Binding of tRNA to Reverse Transcriptase of RNA Tumor Viruses

(1)

0022-538X/78/0026-0214$02.00/0

Binding

of tRNA

to

Reverse

Transcriptase of RNA

Tumor Viruses

AMOS PANET* ANDHADASSAH BERLINER

DepartmentofVirology,The HebrewUniversity,Hadassah MedicalSchool,Jerusalem, Israel Received forpublication28November1977

The interactionof tRNA with the reversetranscriptase (RNA-dependentDNA

polymerase) of mammalian RNA viruses, such as Moloney murine leukemia virus andsimiansarcomavirus, has been studied. Whereas the purifiedreverse

tran-scriptase of mammalian virusessedimented in glycerol gradients as a globular protein with a molecular weight of 70,000, after interaction with tRNA the enzyme cosedimented with aprotein of 150,000 molecular weight. The twofold increase in molecularweight could be a result of either two reversetranscriptase molecules

complexed withatRNA or, alternatively, several tRNA molecules bound to a

single enzymepolypeptide.The enzymecomplexes were dissociated in part upon degradation of the tRNA moiety by pancreatic RNase A. The reverse transcrip-tasereleased from virions ofMoloney murine leukemia virus, simian sarcoma

virus, andavianmyeloblastosis virus, bynonionic detergent, migrated fasteron

glycerol gradients than purified enzyme preparation. This phenomenon was

probablydue tocomplex formation between part of the virionenzyme andthe

tRNA,whichisendogenousinvirions. Addition ofexogenoustRNA wasneeded,

however, to quantitatively complex all the virion reverse transcriptase ofMoloney

murineleukemia virus and simiansarcomaviruses. The reversetranscriptase of

Moloney murine leukemia virus did not show tRNA species specificity in the

binding reaction when glycerolgradients were used for assay. Thus, several tRNA

species ofEscherichia coli, yeast, chicken,andrat originwereable to complex

with theenzyme. The species specificityin the interaction between tRNA and

avianmyeloblastosisvirusreversetranscriptasewasalsoexamined. We

demon-strated thatunderourexperimental conditions,thisenzyme binds differenttRNA

species of E. coli and yeastaswellastRNA of chickenorigin.

Initiation of DNA synthesis in RNA tumor scriptase, shows both DNA polymerase and

viruses isprimed in vitro bya tRNA, which is RNaseHactivities(12, 22).

partof the70SRNAgenomecomplex (1). Two In this study we showthat the reverse

tran-groups ofvirus use different tRNA species of scriptase of two mammalian viruses, Moloney

celloriginasprimer. Thus, tryptophan tRNAis MuLVandsimiansarcoma (SiSV),forns

com-theprimer in avian myeloblastosis virus (AMV) plexes with tRNA. The binding of tRNA

and avian sarcomavirus (6), and proline tRNA changes the molecular weight of the mammalian

has recently been shown to be the primer in enzyme from 70,000 to 150,000, suggesting a Moloney murine leukemia virus (MuLV) (17). complexstructure oftwo enzyme subunits and Theprimer tRNATrP in AMV hastworecogni- atRNAmolecule.

tion sites: it is boundat a specific site to the

template35S RNAgenomesubunit(21), and it MATERIALS AND METHODS

complexeswithhighaffinitytothereversetran- Virusesandenzymes. Moloney MuLV was grown

scriptase (16). ThebindingoftRNAT"" topun- inSwiss mousecells(clone 1)and purified as described

fied AMVreversetranscriptaserequires the hol- before(3).AMVwas agiftfromJ.W.Beard ofLife

oenzyme,whichconsists oftwopolypeptides (a SciencesInc., St. Petersburg,Fla., and SiSVwas

ob-and

I?).

Theisolatedsmallerasubunit(molecu- tamed from J. Gruber of the National Institutes of

larweight, 65,000), which manifests both DNA Health, Bethesda, Md. Reverse transcriptasewas

pur-polymeraseand RNase H activities (4), cannot ifiedfrom these three viruses by sequential column

formastablecomplex with tRNATrP (5, 7). e- chromatography on DEAE-Sephadex followed by

forme

aian

transcriptamisolated from mamma

phosphoceliulose

(23).

All

buffersusedthroughoutthe

verse transcriptase isolated from mammalian purification procedure contained 20 mM potassium RNA tumor viruses is asinglepolypeptidewith phosphate, pH 7.5,ratherthan

Tris-hydrochloride,

pH

a molecular weight of 70,000 (19). This poly- 7.9,since we found that phosphate bufferssignifcantly

peptide,like theasubunit of AMVreversetran- increased the stability of the reverse transcriptase

* ~~~~~~~~214

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

duringpurification. The purified enzymewasdialyzed between enzyme andtRNA,we first sought

con-extensively against 50 mM Tris-hydrochloride (pH ditions to separate the complex from the

un-7.5), 0.1 M KCI, 0.1% (vol/vol) Triton X-100, 20% bound reverse

transcriptase.

Glycerolgradients (vol/vol)ethyleneglycol, 2%(vol/vol) glycerol,and 10 conting ethylene glycol were found to be most

mM mercaptoethanoL Pancreatic RNase A, electro- t bl

phoretically purified, was purchased fromWorthing- mixe with purified an after short

ton Biochemicals Co.,Freehold,N.J. mixed with

purified

enzyme, and after short Reagents.Chlcken liver tRNA and rat livertRNA incubation in thecold the mixture, togetherwith were isolated as described by Rogg etal. (18). Yeast twointemalprotein markers,waslayeredonto tRNAwas a product of Schwarz/Mann, Orangeburg, the gradient. After centrifugationfor 27h, the

N.Y., and Escherichia coli WtRNA was a gift from positionsin the gradient of the reverse

transcrip-Y. Lapidot, The Hebrew University. These tRNA tase and the protein markers bovine serum

al-preparations were further purified onDEAE-celiulose

bunin

and

human

IgG were determined.Figure columns (Whatman, DE-52). The tRNA was loaded 1

shows

that tRNA affects the sedimentation

ontothecolumns,which werethen washed stepwise coefficientof thereverse

banscriptase.

Whereas

withpotassiumacetate buffer, pH 5.0, containing 0.1

and 0.3MNaCl. The tRNAwas theneluted with 1.0 free enzyme cosedimented with bovine serum M NaCland precipitated with 2 volumes of ethanol albumin (4.7S; molecular weight, 70,000), addi-Purified E. colitRNAVI and yeasttRNAph were tion of 30 pg oftRNA shifted the polymerase products of Boehringer Chemical Co., Mannheim, Ger- activity to the position of the IgG marker (7.2S; many. These purified tRNA preparations were found molecular weight, 156,000). A twofold increase to lack acceptor tRNA for proline, tryptophan, and in sedimentationrate of the enzyme cannotbe methionine.One absorbance unit of tRNA at 260nm accounted for by equimolarbinding of one en-wasassumedtobe 50

pg,

or 1.67 nmoL Poly(A)and zyme molecule (molecular

weight,

70,000) with poly(dT)IOweregiftsfrom Y.Lapidot; [3H]dTTP, 48

siyme

tNmolecule (molecular weight,7,0)wt

Ci/mmol, was obtained from the Radiochemical a single tRNA molecule (molecular weight,

Centre, Amersham, Engld; bovineserum albumin, 25,000). Such acomple

is

more likelytoconsist

electrophoretically purified,wasfromCalbiochem,L of twoenzyme polypeptides and one tRNA

mol-Angeles, Calif.; andpurifiedhumanimmunoglobulin eculeor, alternatively, several tRNA molecules G(IgG)was agift from G. Spira, The Hebrew Univer- bound to a single polypeptide. Addition ofan

sity. even larger excess of tRNA does not further

DNApolymerase assay. Reaction mixtures (100 increasethe sedimentation rate ofthe enzyme 1) contained50mMTris-hydrochloride (pH 8.3),10 (data notshown).However,smalleramounts of

mMdithiothreitol,1ugofpoly(A)-oligo(dT),( 10 of tRNA (3

ug)

cause themajority of reverse

tran-bovineserumalbumin6mM MgC12, tM

~[scriptase

moleculesto

migrate

at an

intermedi-(350cpm/pmol),andsamples(usually30

Al)

from the

glycerol gradient. Reaction mixtures were incubated ate position between the two internal protein

for 60min at37°C, andtrichloroacetic acid-insoluble

radioactivity wascollected on glass fiber filters (What- _

man). One unit of activity is the amount of reverse I 6

BSA

transcriptase needed tocatalyze theincorporation of 7

1pmol of[3H]dTMPpermin under the above condi- . 0 4

tions. ^ 8 -

~~~~~~~~~~~~~~~A

/f.a

tiNA.,

38jol 0.4 E

tions. ' 8

tRNAbindingassay. Mixtures (200

pil)

contained £.4o 0.2 50mMTris-hydrochloride (pH7.5), 5 mMMgC2, 10

mM dithiothreitol, and 0.2% (vol/vol) Triton X-100. a U

0.0

After 10min of incubation at 4°C, theinternalprotein 8 B3A,3, 0.4

markers bovine serum albumin (0.6 mg) and human I z

IgG (0.6 mg) were added, and the mixtures were lay- X 4 I 0.2 ered onto5-ml glycerolgradients (5 to 25%, vol/vol) in o a 30 50 mMTris-hydrochloride (pH 7.5), 0.1 M KCI, 0.1% z o.oS

(vol/vol)TritonX-100, 10 mMmercaptoethanol, and 8C c a

20% (vol/vol) ethylene glycol. Gradientswerecentri- -0.4 z

fugedfor 27 hat49,000rpmat4°C inanSW 50.1rotor _ -0.2

i-ofaBeckman

ultracentrifuge

andfractionated from 1 2 30

the bottom (usually 32 0.16-mi fractions were col- I -'0_

lected). Samples (30 1d) were taken for the DNA FRACTION NUMBER polymerase assay, and 30 p1 was taken for protein

determination (11). Proteins were first precipitated FIG. 1. Binding oftRNA to Moloney MuLV re-with 10%(vol/vol)trichloroacetic acid to remove ma- versetranscriptase. Binding reactions and

centrifu-terials that interfere with theLowry assay(11). gation inaglycerol gradientwereperformedas

de-scribedin thetext. (A) Fortyunits ofreverse

tran-RESULTS scriptaseand 30 pg of rat liver tRNA;(B)40 units of

reversetranscriptase and3pg ofratlivertRNA; (C)

Binding of tRNA to Moloney MuLV re- 40 units of reversetranscriptase.A6w,Absorbance

versetranscriptase.Tostudythe interaction at 660 nm.

http://jvi.asm.org/

[image:2.509.285.431.423.598.2]

(3)

216 BERLINER VIROL.

markers (Fig. 1B).The formation ofan appar- byRNaseA.The factthat RNaseAdissociates

entlysmaller enzyme

complex

may either be the preformedcomplexes (Fig.

2B)

suggests that the

result ofpoor separation between the

complex

reverse transcriptase-bound tRNA is not

pro-and free enzymeor

perhaps

limitedamountsof tected from RNaseAaction.

tRNA induce formation ofa smaller

complex.

Specificity of the binding reaction. We

Inclusion of only bovine serum albumin as a havestudied tRNAspeciesspecificityinbinding

marker in the gradient

(Fig. 1B)

demonstrates tothereversetranscriptase ofMoloney MuLV

that the purifiedreverse

transcriptase

doesnot and AMV by using unfractionated or purified

contribute enoughproteintointerfere with the tRNA species ofdifferent organisms. Figure 3

exactpositioningof the markers. shows that Moloney MuLV reverse transcrip-Toprovethat thereversetranscriptasecom- tase can interact with unfractionated chicken

plex indeed contains tRNA, we treated such livertRNA, yeast tRNA,or E. colitRNAwith

complexes with pancreatic RNase A, followed asubsequent increase in the sedimentationrate

bycentrifugation inglycerol gradients. RNaseA of theenzyme. Each of these tRNApreparations

wasmixedwith tRNA either before(Fig.

20)

or contains proline acceptorspecies, but the

nu-after (Fig. 2B) binding to the enzyme, and in cleotidesequence of

tRNA'°

of mammalian

or-bothexperimentsmostofthereversetranscrip- igin,whichservesas aprimer for DNAsynthesis

tase sedimented as free enzyme. Some of the in Moloney MuLV (17),differs from that of E.

complexes after RNase treatment sedimented coli

tRNAP'ro

(J. Dahlberg, personal

communi-faster than the free reverse transcriptase (the cation). Purepreparations ofyeast

tRNAPhe

and

position ofbovine serum albumin marker) and E. coli

tRNAvaI,

whichcontain no proline and

similartocomplexesmade withasmallamount tryptophanacceptor tRNA, also change the

sed-of tRNA (Fig. 1B). These enzyme molecules imentation coefficient ofthe reverse

transcrip-may still contain some intact, or partially de- tase (Fig.4).

graded, tRNAmolecules,since underour

exper-imental conditions 5 to 10% ofthe tRNA was

notconvertedintoacid-soluble

oligonucleotides

3-

19G

BSA

2- A 4 ' E Coli flNA

196 BSA w 1

o E

4 *0 20 E0, *

A0E.

u 2 tDNAYeast

MoloneyMuLVreveseransripase40nit

glceo grdin cetiuainwr aredota * _

Cr4C

0I4

decieihet.()Ezyead5 zofra

lieRA. (B

Enyead50u

frtlvrtN RCINN

werinuae for

30mna

°,adte Ns

s

_0m

oa w c ChickentrNA

_ C~~~~~~~~wL

0O 10 20 30 4 4

[image:3.509.293.437.336.581.2]

FRACTION NUMBER 2

FIG. 2. Dissociation of reverse transcriptase- 2

tRNAcomplexesbyRNaseA.Bindingreactionswith 1Mo

MoloneyMuLVreverse transcriptase(40units) and

glycerolgradient

centrifutgation

werecarriedoutas C (A

described in thetext. (A) Enzymeand 50

pig

ofrat 0 10 20 30 livertRNA.(B) Enzymeand50pgofratlivertRNA FRACTION

NUMBER

wereincubatedfor10minat40C,andthenRNase A

(10pgJwasadded.After30minat40C,dithiothreitol FIG. 3. Reverse transcriptase complex

formnation

was addedto 10mM, andthe mixture was loaded with

unfr-actionated

tRNA of various organisms. ontoagradient.(C)A50-pugamountofratliver tRNA Conditions for binding reactions with Moloney

waspreincubated for15minat370CwithRNaseA MuLVreversetranscriptase (40 units) andglycerol (10

p.gJ

in the binding mixture, and then reverse gradientcentrifugationaredescribedinthetext.(A) transcriptasewasadded.Afteranadditional10min Enzymeand 29

pg

ofE. colitRNA; (B)enzymeand at 4°C, the mixture was layered onto agradient. 34pgofyeasttRNA;(C)enzymeand

33

pgofchicken

A,m ,Absorbanceat660nm. livertRNA; (D) enzyme.

http://jvi.asm.org/

[image:3.509.92.231.363.528.2]

(4)

lgG

BSA

tRNATr

and

tRNA4Mt

of origin.

60 - Thus, total yeast tRNA, which contains

A I\ Yeast

tINAPhe

tRNATrp with different

primary

structures

(6,

8,

o0 _ /

\10)

orpure E. coli

tRNAVaJ

andyeast

tRNAPhe,

020 I! \ can complex with AMV reverse transcriptase

and

change

its

sedimentation

coefficient from

E

7.2S

(IgG

marker) to9S

(Fig.

5).

30 - \ Structureofreverse

transcriptase

in

dis-d20 B EColi

tUNA"'

rupted virions of Moloney MuLV, SiSV, and

w ! \ AMV. Some 30% of the total RNA in virions of

< 10

Moloney

MuLV consists of small RNA (4-7S)

0 ₁₅0

containing

15

species

of host

cell

tRNA (20).

_ + Someof the small RNAmoleculesareboundin

o C s d11 the 70S RNA complex, and the rest arereleased

Z-10

_ d free in solution after

disruption

of

virions

with

a. /.

\nonionic

detergent.

We have

investigated

X 5 - 1 \ whether the "free tRNA" is bound to the reverse

- o

.transcriptase,

l\_ | which isalso released from virions

I _

by

detergent

treatment. The results in

Fig.

6

30 - D illustrate that the enzyme in disruptedMoloney

20 - MuLV

migrates

in

glycerol

gradients

faster than

bovineserumalbumin, whichmarks theposition

10_ / of thepurifiedreversetranscriptase.Addition of

exogenousratliver tRNAfurther increases the

C0 ₀ 10₁₀ _~2020 30 molecular

weight

of virion

polymerase,

with the

30 FRACTION NUMBER

FIG. 4. Binding ofreverse transcriptase to purified

tRNAspecies. Conditionsfor bindingreactions with A 19g

MoloneyMuLVreversetranscriptase(60units) and

glycerol gradientcentrifugationaredescribed in the 4 E t tUNA"'Val 0.4 c

text. (A) Enzyme and 20 pgof yeast tRNAPhe; (B) 2r0

enzymeand25 pgofE. colitRNAVal; (C) enzyme and

10pg of oligo(dT);(D)enzyme.

0,

c-

o

.0°

64-B * YeasttUIA"o

From these results we conclude that under E 2

N

the

binding

conditions

used,

Moloney

MuLV

Q.

O

reverse

transcriptase

doesnot

selectively

binda, °

unique tRNA species. In contrast, synthetic C Y.at toNA Z

oligo(dT),which isanefficientprimerfor DNA 4- /

synthesis with poly(A) as template, does not

o0

* . - w

affect the sedimentationrateof theenzyme

(Fig.

8 D i+ tUNA 04

4). 0

Tofurther

investigate

the

question

of

specific-

Z 4- 0.2

ity,wereexamined the tRNAbinding specificity a. - o

of AMV reverse transcriptase by the use of 2 .0

1-- E

glycerol gradients. In a previous study (16) it 4

-was shown that outof unfractionated chicken

cell

[32P]tRNA,

only two species, tRNATrP and 2

tRNA4Met, were able to bind to AMV enzyme o

withhigh affinity.The

analysis

of

complex

for- 0 10 20 30 mation wascarriedoutbyfiltration of the mix- FRACTION NUMBER

turesthrough SephadexG-100 columnsthatsep- FIG. 5. Interaction of various tRNA specieswith arated the enzyme-bound [32P]tRNA from un- AMV reversetranscriptase. Conditions for binding

bound[PtRNA

(molecular weight, 25,000). reaction and glycerol gradient centrifugation with

bound

[lycerolgadin

etfgtorte AMVreversetranscriptase (50 units)aredescribedin

When glycerol gradient

centrifugation,

rather thetext.

(A)

Enzymeand

20pg

ofE.colitRNAvalJ(B)

than gel filtration, was used for analysis ofA.MV enzyme and 15pg ofyeast tRNAPhe; (C) enzymeand

reversetranscriptase-tRNA complex formation 52pgof totalyeasttRNA; (D) enzyme and47pgof

(Fig. 5), we found that the AMV polymerase totalchickenliver tRNA; (E) enzyme.A66,

Absorb-could also bind tRNA species other than ance at660 nm.

http://jvi.asm.org/

[image:4.509.82.224.63.335.2] [image:4.509.283.426.337.578.2]

(5)

218 PANET AND BERLINER J. VIROL.

I 196 BSA the reverse transcriptase in virions of AMV.

30- A f Figure8A shows that all the reverse

transcrip-o 20 *; MvLV tasereleased from AMV after detergent

treat-x )'7\/ mentmigratesonglycerol gradients as a protein

E 10 i \_ $ with asedimentation coefficientof 9S,consistent

o

witha

complex

ofenzyme(7.2S)and tRNA

(Fig.

i20

B ° 8C and

D).

Addition ofexogenoustRNAtothe

MuLV+

tIUA

virus

does notfurther increase the

sedimenta-10

lo

if

\ z tionrate of thecomplex (Fig. 8B).Itis

interest-o '--.--.-. w ing to note that a virion of AMV contains

be-z 6- C a tween20and80molecules ofreverse

transcrip-4ulV

enzyme+

tUNA tase (14, 15) and some 35 to 70 molecules of 15

MuLY 0

_4. tRNAspecies; ofthese, only 7 to 10 molecules

X2 / i 9Z \ 0.2 o are the primer tRNAT'P (20). The observation

I

0 I~~m S -3,00that

all

the released virionenzymeis

complexed

0 10 20 30 to tRNA further strengthens the notion that

[image:5.509.87.246.49.239.2]

FRACTION NUMBER many tRNA species, besides tRNATrP, can bind

FIG. 6.Binding of tRNA to reverse transcriptase to the reverse transcriptase when glycerol

gra-in virions. Conditionsforbinding reactionsare de- dients are beingused as an assay system.

scribed in the text. (A)MoloneyMuLV (250agJ was

treated with0.2%(vol/vol)TritonX-100 in a binding DISCUSSION mixture. After incubation for 10 min at 4°C, the

mixture wascentrifuged for 20 min at 6,000 x g, and In this study wehave shown that the reverse the clearsupernatant was layered onto a glycerol transcriptaseof mammalian RNA tumor viruses

gradient. (B) Virions(250gj were treatedwith Triton exists in two forms; the purified enzyme is a

X-1OO)asdescribed in(A)in thepresence of30()

lg

of single polypeptide with a molecular weight of

rat livertRNA. (C)Forty unitsofpurifiedMoloney 70,000,whichisconvertedinto a high-molecular-MuLV reverse transcriptase and 30 pg of rat liver weight complex after binding of tRNA. The tRNA. Protein determinations were not done with exactstructure of this complex is not yet

known,

gradients A and B, since virionproteins interfere butaccording to its molecular weight

(150,000)

with the assayofthemarkers.A66o

,.

Absorbance at twoarrangementsarepossible:

i)

anaggregate 660 nm.

result that it sediments at the position of the A BSA

purified reverse transcriptase-tRNA complex 3 A

(Fig. 6B andC).Theseobservations suggest that X S

S'

some polymerasemolecules released fromviri- E 2 E

onsare

complexed

withtRNA and thus

migrate

1 - / > .

faster in

glycerol gradients.

However, to quan-_ c

titatively complex

all of the virion enzyme, ad- i o

-ditional exogenous tRNA is needed. It ispres- o + 4

entlyunknownwhether the enzyme is boundto a. 4 / z

tRNA in the virion or complexes are formed ° 2

subsequenttovirusdisruption. z p

w

To determine whethermammalian RNAtu- C a

mor virus reverse

transcriptase

otherthan the 2 C 4

sisY

@RFme

0.4z

Moloney MuLV enzyme can also bind tRNA, X

we chose a primate virus, SiSV. The results 10.20

presented

in

Fig.

7show that whereas

purified

,.o

0

SiSVreversetranscriptasemigratesas aprotein 0 10 20 30

withamolecularweightof70,000 (usingbovine FRACTION NUMBER

serumalbuminas

marker),

theenzymereleased FIG. 7. Interaction between SiSV reverse

tran-fromSiSVvirions is somewhatheavier(Fig.7A). scritaseand tRNA. Conditionsforbindingreactions After addition ofexogenous tRNA, the DNA andglycerolgradientcentriAgationaredescribed in

polymerase of SiSV sediments as a proteinof the text. (A)SiSV(125 ag)wastreated withTriton

X-p1(Xecunr

_150,00_mlecuar_{wigh (Fi. 7B. Ths}the

_exer-in

_thed_legendlayered_toonto_Fig.

_6;

aglycerolgradient_(B)

_SiSV

₍₁₂₅

_pg)as_wasdescribed_treated

iment demonstrates that thereversetranscrip- withTriton

X-.(X)

inthepresence of

300jig

ofrat liver taseofa

primate

viruscanbindtRNA

similarly

tRNA asdescribed in the legend to Fig.

6;

(C)purified

totheenzymeofamurinevirus. SiSVreversetranscriptase(25units).

A6w,,

Absorb-We havefurtherinvestigatedthestructureof ance at 660nm.

http://jvi.asm.org/

[image:5.509.286.431.384.565.2]

(6)

of two subunits with one or two tRNA molecules, to the enzyme. (ii) Thepattern of the

sediment-or(ii) several tRNA molecules bound to a single ing enzyme complex suggests a unique species

polypeptide. If the mammalian virus reverse with a molecularweight of 150,000; nonspecific

transcriptase-tRNA complex is indeed a dimer, aggregation is more likely to produce multiple

then it maybecomparedtotheholoenzyme of species. (iii) Proteins such as bovine serum

al-avian sarcoma leukosis viruses. The avian hol- bumin, IgG, oreven the isolated a subunit of

oenzyme is composedoftwosubunits, aand /1 AMVreversetranscriptase (5, 7)do notcomplex

(4), or

,B,,

in Rous sarcomavirus propagated in with tRNA. (iv) Reverse transcriptase of Rous

duck celUs (9). In experiments in which tRNA sarcoma virus temperature-sensitive mutant

concentrations were low, the reversetranscrip- ts335is thermolabile for both DNA polymerase

tasecomplex sedimented at an intermediate po- activity (24) and the tRNAbinding function (A.

sition. Such sedimentation may reflect forma- Panet,G. Weil, and R.Friis, manuscript in

prep-tion ofa complexwith adifferent structure or aration). (v) Inhibitors of reverse transcriptase

dissociation of the dimer during centrifugation. activity such as monospecific antibody and

N-It may be argued that the association of re- ethylmaleimide also inhibit the tRNA binding

verse transcriptase and tRNA represents non- function (7).

specific aggregation of protein and nucleic acid; The fact that crudereverse transcriptase

re-there are,however,several lines of evidence that leased from virionssediments faster than

puri-indicatespecificity in this interaction. (i) Com- fiedenzymesuggests that it is bound to tRNA,

plexformation requires intact tRNA, whereas and, during enzyme purification, the tRNA is

fragments of tRNA(7), oligo(dT), or 16S rRNA removed and the two-subunitenzyme of

mam-of E. coli(unpublished observation) do not bind malian viruses dissociates. On the other hand,

thetwosubunits of theavian virus enzyme may

hold

togetherevenwithout tRNA. It is not clear

60- 196 touswhy inaprevious studymouseliver tRNA

fi

A

AMVdid

not interact with MuLV reverse

transcrip-T40

_A

A tase (7). The quality of the tRNA preparation

o /, or the differentcomposition of the glycerol

gra-x 20 - dients used for theassay may have been

respon-Efi

O

sible for the

seeming

lack of interaction.

u There are several studies in the literature

d 40 B AMV +tllA

regarding

thestructureofthereverse

transcrip-2 / tase in virions and

cells.

Mondal et al. (13) have

<a

20- /

\reported

the existence of two enzymes in

lym-2

0 I .

./

phosarcoma cells

productively

infectedwith

gib-u c Al enzym the reversetranscriptase isolated from

virions,

z5 / *^ with a molecular weight of 70,000; and (ii) a

cL

5

high-molecular-weight

enzyme

(140,000)

that is x .__v._/able~ to copy the 70S RNA genome very

effi-1 C

ciently.

Thislatter form could bedissociated

by

1 2 D r>t"1e

high-salt

and detergent treatment to yield the

w

\70,000-molecular-weight

enzyme. Based on the

1 _, results presentedhere, we suggest that the

high-I.

molecular-weight

reverse

ranscriptase

found in

00 10 20 30

productively

infected

cells

(13)

orinRous

sar-FRACTION NUMBER coma virus-disrupted

virions

(2) is indeed an

[image:6.509.83.224.307.544.2]

enzymecomplexedtotRNA.The association of

FIG. 8. Binding of tRNA toreverse transcriptase tRNAwith the reverse transcriptase in

produc-in disrupted virions ofAMV. Conditionsfor tRNA tively infected cells could provide the

mecha-bindingare described in the text. IgGmarkerwas nism responsible for the incorporation ofcertain added to reactions (C) and(D) only, and glycerol

cell

tRNAspecies into the

budding

virus. Such

gradientswerecentrifuged for 20h (A)

MAMV(50)iw9)

a

mechanism,

however,

required some tRNA

was treated with Triton X-100 asdescribed in the * * ' * * r

legendtoFig. 6;(B) AMV

(5() g)

was treatedwith

species

specificity lIn

thebidig reaction,

isince

TritonX-1IK)in thepresence of125pgof chicken liver a specific subset of the celi tRNA's is incorpo-tRNA as described in thelegendtoFig. 6;(C) purified rated into virions (20).

AMVreverse transcriptase (50units) and 50pg of AMVreversetranscriptasewasshowntobind chicken livertRNA; (D) purified AMV reverse tran- only tRNATrp andtRNA4Met withhigh affinity

scriptase

(50

units).

(16). In the present study we found that the

http://jvi.asm.org/

(7)

220

AMVenzymecanalso bindother tRNAspecies, 9.Hizi, A., J. P. Leis, and W. K.Joklik. 1977. RNA althoughpossiblywith lower

affinity.

Such bind- dependent DNA polymerase of ASV. Comparison of

ing

was observed when

high

concentrations of the catalytic properties of the a, /2 and 8 enzyme

ingwasobservedwhen high ~~~~forms.J. Biol. Chem.252:2290-2295.

tRNAwereaddedtothepurifiedAMVenzyme 10.Keith, G.,A.Roy,J. P.Ebel,and G.Dirheimer. 1971.

andcomplexformationwasassayedusing glyc- The nucleotide sequence oftwo tryptophan tRNAs

erolgradient centrifugationrather thanSepha- from brewer yeast. FEBSLett.17:306-308.

dex gel filtration (16). This apparent lack of 11.Lowry,0.H.,N.J.Rosebrough,A. LFarr,and R. J.

dex.gelifitration(16).

This apparent lackof Randall. 1951. Proteinmeasurement with theFolin

specificity isreminiscent of the interaction be- phenol reagent.J.Biol. Chem.193:265-275.

tweenaminoacyl-tRNA synthetasesand tRNA. 12. Moellng,K. 1974. Characterization ofreverse transcrip-The latterenzymesbind theircognatetRNA's taseand RNase H from Friend-murine leukemiavirus.

tighlyut an lsoassociatewithother tRNA

1.Virology

62:465-59.

tightly but can alsoassociate with other tRNA 13

Mondal, H.,

R. E.

Gallagher,

andR.C.

GaBo.

1975.

specieswith loweraffinity (25).

Furthermore,

we RNAdirected DNApolymerasefromhumanleukemic

couldnotdemonstratetRNAspeciesspecificity bloodcells and fromprimate type-Cvirus-producing

inthecomplexfonnedwith MuLVreversetran- cells:high and low molecularweight forms withvariant

scriptae.Ifideedte affinty of te MuLV biochemicalandimmunological properties.Proc. Natl.

scriptase. If indeed the affinity of the MuLV Acad. Sci. U.S.A.

72:1194-1198.

enzymetoitsprimer tRNAP"oishigherthanto 14. Mosser,A.G.,R.C.Montelaro,andR. R.Rueckert.

other tRNA species, otherexperimental condi- 1977. Proteins of Rous-associated virus type61:

poly-tions willhave tobesought.Sephadex gelfiltra- peptide stoichiometry and evidence thatglycoprotein

tion, for example, failed to show any complex

gp35

is not a cleavage product of

gp85.

J. Virol.

fonnationetween[3p]tRNA an the MuLV 23:10-19.

formation between [32P]tRNA and the MuLV 15. Panet, A.,D.Baltimore,and T.Hanafusa. 1975. Quan-reversetranscriptase. titation of avian RNA tumor virusreversetranscriptase

byradioimmunoassay.J.Virol.16:146-152.

16. Panet,A.,W.A.Haseltine,D.Baltimore,G.Peters, ACKNOWLEDGMENTS F.Harada,and J. E.Dahlberg.1975.Specific binding This workwassupportedbygrantsfromStiftungVolkswa- oftryptophan transfer RNAto avianmyeloblastosis genwerk and The IsraelCommissionforBasicResearchu virusRNA dependent DNA polymerase (reverse tran-Wethank N.Goldblum for hishelpf scriptase). Proc. Natl. Acad. Sci. U.S.A.72:2535-2539.

Wethak

NGdbumforhihep.17.

Peters,G.,F.Harada,J. E.

Dahlberg,

A.Panet, W.

A.Haseltine,and D.Baltimore.1977.

Low-molecu-lar-weightRNAs ofMoloney murineleukemiavirus:

LITERATURE CITED identification of the primer for RNA-directed DNA 1. Dahlberg,J. E., R. C. Sawyer,J. M.Taylor,A. J. synthesis. J. Virol.21:1031-1041.

Faras, W.E.Levinson,H. M.Goodman,and J. M. 18. Rogg,H.,W.Wehrli,and M.Staehelin.1969. Isolation Bishop.1974.TranscriptionofDNA from the 70SRNA ofmammalian transferRNA.Biochim.Biophys.Acta ofRoussarcomavirus.I.Identificationofaspecific4S 195:13-15.

RNA which serves asprimer. J. Virol. 13:1126-1133. 19. Ross, J., E. M. Scolick, G. J. Todaro,and S. A. 2. Duesberg, P.,K. V. D.Helm,and E.Canaani. 1971. Aaronson. 1971. Separationofmurine cellular and Properties ofasoluble DNApolymeraseisolated from murineleukemiavirus DNApolymerases.Nature (Lon-Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. don) New Biol. 231:163-167.

68:747-751. 20. Sawyer, R.C.,and J. E.Dahlberg.1973.Small RNAs 3. Fan, H.,and M. Paskind. 1974. Measurementofthe ofRoussarcomavirus:characterization by two-dimen-sequencecomplexityofclonedMoloneymurineleuke- sional polyacrylamide gel electrophoresisand finger-mia virus 60 to70sRNA: evidenceforahaploidge- printanalysis. J. Virol.12:1226-1237.

nome.J.Virol.14:421-429. 21.Taylor,J.M.,and R.Illmensee.1975.Site on the RNA

4. Grandgenett,D.P., G.F.Gerard,and M. Green. 1973. ofanaviansarcomavirusatwhich primer isbound. J. Asinglesubunit from avianmyeloblastosisvirus with Virol. 16:553-558.

bothRNA directed DNApolymerase andribonuclease 22. Verma, I. M. 1975.Studiesonreversetranscriptase of Hactivity. Proc. Natl. Acad. Sci. U.S.A.70:230-234. RNAtumor viruses.III.Properties of purified Moloney 5. Grandgenett,D.P.,A.Vora,andA. J.Faras.1976. murineleukemia virus DNApolymerase and associated

Different states of avian myeloblastosis virus DNA RNase H. J. Virol.15:843-854.

polymerase and their binding capacity to primer 23. Verma, I. M., and D.Baltimore. 1973. Purification of tRNA'P.Virology 75:26-32. theRNA-directedDNA polymerasefrom avian

mye-6. Harada, F., R. C. Sawyer, and J. E. Dahlberg. 1975. loblastosis virus and its assay withpolynucleotide

tem-Aprimer ribonucleic acid for initiation ofinvitro Rous plates. MethodsEnzymol.29:125-131.

sarcomavirusdeoxyribonucleicacidsynthesis.J.Biol. 24. Verma, I. M., W. S. Mason, S. D. Drost, and D. Chem.250:3487-3497. Baltimore. 1974. DNA polymerase activity from two

7. Haseltine,W.A.,A.Panet,D.Smoler,D.Baltimore, temperature sensitive mutants of Rous sarcoma virus is G.Peters,F.Harada,and J. E.Dahlberg.1977.The thermolabile. Nature (London) 251:27-31.

interaction oftryptophan tRNA and avian sarcoma 25. Yarus, M. 1972. Solvent and specificity. Binding and virus reversetranscriptase: furthercharacterizationof isoleucylationofphenylalaninetransfer ribonucleic acid thebinding reaction.Biochemistry16:3625-3632. (Escherichia coli) by isoleucyl transfer ribonucleic acid

8. Hirsh, D.1971.Tryptophantransfer RNA as theUGA synthetase from Escherichia coli. Biochemistry suppressor. J. Mol. Biol.58:439-458. 11:2352-2361.

http://jvi.asm.org/