000 molecular

weight arecomparedtothose ofthe avian asubunit andthe avian a/3enzyme

complex. Theintact murine enzymeresembles the avian /3-containing enzyme

complex andisdifferent from a inthefollowing respects: (i) itbindsto native

RNA templates; (ii) ittranscribesnativeRNAtemplatesinto DNA, areaction

whichcanbe inhibitedby actinomycin D; (iii) RNaseHactivity behaves likea

processive exonuclease; and (iv) analysis of the RNase H digestion products

reveals oligonucleotides approximately four basesin length.

TheRNA-dependent DNApolymerase

activ-ity from mammalian viruses, particularly the

murine ones, have been characterized by

sev-eral laboratories withconflictingresults. As far

asthestructureof theenzyme isconcerned, the

number of subunits identifiedon sodium

dode-cyl sulfate-polyacrylamide gels afterextensive

purification ranges from one to three. The

en-zymesfromFriendmurineleukemiavirus

(Fr-MuLV) and from Moloney murine leukemia

virus(Mo-MuLV) have beendescribedas a

sin-gle polypeptide of 80,000 to 84,000 molecular

weight (8, 12). In other reports the Fr-MuLV

enzyme has been characterized as a

two-poly-peptide complex (15), and the Mo-MuLV

en-zyme has been shown to consist ofthree

sub-units(2).Thetwopolypeptides observed for Fr-MuLV (15)resemble those of the hamster viral enzyme (13).

As the avian viral reverse

transcriptase-RNaseHcomplex consists of twopolypeptides,

aand

/3,

one ofwhich can be shown toarise by

proteolytic cleavage from the other

(Moell-ing,Virology,inpress),the questionaroseasto

whether some of themurine viral enzyme sub-units represent degradation products as well. The answer ofthis question wasapproached in two ways: (i) the purified Fr-MuLV enzyme, consisting of a single polypeptide, was

sub-jectedto mild protease treatment in vitro; (ii)

Presentaddress: Max-Planck-Institut fur Molekulare Genetik,D-1Berlin-33 (West),Germany.

an enzyme preparationwasextracted from

Fr-MuLVundernonoptimal conditions for

conser-vation of the enzyme activity. Both experi-ments (trypsin treatmentand intentional

deg-radation) generated several polypeptides.

Structural changeswere correlated with

enzy-matic alterations that reproduce and explain

some of the conflicting data published on the

structure and enzymatic properties of

mam-malianviralreversetranscriptases.

Thesizeof the largestmurine viral enzyme

polypeptideranges between thesizeof thetwo

avian enzymesubunits, leavingopenthe

ques-tionof whetheritcorresponds to aor/3. There

is evidence in favor of either: the ability to

transcribe nativeRNA observed insome cases

(2, 8, 12)resembles thepropertiesof

/,

whereas

absence ofRNA-dependent DNAsynthesis (1,

14) is similarto a. The RNase H activity has beencharacterizedas a randomexonuclease (3)

identicalto thatexhibited by the avian a

frag-ments (3), and theRNase Hdigestionproducts (12) have been described as being larger than

expectedby analogy toa /3-containingenzyme

preparation. Furthermore, it has been observed that protease treatment of the avian

a,8

en-zymecomplex in vitro can completely remove

,/,

whereas a is much more resistant to this treatment. Only more stringent conditions

al-low digestion of a as well (Moelling,

Virol-ogy, in press). The possibility exists, therefore,

thatthelargestmurineviral proteindescribed

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sofar is adegradation product ofan evenlarger one.

To find out whether or not the 84,000-molecu-lar-weight polypeptide exhibits the properties of a real viral reverse transcriptase-RNase H complex, several of its enzymatic properties werecompared to those of the avian asubunit,

asrepresentative of a degraded enzyme, and to the

a,/

avian enzyme complex. As all previous attempts to isolate /3 alone have failed (Moell-ing, in press), the avian a,3 enzyme complex waschosen for comparison, since it is the best representative of a functioning reverse

tran-scriptase-RNaseHcomplex presentlyknown.

MATERIALS AND METHODS

Virus. Avianmyeloblastosisviruswasisolatedin partinthis laboratory andwas inpartagenerous gift of J. Beard. Fr-MuLV was grown inthis labo-ratory. Theviruswasoriginallyproduced in STU-mouse cells (10) that have sincebeenestablished as as permanent line (Eveline cells). The cells that were growninsuspension culture wereadapted to substrate-dependent growth by G. Pauliinthis lab-oratory. Approximately 107 cells were seeded in roller bottles (5-cm diameter, 50-cm long) and grown at37C with25mlof Dulbecco modifiedEagle growth mediumin the presence of5% heat-inacti-vated calf serum (56 C, 45 min) and 10% tryptose phosphate broth.

Reagents. 3H-labeled poly(rA) and poly(dT) were obtained from Miles Laboratories. Oligonucleotides (A)A, (A)6A, (A)4A,and(A)2Awerepurchased from Boehringer (Mannheim, WestGermany). Acrylam-ide and methylenebisacrylamide, electrophoresis grade, were bought from Bio-Rad Laboratories

(Richmond, Calif.). Soluene-350 originated from Packard Instrument Co. (Zurich,Switzerland). Dul-becco modified Eagle medium was purchased from Flow Laboratories (Bonn, West Germany), and tryptosephosphate broth was from Difco Laborato-ries (Detroit, Mich.).

Enzymepurification. The reverse transcriptase-RNase H complex from avian myeloblastosis virus and Fr-MuLVwasisolatedbypublishedprocedures (8, 9), anditspurity wasdetermined on SDS-poly-acrylamide gels. Subunit a was recovered from a

phosphocellulose column at 0.11 M KCl. For isola-tionoflarge amountsof a, theenzyme attachedto the DEAE column was left at 4C for 1 to 2 days before elution. The same treatment wasappliedto theFr-MuLVenzymetodeliveradegraded enzyme. Enzyme activity assay. DNA polymerase and RNase H were determined aspreviously described (8, 9). The avianenzyme assayswereperformed in the presence of 8 mM MgCl2, and the Fr-MuLV assays wereperformed in the presence of 0.4 mM MnCl2. Thespecific radioactivity ofonelabeled

de-oxyribonucleotide was 5,000 counts/min per pmol.

Theassayfor RNase Hcontained10,000counts/min ofphage fd DNA-RNAhybrid,withspecificactivity of3,500 counts/min perpmolof[3H]UMP.

Unit of activity. A unit of enzyme activity is

defined as the amount of enzyme required to incor-porate 30pmol of TMP at 39C in 30 min with the synthetic templatepoly(rA)-oligo(dT) inastandard polymerase assay mixture, as this reaction is not affected by any degree of enzymedegradation. Com-parative studies ofenzyme activities were always based on identical units input.

Binding of enzyme to RNAtemplates. In a total volume of 100 ,l, 20 tkl of the murine or avian

/3-containing enzymes were mixed with an excess of RNA (5jigof70S AMV RNA)inthepresence of ions asusedinenzyme assays.Forbinding analysisofa, 100,ul of enzymewasmixed with 5 ,ug of70S AMV RNA under assay conditions in 200 ,l (final vol-ume). Withoutfurther incubation the material was layered on top ofpreformed glycerol density gra-dients (10 to 30% glycerol in TNE [0.01 M Tris-hydrochloride-0.1 MNaCl-1 mM EDTA], pH 7.4-5 mM dithiothreitol-0.2% Nonidet P-40). After cen-trifugation for 90 min at 50,000 rpm and 4 C in a SW50.1Beckman rotor, thegradients were fraction-ated. Position of the enzyme was determined by testing an aliquot (20,u)of eachfraction for enzyme activitybythe addition of fourdeoxyribonucleoside triphosphates and ions toestablish regular enzyme assay conditions. Alternatively, poly(rA) oligo(dT)

andL3H]TTPcan beadded to each fraction for

deter-mination ofresponse tosynthetictemplate-primers (in thecase ofathistesthastobeapplied, since a

does not transcribe native RNA).

Chromatography of 3H-labeled poly(rA) diges-tion products. 3H-labeled poly(rA) oligo(dT) (5,000 counts/min per pmol of AMP) was hydrolyzed by viral RNase H activities in standard assay mix-tures.After the reaction themixturewasspottedon strips (50-cm long) of Whatman no. 1 paper and

chromatographed in n-propanol-ammonia-water (55:10:35) for48 h to separate variousoligomers of AMP (5). Portions (50 to 100 ,ug) of (A),A, (A)6A,

(A)4A, and (A)2A were added as standards to the reactionmixturesbefore spotting. The paperwasair dried after chromatography, the position of the standardswaslocatedby UV light, and1-cmstrips were cut. Radioactivity wasdeterminedin toluene-based scintillation fluid. Thestandardsappliedhere donot have terminal phosphates and therefore do notmigrateexactly likephosphorylated productsof RNase H digestion. Therefore no precise assign-ments for thelength of the oligomerswerepossible.

Polyacrylamide gel electrophoresis. Enzyme

polypeptides wereanalyzed by sodiumdodecyl

sul-fate-polyacrylamide gel electrophoresis accordingto amodified method ofShapiroetal. (11)asdescribed byKacian etal. (4). Beforeapplying proteinstothe

gel, concentration was achieved by precipitation

with50%ethanol (for solubilizationof thedetergent NonidetP-40) and 10% trichloroacetic acid (-20C,

10h). Protein waspelletedinaSorvallHB4rotor at

10,000 rpmfor 30 min,dried, andsuspendedin

sam-ple buffer for electrophoresis.

[3H]glucosamine-la-beled virus (Prague A [PR-A] strain ofRous sar-coma virus) was used as internal standard (1,000

counts/min per gel). The so-called gp85 and gp37 glycoproteins migrate more slowly in the buffer system used (13) than their names indicate. The

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420 MOELLING

gels were stained with Coomassie blue, and the positions of thepolypeptides weredeterminedbya Gilford scanningapparatus at 600nm wavelength. Afterwards the gels were frozen, sliced (1 mm),

andincubated in 200

A.l

of soluene for 2 h at 60 C. After the addition of 2 ml of scintillation fluid, radioactivity was determined in a liquid scintilla-tioncounter.

RESULTS

Structural properties of the Fr-MuLV

re-versetranscriptase-RNaseHcomplex. The re-verse transcriptase-RNase Hcomplex was

iso-lated from purified Fr-MuLV by

DEAE-cellu-loseandphosphocellulosecolumn

chromatogra-phyaccordingtopublished procedures (8).The

purifiedenzymeconsists ofasinglepolypeptide

with anestimated molecularweightof84,000,

basedonthe sizeof thetwoavianenzyme

sub-units 13andabeing 110,000 and70,000(4)(Fig.

1AandE). Mild proteasetreatmentunder

con-ditions thatallow increase ofa atthe expense

of

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inthe avian system(7)generated,fromthe

84,000-molecular weight polypeptide,new poly-peptidesof 78,000, 69,000,and60,000molecular

weights, andalargeamountof

low-molecular-weightmaterial. Increasingamountsoftrypsin

(5 to 30

/ig/ml)

predominantly resulted in an increase in theproportion ofthe

78,000-molecu-lar-weightpolypeptide (data notshown).

Fur-thermore, the enzyme wasextracted from the

purified virus under conditions that allowed

transfer of the avianenzyme to a preparation

consistingpredominantlyofpure a.Thiscanbe

achieved by binding the enzyme to a DEAE

column for1 to2daysat4Cbefore elution with

acontinuoussaltgradient (Moelling, Virology,

inpress).Suchatreatmentcauseddegradation

of themurine enzyme aswelland givesrise to a

preparation of 84,000-, 78,000-, 69,000-, and

60,000-molecular-weight

polypeptides in

addi-tion to smaller material (Fig. 1C). This poly-peptide patternwas obtainedtwice. Inathird

case the 78,000 polypeptide wasmissing (data

not shown). Storage at -20C increased the

69,000polypeptidepreferentially.Elutionofthe

enzyme from phosphocellulose by means of a continuous salt gradient inaregular

purifica-tion procedure consistently revealed a small

peakof activity at a low salt concentration(0.11

M

KCl)

inadditiontothe main peak at 0.22 M

KCl

(notshown). The firstpeak correspondsto

the region from which the subunit a in the aviansystem can berecovered (3). Its

polypep-tide composition (Fig. iD) consisted of

78,000-and69,000-molecular-weight bandsinaddition

to the84,000 andsmaller ones. Purified avian

af-containing

enzyme is shown for control.

Figure iF shows the isolated avian a subunit

x

C-)

E

0.3-Fr-MuW

"degraded'

0.5

U)

.0

migration

FIG. 1. Scanning profiles of murine and avian viral enzymepolypeptides after gel electrophoresis. (A) PurifiedFr-MuLV reverse transcriptase-RNase H; (B)purified Fr-MuLV enzyme after trypsin treat-ment (25

H.g/ml,

15

min,

35 C); (C) purified Fr-MuLV reverse transcriptase after long-lasting col-umn chromatography (attachment to DEAE-cellu-lose for 1 or2days at4 Cbeforeelation);(D)marine

materialelatedat0.11MKC1from the phosphocellu-lose column(corresponding to theelationpoint of the avian asubunit); (E)purifiedavianaf3enzyme prep-aration; (F) avian subunit a aselatedfrom the phos-phocellulose column and also obtained after in vitro protease treatment of the purified af3complex (40pg

oftrypsin, 35 C, 20 mm). The enzymepolypeptides were subjected toelectrophoresis in the presence of [3H]glucosamine-labeled virus (2,000

counts/mmn;

Rous sarcoma virus, Prague A) used as internal standard in all gels except for Fr-MuLV "a." The gels were stained first and subsequently processed for determination ofradioactivity as described. In

(A)thedottedline indicates theradioactivemarker

which is onlyshown by arrows in(B)through (F).

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REVERSE TRANSCRIPTASE-RNase H COMPLEX 421

thatcanberecoveredassuch from the

phospho-cellulose column at low ionicstrength. It can,

however, also be generated from the

a/3-con-tainingenzyme complex by protease treatment,

which completely removes 13 (Moelling,

Vi-rology,inpress).Onlymore stringentdigestion

conditions will also digest a. This effect is

shown since itdemonstratesthat thereis areal

possibility of preferentially losingone subunit

andonlydetectingacomparativelymorestable breakdown product.

Binding to natural RNA and RNA

tran-scription. The Fr-MuLVreverse

transcriptase-RNase H complex was tested for its ability to

bindtonative RNAtemplates, and itsbinding

propertieswerecomparedtothose of the avian

subunit a and the avian a13 complex (Fig. 2).

The murine enzyme, just like the avian a,8

enzyme, bound to the high-molecular-weight 70S AMV RNA and could be sedimented

through a glycerol density gradient with the

viral RNA. It was then detectable by enzyme

activityassay intheregionof the

high-molecu-lar-weight RNA. Incontrast, acould notbind

toRNA andstayedontopof thegradient under

these conditions. Asexpected from theabilityof

the murine enzyme to bind to native RNA, it

was also able to transcribe native RNA into

DNA (Fig. 3). Addition ofoligo(dT) stimulated

DNA synthesis severalfold. Both reactions

could beinhibited by actinomycin D, with the

reactionwithoutoligo(dT) inhibited about50%.

ActinomycinDinhibitionhasbeenshown tobe

indicative of synthesis of double-strandedDNA

(6).

Native RNAtranscription was also analyzed

with the degraded murine enzyme (Fig. 1C).

RNA-dependent DNA synthesis with native

RNAwas not seen(Table 1).Onlytheaddition

ofoligo(dT) overcame this defect. Actinomycin

Ddidnot inhibitthis reaction. The same phe-nomena have been observed for the avian a subunit(Moelling, inpress)and arelisted here for comparison. The properties of the

unde-graded murine enzymeand those of the avian

a,/

complex are shown in Table 1 as well.

RNase H activity of the degraded murine

en-zyme was reduced by about 30% (not shown)

comparedtothe undegradedone.

Mode of action of murine viral RNase H

activity. Themurine viral RNase H presentin

the 84,000-molecular-weight polypeptide has

been characterizedas anexonuclease (8, 12).If

this RNase H behaves like the one from the

avian a,3 complexit should degrade one RNA

strand completely beforeproceedingtoanother

one. In contrast, the RNase H of the avian a

subunitattacks a new RNA strand after each

bondscission (3).

The RNase H activities of the Fr-MuLV

en-zyme consisting only of the

84,000-molecular-weight polypeptide and of a degraded murine

viralenzyme preparation weretested fora

ran-domorprocessive mode ofaction. Behavior of theavian a-anda/3-containingenzyme prepa-rations are shown forcomparison (Fig. 4).

Ra-dioactively labeled hybrid was hydrolyzed by

RNase H from enzymes of both murine and

avian viral origin. If the radioactively labeled

hybrid was competed for with tenfold molar

20 200 10

E

I~~~~~~~~~~~I

10 20 10 20 10 20

factions

rVIG. 2. Bindingproperties ofthe murine enzyme and avian aand af3 toRNA. Purifiedenzymes were

mixedwith an excessofnativeviral RNAandsedimentedwithoutfurther incubationunderconditions which allowthe RNAtomigratetwo-thirds down thegradient (arrows).Thegradientswerefractionated,and each fractionwastestedfor thepresenceofenzymeactivitybyresponsetosynthetictemplateprimers(seeMaterials andMethods)inthecaseofAMVaandaf3.RNA-dependentDNApolymerase activityinthecaseofFr-MuLV wastestedbytheadditionof fourdeoxyribonucleoside triphosphates (andnoadditionaltemplate).

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x

E

IL

I

0

I

10 20 30 minutes

0

I

-o

v

x

E

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I

FIG. 3.Kinetics of RNA transcription by purified AMV

a,-containing

enzyme, pure a,andpurified Fr-MuLV reversetranscriptase-RNaseH.The three enzymepreparationswereincubated with2Mgof70S AMV RNAinthe absenceand presenceofoligo(dT)primersunderstandard assay conditions().Furthermore,

DNAsynthesis ofthese reactions was investigated in theabsence (-) and presence (+)ofactinomycin D (100 pg/mi). Aliquots were withdrawn from the reaction mixtures at the times indicated andprocessed for

determination of acid-insoluble radioactive material.

TABLE 1. RNA-dependentDNA-synthesisofdegraded and undegradedFr-MuLV reverse transcriptase (RT) compared with that of theavian aandaf3enzymesa

Template Degraded Undegraded Avian a Avian a/3

Fr-MuLV Fr-MuLV

RT RT

RNA 0.4 6 0.05 5.5

RNA +oligo(dT) 18 25 5.5 11

RNA +ActD 0.5 2.5 0.05 2.4

RNA +oligo(dT) +ActD 19 17 5.4 6

aFr-MuLV reversetranscriptase was purified under nonoptimal conditions(long-lasting column

chroma-tography),and its ability to transcribe native RNA was compared with that of undegraded Fr-MuLV reverse transcriptase (see Fig. 1)and to that of avian a- and to aviana,8-containingenzyme. Identical enzymeunits,

defined in Materials and Methods, were applied. A1-Mugsample of70S avian myeloblastosis virus RNA was used astemplateineach reaction, and 1 Mugofoligo(dT) and 100Mugofactinomycin D (ActD) per mlwere

added to the reactions asindicated. The amount of acid-insoluble material was determined and expressed as picomoles of[3H]dGMPincorporated.

excessof unlabeledhybrid2minafterthestart

of thereaction, therateof RNAdegradation by

theRNaseHfrom theintactFr-MuLV enzyme and the avian a,8 enzyme was not affected, whereas the reactions catalyzed by the

de-gradedmurine enzymeorbythe avian awere

severelyreduced. Addition ofexcess unlabeled

hybrid to the reaction mixtures prior to the

enzymesdidnotreveal any RNase H activities. The intact murine enzyme, once bound to its

substrate, proceeded to hydrolyze its RNA

strand,justlike the aviana,3complex,whereas

a degraded murine enzyme behaved like the

avian asubunit. This reactionwascarried out withasynthetichybrid,

[3H~poly(rA)-poly(dT),

as well as with a phage fd 3H-labeled

RNA-DNA hybrid (not shown), with identical

re-sults.

Murine viral RNase H digestion products. L3H]poly(rA)-poly(dT)wasincubated with iden-tical units ofFr-MuLVenzyme,avian af, and

a under conditions that convert 70% of the

[3H]poly(rA) into acid-soluble material. The

size of the digested products was analyzed by paperchromatography, which allows the sepa-rationofmononucleotides andoligonucleotides

up to eight bases in length, the position of

whichcanbeestimated bytheuse of

fluoresc-ing standards. Fr-MuLV and the avian af3

RNase H activities gave rise to similar

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amounts of small-sizedoligomers (possibly tet-ramers; see above), whereas no monomers could be detected (Fig. 5). In contrast, a did not

giverise to smalloligonucleotides but all of the

material remained at the origin, indicating a

sizelarger than eight bases. The untreated

con-trolalso did not migrate. Solubilization of 20 or

90%ofthehybrid or use of the phage fd

DNA-RNA hybrid gave the same results (not shown).

DISCUSSION

The Fr-MuLV reverse transcriptase-RNase

H complex has been previously described as a

single polypeptide of 84,000 molecular weight

(8).The present study shows that this

polypep-tide reveals all the properties known for the

reverse transcriptase-RNase H complex of the

avian viruses. These properties are as follows:

(i) the ability to bind to natural RNA and to

transcribe it without the addition of synthetic primers; (ii) inhibition of DNA synthesis by

actinomycin D, indicating double-stranded

DNAsynthesis;(iii)co-purification of an RNase

H; (iv) exonucleolytic mode of action of the

RNase H activity; and (v) oligomers,

approxi-mately tetranucleotides, as the main RNase H digestion products and the absence of mononu-cleotides.

The Fr-MuLV reverse transcriptase-RNase

Henzyme complex undergoes spontaneous

deg-radation inside the virion due to freezing and thawing or during purification by long-lasting

column chromatography, giving rise to

addi-tional polypeptides of 78,000, 69,000 and 60,000

molecular weight (Fig. 1). Degradation of the

enzyme can also be achieved artificially byin

vitrotreatmentwith trypsin, whichresultsina

similar polypeptide pattern.

Enzyme preparations consisting of two or

threepolypeptides have been described by

oth-ers(2, 13, 15), with sizedeterminationssimilar

totheones observed here. With such degraded

murine viralenzymepreparations it was

possi-ble to reproduce some of the deficiencies

de-scribedfor mammalian viralreverse

transcrip-tases; degradation of an enzyme preparation

causes loss of the ability to transcribe native

RNAtemplates (Table 1). It can, however, be

drastically stimulated to synthesize DNA if oligo(dT) primers are added to the natural RNA

template. These results obtained with a

de-graded enzyme preparation explainpreviously

published results on mammalian reverse

tran-scriptases (1, 13, 14). Response to synthetic

template primers is unaffected by any degree of

enzymedegradation. Reduction of RNaseH

ac-tivity during degradation has been observed

consistently in these studies; however,

com-plete loss of RNase H couldneverbe achieved

with theRNase H assay applied here.

Only two studies have beenpublished

show-minutes

FIG. 4. Competition ofhydrolysis ofradioactively labeled hybrid by the addition ofexcess unlabeled

hybrid.Purifiedreversetranscriptase-RNaseH activities wereincubated with[3H]poly(rA)-poly(dT) under standard assay conditions. At thetimesindicatedportionswerewithdrawnfromthe reaction mixtures and

processedfordeterminationofacid-insoluble material(C).Inaparallelexperimenta10-foldmolarexcessof

unlabeledpoly(rA)-poly(dT)wasadded2 minafterthestartofthe reaction(-).Inacontrol reactiona10-fold

molarexcessofunlabeledhybridwasalreadyaddedtothereaction mixturepriortotheenzyme(A).

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C13

x 20

AMV a

1

20!

AMVa

3 -iContro

10

20

30

40

50 fractions

FIG. 5. Chromatography of digestion products. Fr-MuLVRNaseHdigestion products obtained from

areactionthat transferred70%ofthe

[3H]poly(rA)-poly(dT) hybrid into acid-soluble material were

chromatographedto separateoligomers ofAMP. The

sameexperimentwasperformedwith identicalunits

ofavian aandaf3enzyme inputs. Untreated hybrid

is shown as control. Included standards were

lo-catedbyUVlight.

ingamurine enzymepreparation consistingof a single polypeptide around 80,000 molecular weight (8, 12); in athirdcasethispolypeptide

appearsamongothers (2). In these three

stud-ies natural RNA transcription has been ob-served. Therefore, the84,000-molecular-weight polypeptide appears to be necessary for

tran-scription of native RNA templates-itdoesnot

seemtobe sufficient, however, sinceadegraded

enzyme may retain some of it without being

able to transcribeRNA (Fig. 1C and Table 1).

An analogous lack of RNA transcription was

observed with the 84,000-molecular-weight

en-zymefromreticuloendotheliosisvirus (9). The results obtained here with the

unde-graded and degraded murine viral enzyme

preparations strongly resemble those obtained

with the aviana,3enzymecomplex andisolated

a, respectively. It was shownrecently

(Moell-ing, Virology, in press) that transcription of natural RNAcanonlyoccur inthepresenceof

,8. Subunit a wascharacterized as a

degrada-tion product of /3 that doesnotbindto natural

RNA and is unabletotranscribeit. This defect

canbeovercomeby addition ofexcessoligo(dT)

primers. DNA synthesis, then, appears to be

predominantly single stranded asjudgedfrom insensitivity ofthe reaction to actinomycin D.

It hasbeen shown previously thatthe RNase

H of the a subunit behaves like a random

exonuclease (3) that is in agreement with its

bindingdeficiency. The random mode ofaction

of the Moloney murineviral RNase H activity

and its large digestion products described by

Verma (12) may then be a result of enzyme

degradation as well. The

84,000-molecular-weightpolypeptide doesnotreveal these

prop-erties (Fig. 4 and5) but behaves asone would

expect, in analogy to the 18-containing avian enzyme.

Nolonger does the murineviralreverse

tran-scriptase-RNase Hcomplexappear tobe

excep-tional in its enzymatic and structural

proper-ties; instead, itbehaves identically to whatis

considered to be characteristic ofa real viral

reverse transcriptase-RNase H complex, from

experience with the avian enzyme as it

tran-scribes natural RNA in the absence of

syn-thetic primers and the RNase behaves like a

processive exonuclease. Itrather looksasifthe

avian enzyme is exceptional in respect to its

structure, since itnormallycanonly be isolated

asacomplex of the actualreversetranscriptase

molecule,

/3,

accompanied byusuallyequal mo-lar amounts of its degradationproduct, a. The reason for this effect is notunderstood, but it may reflect a tendency of the enzyme to form dimers or oligomers. Appearance of equal mo-lar amounts of a prevents 8 from decaying further.

Careshouldbe taken infurther enzyme

char-acterizations not to analyze and describe the properties of a degraded enzyme preparation.

Justonefreezingandthawing ofthe virus can

be sufficienttodestroytheability of the enzyme

totranscribeRNA and canprevent asuccessful

simultaneous direction assay, as was the case

withreticuloendotheliosis virus (9).

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It seems verylikely that theundegraded re-verse transcriptase-RNase H enzyme complex exists inall RNAtumorviruseswiththeabove mentioned enzymatic properties, possibly con-sisting of a single polypeptide. It will be of interesttoelucidate itsmode of action.

ACKNOWLEDGMENT

Ithank H. Bauer for his support and interest in these studies.

These studies were supported by the Deutsche For-schungsgemeinschaft (SFB 47). s

LITERATURE CITED

1. Abrell, J. W.,and R. C. Gallo. 1973. Purification, char-acterizationand comparison of the DNApolymerase from two primate RNA tumor viruses. J. Virol. 12:431-439.

2. Gerard, G. F., and D. P. Grangenett. 1975. Purification and characterization of the DNA polymerase and RNaseHactivitiesinMoloneymurine sarcoma-leu-kemia virus. J. Virol. 15:785-797.

3. Grandgenett,D.P.,and H. Green. 1974. Different mode of action ofribonucleaseH inpurifiedaand/3 RNA-directed polymerase from AMV. J. Biol. Chem. 249:5148-5152.

4. Kacian,D. L., K.R.Watson, A. Burney,and S. Spie-gelmann. 1971. Purification of the DNApolymerase of avian myeloblastosis virus. Biochim. Biophys. Acta246:365-383.

5. Lapidot,Y., andH.G. Khorana. 1963. Studieson poly-nucleotides. VVIX. Thespecificsynthesis of C'3-C3'-C,'-linked ribonucleotides. J. Am. Chem. Soc. 85:3857-3862.

6. McDonnell, G. P., A.-C.Garapin,W.E.Levinson, N.

Quintrell, L. Fanshier, and J. M. Bishop. 1970. DNA polymerase of RSV: delineation of two reactions with actinomycin. Nature (London) 228:433-435. 7. Moelling, K. 1974. Reverse transcriptase and RNase H:

present in a murine virus and in both subunits of an avian virus.Cold Spring Harbor Symp. Quant. Biol. 39:969-973.

8. Moelling, K. 1974. Characterization of reverse tran-scriptaseand RNase H fromFriend murine leukemia virus.Virology 62:46-59.

9. Moelling, K., H. Gelderblom, G. Pauli, R. Friis, and H. Bauer. 1975. A comparative study on avian reticulo-endotheliosis virus: relationship to murine leukemia virus and viruses of the aviansarcoma-leukosis com-plex. Virology 65:546-557.

10. Schafer, W., andE. Seifert.1968. Production ofa po-tentcomplement-fixing murine leukemia virus anti-serumfrom the rabbit and its reactions with various types of tissue culture cells. Virology 35:323-328. 11. Shapiro, A. L., E. Vinuela, and J. V. Maizel. 1967.

Molecular weight estimation ofpolypeptidechainsby electrophoresis in SDS-polyacrylamide gels. Bio-chem.Biophys. Res. Commun. 28:815-820.

12. Verma, J. 1975. Studies on reverse transcriptase of RNA tumor viruses. III. Properties ofpurified Molo-ney murine leukemia virus DNA polymerase and associated RNase H. J. Virol. 15:843-854.

13. Verma,J. M., N. L. Meuth, H. Fan, and D. Baltimore. 1974. Hamsterleukemia virus: lack of endogenous DNA synthesis of its DNA polymerase. J. Virol. 13:1075-1082.

14. Wang,L.-H.,and P.H.Duesberg.1973.DNA

polymer-aseof murinesarcoma-leukemiavirus:lack of detect-ableRNase H and low activity withavianviral RNA and natural DNAtemplates. J.Virol. 12:1512-1521. 15. Weimann, B. J., J. Schmidt, and D. J. Wolfrun.1974.

RNA-dependent DNA polymerase andribonuclease

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