In Vitro Transcription of 70S RNA by the RNA-Directed DNA Polymerase of Rous Sarcoma Virus: Lack of Influence of RNase H

JOURNAL OF VIROLOGY, Jan. 1976,p.291-295 Copyright01976 AmericanSocietyforMicrobiology

Vol. 17, No. 1 PrintedinU.S.A.

In

Vitro

Transcription

of

70S RNA

by the RNA-Directed DNA

Polymerase of Rous

Sarcoma Virus: Lack

of

Influence

of

RNase H

MARC S. COLLETT AND ANTHONY J. FARAS*

Department ofMicrobiology, Universityof Minnesota MedicalSchool,Minneapolis,Minnesota55455

Received forpublication8August 1975

The influence of Roussarcomavirus(RSV)-associatedRNaseHontheinvitro

synthesisofDNA by the RSV RNA-directed DNA polymerase wasdetermined

under conditionswherebyRNase Hactivitywasselectivelyinhibited with NaF.

Notonly were weunable todetect any effect onthe size, structure, orgenetic

complexity of the DNAproductsynthesizedinthe absence of RNaseH activity,

but the displacementofDNAfromthe70S RNA:DNAhybridstructureswasalso

unaffected. The suitability of70S RNA:DNA hybrid structures synthesized in

vitroto serve as asubstrate for RNase H is discussed.

We have beenstudying theinvitro

transcrip-tionoftheRoussarcoma virus(RSV) 70S RNA genome in an effort to elucidate the mech-anisms by which the RNA-directed DNA

po-lymerase synthesizes DNA. To date, several

apparentlimitations exist regardingtheability

ofthe DNApolymerase toefficiently transcribe

the RSV genome in vitro. These include the

relatively small size of the bulk of the DNA

product compared with that of the RSV

ge-nome, the inefficiency of transcription ofRNA

intoDNA, and the limited transcription ofthe

genome intodouble-stranded DNA (10, 13, 20). In our attempts to understand the nature of

theselimitations, and therefore the capabilities

ofthe RNA-directed DNApolymerase, we

ana-lyzed the effect of RNase H on the in vitro

synthesis ofDNA since this particularactivity

appears toresideonthesamepolypeptideasthe

DNA polymerase activity (12, 22, 23) and is

thought to play a role in the synthesis of

pro-viral DNA in vivo (2, 7, 11, 13, 15-17). These

studies were facilitated by the recent

demon-strationthat the RNaseH activityof

ether-dis-rupted virions of avian myeloblastosis virus

(AMV) can be selectively inhibited by NaF

without appreciably affecting the DNA

polym-erase activity (2, 7). We obtained similar data

employing detergent-disrupted preparations of

RSV. For instance, when NaF isincluded at a final concentration of 30 mM instandard reac-tionmixtures(2a, 3, 5,6)greaterthan90% ofthe RNase H activitywas inhibited, asdetermined

bysolubilizationof

[3H]poly(rA)n

.(dT)

12-18(24),

whereas the DNA polymerase activity (8) was

virtually unaffected. Utilizingthis selective

in-hibition procedure, we compared the nature of

the DNA product synthesized in vitro in the

presence (-NaF) and absence (+NaF) of

RNase H activity. Under reaction conditions

thatpromoteextensivetranscription of theRSV RNAgenomeinto DNA (2a),no majoreffects on

either the size (a result analogous to that

ob-tainedrecentlyby Brewer and WellswithAMV

[21)orthesecondarystructureofthe DNA

prod-uct canbe detected (datanotshown).

Inadditiontostudiesonthesizedistribution

of the product DNA, we also examined the

genetic complexity of the DNA synthesized

during NaF inhibition of RNase H activity by

twomethods. First, the total DNAproduct was

analyzed for its ability to protect radiolabeled

70S RNA frompancreatic RNase hydrolysisto

determine theextentoftheRSVgenome

repre-sented by the DNA transcripts (9). The data

(Fig. 1) demonstrate that theextent of

nucleo-tide sequence representation of the RSV ge-nome in the total DNAproduct was unaffected

by the selective inhibition ofRNase H during

enzymatic synthesis of DNA. Secondly, the

kinetics of reassociation of denatured duplex

DNAwasanalyzed in an effort to determine the geneticcomplexity of the double-stranded DNA

(21). Similarly, no difference in the

reassocia-tionkinetics ofthedouble-strandedDNA

prod-ucts synthesized in the presence or absence of

NaFinhibitionofRNaseH activity was

demon-strable (data not shown).

We have also attempted to determine the effect, if any, ofRNase H on the integrity of the

291

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XR60 _

40

10° 101 102

DNA/RNA RATIO

FIG. 1. Extent of RSV genome represented by

RSV-specific DNA synthesized in the presence or

absenceofRNase Hactivity.3H-labeled DNA

synthe-sizedbydetergent-disruptedRSV in thepresence(30 mM) orabsence ofNaF was purified as described

previously(2a, 3).Approximately0.5 ng(2g200counts/

min) of 32P-labeled RSV 70S RNA (4, 10), 40;tgof yeast RNA, and varying amounts ofthe denatured

'H-labeledDNAproductsin 0.1 mlof3xSSC(Ix =

0.15MNaClplus0.015Msodiumcitrate)were

over-laidwithparaffinoil andincubatedat68 Cfor36h.

The extent of hybridization was measured by

hy-drolysiswithpancreaticRNaseasdescribedelsewhere

(3, 9). The results are plotted as a function of the

ratio of DNA to RNA present in each sample.

Symbols: *0,DNAproductsynthesizedin theabsence

of NaF;O. DNAproductsynthesizedin the presence

ofNaF.

70S RNAgenome and the releaseofDNAfrom

70S RNA:DNA

hybrid

structures

during

en-zymatic

synthesis

ofDNA. These studies were

performed

with reconstructed reactions

con-taining

purified

RNA-directed DNA

polymer-ase and 70S RNA (5) to obviate the apparent

problem

of

degradation

of

template

RNA by

virion-associatednucleases other than RNase H

during enzymatic

synthesis

ofDNA(1, 5, 14, 18,

19). The

specific

effect of RNase Hactivityon

the 70S RNA template was determined by

comparingthe profiles, in

polyacrylamide

gels,

ofdenatured 70SRNAobtained from reactions that lackeddeoxynucleoside

triphosphates

with

denatured 70S RNAobtained from enzymatic

reactions containing

deoxynucleoside

triphos-phates.

Since the70SRNAtemplate obtained

from reactions containing

deoxynucleoside

tri-phosphates

is in the form of an RNA:DNA

hybrid structure, itwasconceivable that these

structures might provide a suitable substrate

for RNaseH. However,the profiles of denatured

70SRNA obtained from eitherofthereactions

were quite similar (Fig. 2), indicating that

RNase H did notexhibit adramatic effect on

the integrity of the template RNA during the

enzymatic synthesis of DNAinvitro.

Finally, we tested the effect ofRNase H on

the release of the DNA product from 70S

RNA:DNA hybrid structures synthesized in

vitro since previous pulse-chase experiments

performed in our laboratory indicated that

DNA initially associated withviral70S RNAis

ultimatelydisplaced from the hybridstructures

bysome asyetundescribed mechanism (4, 19). We performed similar pulse-chase studies with

detergent-disrupted virus under conditions in

which RNase H activityisselectively inhibited

with NaF. The same amount ofpulse-labeled

DNA was chased out of the 70S RNA:DNA

hybrid structuresirrespective ofwhetheror not NaF was present in the reaction mixture (Fig.

3). Therefore,although much of the DNA

prod-uct may have been displaced from the 70S

RNA:DNA hybrids during thecourse of

pulse-chase experimentsbythe action of other

virion-associated nucleases, RNase H did notappear

toincrease either the rate (datanotshown) or

amountofDNA releasedfrom 70S RNA:DNA

hybrids significantly (Fig. 3). This contention

was substantiated by the apparent lack of

release of DNA from purified 70S RNA:DNA

hybrids added toreaction mixtures containing

the purified AMV DNA polymerase (Collett

andFaras,unpublished data).Furthermore,no

displacement of DNA product from 70S

RNA:DNAhybridstructures could bedetected

during the synthesis of DNA in enzymatic

reactions containing purified 70S RNA and

AMV DNApolymerase (datanot shown).

Although theRNase H activity ofthe

RNA-directed DNApolymerase of oncornaviruses is

anintegralpartof theenzyme structure (12,22,

23) and is probably required to facilitate

the

synthesis ofproviral DNAinvivo, we havenot

yetbeen able todetectany significant effect on

the synthesis of DNA in vitro. From these

studies it appears unlikely that the enzyme

activity is responsible for any of the major

limitations andrestrictions of reverse

transcrip-tion in vitro. The reasons for our inability to

detect any effect of RNase H during DNA

synthesis are not clear, but mayreflect either

the structure of the 70S RNA:DNA hybrid

molecules synthesized invitro orsome specific

requirement ofRNase H activity, orboth.For

example, since the enzyme is a processive

exoribonuclease requiring unblockedtermini of

the RNA moiety of RNA:DNA hybrids for

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NOTES 293

o$J

a.~~~~~~~~~~~

20

40

60

80

DISTANCE

MIGRATED

(mm)

FIG. 2. Effect ofRNase H on 70S template RNA during the enzymatic synthesis of DNA. Theeffect of

RNase Hactivity on the 70S RNA template during enzymatic synthesis of DNA wasdeterminedasfollows. Deoxynucleoside triphosphateswereomittedfromcontrolreactions to inhibit DNA synthesis and thus prevent

the formation of a potential RNA:DNA hybrid substrate for RNase H. Conversely, reactions containing

deoxynucleoside triphosphates synthesizeRNA:DNA hybrid structures, which were tested for theirpossible

utilization by RNase Has asubstrate. Both reactions contained 3H-labeled 70S RNA (50,000 counts/min),

purified AMV DNA polymerase, and 32 ug of bentonite per ml, which inhibits all traces of nonspecific

nucleolytic activitypresent in the AMV DNApolymerase preparations withoutappreciably affecting either RNase H or DNA polymerase activity under the conditions employed (Collett and Faras, manuscript in

preparation).Afterincubation at 37 Cfor1 h, 70S RNA or 70S RNA:DNA hybrid structures were extracted

with sodiumdodecyl sulfate-phenol (19), precipitatedwithethanol, denatured by heat, and analyzed in 2.25%

polyacrylamide gels (0.4 by 10 cm) as described previously (2a, 6, 19). The solid line representsdenatured 70SRNA incubated in reaction mixtures lacking deoxynucleoside triphosphates. The broken line represents 70SRNA:DNAhybrids synthesized in reaction mixturescontainingdeoxynucleoside triphosphates.

Differen-tially labeled 28S and18SrRNAwere includedasmarkers in thegels. The twoelectropherograms are

super-imposed for comparativepurposes.

A.

8.

activity (12, 16), it is possible thatvery few, if

s - any, of the 70S RNA:DNA hybrid structures

synthesized

in vitroexhibit thenecessary

struc-ture required for activity. However, although

6-6

'o r \ \ l t \ l t concentrationof3.5 x 10-6and 1.8 x 10- 6M, respec-tively. Each reaction was divided into two aliquots

N4 and incubatedfor 15minat37C.Afterthe

incuba-tionperiod,oneofthe reactions(pulse labeled) was

z

stopped by

the addition of 0.5% sodium

dodecyl

2 I w I \ . , x SY \ sulfateand incubated with 500 Mgof Pronase per ml for 30minat 37 C. The other reaction(pulse-chase)

was incubated for an additional 105minat 37 C after

.RACTION theadditionofa140-foldexcessofunlabeled TTP and

I0

OU2BER

20

a

50-fold

excess

of unlabeled

dATP. The reactionwas

FRACTIONNUMBER

adjusted

to 0.5%sodium dodecyl sulfate and 500

,g

of FIG. 3. Effect ofRNase Hactivity on the displace- Pronase per ml and incubated for 30 min at 37 C. ment ofDNA from 70S RNA:DNA hybrids during After phenol extraction and ethanol

precipitation,

enzymatic synthesis of DNA by detergent-disrupted samples were analyzed byrate-zonal centrifugation in RSV. Standard reaction mixtures containing deter- anSW50.1rotor for100minat50,000 rpm. A and B

gent-disrupted virus were incubated at 37 C either represent the enzymatic productsynthesized in the in the presence or absence of NaF. Both unla- absence of NaF and in the presence of the

inhibitor,

beled dGTP and dCTP were present at a final con- respectively.Symbols: 0,enzymaticproduct synthe-centration of 4 x

10-'

M. The labeled precursors sized after a 15-minpulse; 0, enzymatic product

['HJTTP and

['HJdATP

were included at a final synthesized after a pulse-chase. VOL. 17,1976

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[image:3.491.87.425.57.275.2] [image:3.491.37.229.442.587.2]

the precise nature ofthe hybridstructures has

not yetbeen elucidated,wehavecircumstantial

evidence suggestingthat the 5' end of the viral

RNAgenome might be capable of servingas a

substrate for RNase H. First, we and others

have localized the principle RSV RNA primer

molecule near (within 10%) the 5' end of the

RSV genome (K. Staskus, M. S. Collett, and

A. C. Faras, submittedfor publication;J.

Tay-lor, in press; and B. Cordell-Stewart, personal

communication). Secondly, under the reaction

conditions employed in these studies, most of

the DNA transcripts ranged between 900 and

1,500 nucleotides inlength (2a). Therefore, it is

entirely conceivablethat the 5' end of the viral

genome is in the form of RNA:DNA hybrid

structures suitable as a substrate for RNase H

activity. Ifthis wereso, thenonly <10% ofthe

RSVgenomewould be affectedbyRNaseHand,

therefore, wouldnotbedetected inourassay

sys-tem (Fig. 2). Anotherdifficulty is suggested by

the recent demonstration thatthe5' end of the

RSV RNAgenome is blockedby m7Gand

con-sists ofthe sequence m7GpppGmCp (J. Keith,

personal communication). RNase H maybe

in-capableofutilizingthisparticularterminusas a

substrate. Furthermore, avian RNA-directed

DNApolymerases maybe unable toeffectively

transcribethe"capped" 5' terminusoftheviral

genomein vitro and thusprevent the formation

of a suitable substrate for RNase H. Further

studies are currently in progress in our labora-tory to delineate the nature of the 70S RNA:

DNAhybridswithrespecttotheirstructureand

possible utilizationas apotential substrate for

RNase H.

Wethank C.Foxand S. Kanellosforexcellenttechnical

assistanceandP.Plagemannforeditorialassistance.

Thisinvestigationwassupported byPublic Health Service

research grantCA 14790-01fromtheNationalCancer

Insti-tute. M.S.C. was supported in part by a H. Rackham

Fellowshipfrom theUniversityofMichigan. LITERATURE CITED

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Extent of transcription by the RNA-dependent DNA polymerase ofRous sarcoma virus. Nature (London)

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