Binding of tryptophanyl-tRNA to the reverse transcriptase of replication-defective avian sarcoma viruses.

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0022-538X/78/0028-0434$02.00/0

Copyright 0 1978 AmericanSocietyforMicrobiology Printed in U.S.A.

Binding

of

Tryptophanyl-tRNA

to

the Reverse

Transcriptase

of

Replication-Defective

Avian

Sarcoma Viruses

AMOS PANET,'* GILDAWEIL,' ANDROBERT R.FRIIS2

Department of Virology, TheHebrew University-HadassahMedicalSchool, Jerusalem, Israel,'and Institut

fur

VirologiederJustus-Liebig-

Universitat,

Giessen,

West

Germany2

Received forpublication6 June 1978

The ability ofreverse transcriptasetobindto[3H]tryptophanyl-tRNA andto

functionas DNApolymerase wascompared for five temperature-sensitive

mu-tants of avian sarcoma virus. Both activities ofthe reverse transcriptase were

found to be heat labile in LA 335 andLA 336 as compared with the wild-type

parents. For theothermutantviruses, LA 338, LA 343, and LA 672,grownatthe permissivetemperature,thereversetranscriptasewasnearlyasheat stableasfor

thewild-type parents intermsof tRNA binding andDNA polymerase.LA 338, LA343, and LA 672 showed characteristic defects in theirreverse transcriptase

whenpropagatedatthenonpermissivetemperature;namely, tryptophanyl-tRNA binding and DNA polymerase activities were coordinately decreased in these

virions. The reduced enzymatic activities were not entirely due to an inactive

reversetranscriptasepresentin thevirions, however, but rather loweramountsof

enzymeprotein incorporated into the virions contributedtotheeffect, according

toassaysofreversetranscriptase antigen by radioimmune competition.

Three activities can be independently mea-sured withpurifiedreversetranscriptaseofavian sarcomaviruses(ASV), namely,DNA polymer-ase(1, 20), RNaseH(13),andtryptophan-tRNA binding (16). Bindingof theprimer tryptophan-tRNA isviewedas aninitiationfunction in pro-viral-DNAsynthesis, whereas DNA polymerase activity with the polyadenylate oligodeoxy-thymidylate [poly(A) oligo(dT)] template is consideredanelongationfunction (19, 23).

Severalreplication-defective ASV have been investigated for theirreversetranscriptase activ-ities(12, 24, 25). Early replication temperature-sensitive (ts)mutantsLA 335and LA 337 were shown to contain heat-labile reverse transcrip-tase for both DNA polymerase and RNase H, thus indicating that these enzymatic activities arecodedby theviral genome. RSV,a,a noncon-ditional defective mutant, has been shown to completelylack the reversetranscriptase protein (8, 15).

In thepresent study, we haveexamined the DNA-polymerase activity and the binding to tryptophanyl (trp)-tRNA of reverse transcrip-tase from five conditional ASV mutants, two mutantscarryinganearly replicationlesion and three mutants defective in a late replication function.

MATERIALS AND METHODS Materials.[3H]tryptophan,11Ci/mmol,and

[3H]-dTTP, 46 Ci/mmol, were obtained from the

Radi-ochemicalCentre,Amersham, England; Sephadex G-100 was a Pharmacia Fine Chemicals product. Rat serumspecificforpurified avian myeloblastosis virus

(AMV) reverse transcriptase was a kind gift from R. Nowinski. Theimmunoglobulin G (IgG) fraction was

purified asdescribed byWatson et al. (26). Chicken liver tRNAwasprepared according to Rogg et al. (18). Chickenliveraminoacyl-tRNAsynthetases were pur-ifiedby using the procedure of Nishimura and Wein-stein (14). AMV reverse transcriptase was purified fromsucrosegradient-banded virus as previously de-scribed(17).

Preparationof[3H]trp-tRNA.The reaction mix-ture(1ml)contained:20mMTris-hydrochloride, pH

7.5; 10mM MgCl2; 10mM KCl;2mM ATP;0.5mM CTP;2mMdithiothreitol;160t,Ciof[3H]tryptophan;

1 mg of chicken liver tRNA; and aminoacyl-tRNA synthetase preparation (260,ug). After30 min of in-cubationat37°C,sodiumacetatebuffer(pH5.0)was addedtoa 0.1Mconcentration; EDTAwasaddedto 25mM,and thetRNA wasextracted three times with an equal volume ofphenol saturatedwith 0.1 M so-diumacetate (pH5). The tRNAwasprecipitated by

adding2volumes ofethanol,and theprecipitatewas washed twice by resuspending in 66% ethanol-33% sodium acetate (0.1 M), pH 5. The final pelletwas dissolved in 10 mM sodium acetate (pH5)-0.1 mM EDTA.A maximumof0.4to0.5%ofthe crude chicken liver tRNA was routinely charged with [3H]trypto-phan.

DNApolymerase assay. Reaction mixtures (100

p1) contained: 50 mM Tris-hydrochloride, pH 8.3; 6 mM MgCl2; 10 mM dithiothreitol; 10 Lg of bovine serum albumin; 10 MM [3H]dTTP (specific activity, 450cpm/pmol); 0.5 ,g ofpoly(A) oligo(dT); and 20-434

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werefor 30minat37°C. Reactionswerestopped with

5% trichloroacetic acid, and radioactive precipitates

were collected on glass fiber filters. A unit is the

amountofenzymeneededtocatalyzethe incorpora-tion of1pmolof[3H]dTMPin 1minunder standard conditions.

Binding assay. Reaction mixtures (100 Ml)

con-tained: 50 mM Tris-hydrochloride, pH 7.5; 60 mM NaCl;6mM MgCI2; 10 Mgof bovine serumalbumin;

0.2%Triton X-100;5mMdithiothreitol; 16,000cpmof

[IHItrp-tRNA

(unfractionated) (1.3 pmolof [3H]tryp-tophan);andpurifiedvirusasindicated.After 10min ofincubationat22°C,themixtureswereloadedonto Sephadex G-100 columns (0.6 by22 cm) and eluted with buffer A (0.1 M potassium phosphate, pH 7.1; 10% glycerol; 0.1% Triton X-100; 10 mM mercapto-ethanol). Fractions of 0.18 ml were collected; 20-ML

samples were taken for assay of DNA polymerase

activity,and therestwerecounted for[3HItrp-tRNA

in toluene scintillation fluid containing30% Triton

X-100.

Cells and viruses. Chicken embryo cells were

preparedfromfertile whiteleghorn embryosobtained from Heisdorf and Nelson Farms, Redmond, Wash., and tested for the expression ofchick helperfactor according to the procedure of Friis et al. (5). Eagle Dulbecco-modified medium supplemented with 10% tryptosephosphate broth, 5% calfserum, and 1% di-methyl sulfoxidewasusedforvirusstockpreparation.

Chickenhelper factor-negative chicken embryo cells

wereinfectedwiththevarious virusesin thepresence

of 2 MgofPolybrene(Aldrich Chemicals) perml(21), andcultures were maintained withregular transfers

for 10to14daysuntil 30to40 dishes(90 mm)of each wild type or mutantvirus stockhad been prepared. Harvests of virus were then made, usually at 24-h intervals, but,asreportedbelow forts-672,

occasion-allyat 3-h intervals,from cultures maintained

sepa-rately at 35and 42°C. Cultures maintained at42°C

wereharvestedina warmroom on a warmtable held

at42°C. Pooled culture supematantswerefrozen at -70°Cuntilavolume of 3to5 literswasavailable,at whichtime viruswaspelletedinaSpincotype19rotor (19,000rpmfor 2h).All thevirusestested inthis work

were purifiedunder identical conditionsby gradient

purificationoncontinuous 20to50%sucrosegradients.

Biologicalcontrolswereperformedwithallvirus

prep-arationstodemonstrate thatthestocks exhibited the typical temperature-sensitive properties of the

mu-tantsand thecorrectsubgroup.

Thefollowing mutants obtained from the Prague strain (PR) ofRoussarcoma virus (RSV) havebeen used:tsLA335,subgroupC(10);tsLA338, subgroup C (27); ts LA 343, subgroupC (27); and tsLA 672, subgroup A (3). For control purposes, wild-typePR RSV,subgroup C (wt PR-C), has beenused. TS LA 336(formerlycalledts149)(6, 22)wasalsoinvestigated

and its parent, wild-type ASV strain Bratislava 77, subgroupC (wt B77), wasused for controlpurposes.

RESULTS

Binding of trp-tRNA to reverse

tran-scriptase usingdisruptedvirions. In

previ-ousstudies, the interaction of the primer

[32P]-tRNAtP and AMV reverse transcriptase has been demonstrated, using purifiedenzyme prep-arations (7, 16). To be able to measure tRNA complex formation with the enzyme of ASV temperature-sensitive mutants, we sought con-ditions for binding in crude virionextracts. Fig-ure 1demonstratesthat[3H]trp-tRNA bindsto reversetranscriptase in detergent-disrupted vir-ions aswellas to a purified AMV reverse tran-scriptasepreparation. Complexformationis ev-identby the comigration of virion DNA polym-erase and part of the [3H]trp-tRNA, with a sedimentation coefficient of 9Songlycerol gra-dients. Under the same conditions, unbound purifiedreversetranscriptase (not shown) hasa sedimentationcoefficient of 7.2S(150,000 molec-ular weight), similar to that of the protein marker IgG (16).

Toexclude the possibility that[1H]trp-tRNA

binds indetergent-disrupted virionstoproteins otherthan thepolymerase and formsacomplex withasedimentation coefficient similartothat of the reverse transcriptase, we investigated whether known inhibitors oftheviralDNA po-lymerase activity also inhibit tRNA complex formation in disrupted virions. In these experi-mentsbindingof [3H]trp-tRNAwasassayedby

gelchromatography onSephadex G-100, where theenzymecomplex migrates inthe voidvolume (fractions 13 to 15), while unbound [3H]trp-tRNAelutesasthe secondpeak (fractions20to 30)and free[3H]tryptophan,adeacylation prod-uctfromtRNA, is retardedtofractions30to 40 (Fig. 2). Preincubation of detergent-disrupted virions withmonospecific antibody, produced in rats against purified reverse transcriptase (26), orwith N-ethylmaleimide (NEM), apotent in-hibitor ofDNApolymerase and tRNA binding activities in purifiedenzyme preparations (15), abolished both activities in crudeextracts (Fig. 2).These resultssuggestthat of all viralproteins only thereversetranscriptase is able to forma specific and stable complex with trp-tRNA.

Inthiswork[3H]trp-tRNA rather than [32P]-tRNAtr"Pwasusedtoquantitate complex forma-tion withreversetranscriptase, andwefind sev-eral advantages in using the aminoacylated tRNAasasubstrate:(i)specific tagging of crude tRNA preparationwith [3H]tryptophan is easy and does notrequireuseof two-dimensionalgel electrophoresis, which is necessary for the puri-fication of

[32P]tRNA"rP

from a mixture of tRNA's; (ii) the isotopein[3H]trp-tRNA is sta-ble as compared with the in vivo labeled

[32P]-tRNA, and specific activities of [3H]trp-tRNA

preparations are accurately assessed. The one

disadvantageinusing[3H]trp-tRNAisthe rela-tiveinstabilityof theaminoacylesterbond. We therefore ran glycerol gradient centrifugations

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436 PANET, WEIL, AND FRIIS

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FIG. 1. Binding of trp-tRNA in disrupted virions. Bindingof [3H]trp-tRNAwasconductedasdescribed in

thetext,except50mMpotassiumphosphate buffer, pH6.0,wasusedinstead of Tris buffer. After incubation

for10minatroomtemperature,internal protein marker, humanIgG (0.6mg),wasadded,andthemixtures

werecentrifuged for 30 minat5,000xgtoremoveprecipitates. Theclearsupernatantswerelayeredon5-ml

glycerol gradients (5to25%, vol/vol) in 50 mMpotassium phosphate(pH6.0),0.1MKCI,0.1%TritonX-100, 10mM/-mercaptoethanol, and 20% ethylene glycol. Gradientswerecentrifuged for23hat49,000rpminan

SW50.1rotorandfractionated. DNA polymerase activityand trp-tRNA bindingwereanalyzedasdescribed

in thetext, andthelocation of IgG proteinmarkerwasdetermined by assaying the proteincontentinthe fractions after trichloroacetic acid precipitation,accordingtoLowryetal.(11). (A)wtPR-A virions,340pg;

(b)purified AMVreversetranscriptase,100enzymeunits.

and gel fitration columns under conditions

which minimized deacylation of aminoacyl-tRNA,namely, low-pH phosphate buffer.

In all experiments described in this

commu-nication, saturating amounts of [3H]trp-tRNA

were added, and thereforeonly 15% of [3H]trp-tRNA bound to the enzyme (Fig. 2A). Under

conditions of suchtRNA molar excess,any

re-duction of the enzyme-binding activity should

bedetectable,thusenablingustomeasure

bind-ing activityperagiven amount of virus. When

anexcessofvirus,rather thantRNA,wasused in the binding reactions, as much as 35% of

[3H]trp-tRNA bound to the enzyme in crude

virus extracts (results not shown). A similar percentage of binding has been previously

re-ported, using a purified preparation of

[32P]-tRNAtrP (16). No structural differencebetween bound and unbound fractions has as yet been

detected, and it could be that conformational

isomersoftRNAt'rPexplain the two

fractions.-trp-tRNAbindingin LA 335 and LA336 mutants. Temperature-sensitive mutants LA 335 and LA 336 have been shown to be early replication-defective mutants which contain

heat-labile reversetranscriptase (6, 10, 12, 25).

The enzyme in these mutants is defective for

both DNApolymeraseandRNase H(12,24,25). The results shown inTable 1demonstratethat

for both mutants, DNA polymerase activity, measured with poly(A) oligo(dT) template, is

heat labileascomparedtothewild-type viruses

RSV PR-C and ASV B77. Thus, in agreement with theresults of Vermaetal. (25), preincuba-tionat45°C resultedin50%inactivation ofthe

polymerase activity with detergent-disrupted virions of LA 335 and LA 336 after4and2min, respectively.

Since the specific binding of the primer tRNA11' represents yet another activity of the

enzymeprobably needed for initiation of DNA

synthesis onthe viral RNAtemplate, it wasof

interesttocomparethe heatlabilityof the

bind-ing site tothat of the polymerasesite. [3H]trp-tRNA binding activity was estimated, after

preincubation ofdisrupted virionsat45°Cfor 30

min, by gelfiltration of thecomplexformed.In

thecontrolreactions,reversetranscriptaseofwt

PR-C, LA 335, and LA 336 was shown to be

active in binding,as is evidentby the

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FIG. 2. Inhibitionofreversetranscriptasebinding activityindisruptedvirions. VirionsofwtPR-A(340 pg)werepreincubated inbindingreactionmixtures,

with 0.1 mMdithiothreitol:(A)withoutadditions;(B)

with ratIgGmonospecificforAMVreverse transcrip-tase(12pg);(C)with 2 mMN-ethylmaleimide.After

20min, dithiothreitol(10mM) was addedtodestroy

excessNEM,followed byadditionof[3H]trp_tRNA

(16,000 cpm). The mixtures were chromatographed afteranadditional 10minonSephadex G-100 col-umnsasdescribedin thetext.

matography of[3H]trp-tRNAand the DNA po-lymerase activityonSephadexcolumns(Fig. 3A, C, and E). Preincubation at high temperature

hardlyaffected thewtPR-Cactivities,whereas both trp-tRNA binding and DNA polymerase

activitiesin mutants LA 335 and LA 336 were completelyinactivated (Fig. 3B,D,andF).

Heat sensitivityof DNApolymerase and

tRNA binding activities with LA 338, LA 343, and LA 672. The three temperature-sen-sitive mutants LA 338, LA 343, and LA 672 exhibit a commonproperty;all carrylate repli-cation defectswhichdetermine that virions pro-duced at the nonpermissive temperature are

noninfectious (3, 27). Other important differ-ences emerge between LA 338and LA 343,on

the one side, and LA 672on the other. LA338

and LA 343clearlycarry severalgeneticdefects

sensitive ASV DNApolymerases

Half-life (min) for DNA polymer-Virus aseactivity after preincubation at

45°C"

wtPR-C 80

LA 335 4

LA338/350C 40

LA338/42°C 60

wt B77 60

LA336 2

"Mixtures (100

pl)

contained 50 mM

Tris-hydro-chloride,pH 8.3,6mMMgCl2,10,ug of bovineserum albumin, 0.02% Triton X-100, 5 mMdithiothreitol, and virions asfollows: B77 and wt PR-C, 32 enzyme units; LA 336, 25.4 enzyme units; LA 338 grown at 35 or 42°C, 33 enzyme units; LA 335,30enzymeunits. The mixtures wereincubated at45°C, and 10-Mul samples

weretaken into ice. Residual DNApolymerase activity was assayed by adding 90,l of assay mixture as described in thetext.

(2, 9), oneof which affects transformation inde-pendent of virus replication. The other is re-sponsible for, in addition to the late defect in replication, an early defect, first described by Wyke and Linial (27) as a transient essential function for the establishment of transforma-tion. Recentexperiments with LA338 (Molling

and Friis, manuscript in preparation) indicate that this early defect, which affects replication, is indeed only a separate phenotype caused by a reversetranscriptase lesion, perhaps thesame whichdetermines the late defect. Hence,for LA 338, a reverse transcriptase lesion probably

causes both an early and a late defective phe-notype. LA 338harvestedat35°C also exhibited in ourexperiments asignificantly reduced

half-life compared withwtPR-C after incubation at

45°C. LA 672 apparently has only a single ge-netic defect,thataffectingreversetranscriptase (4). Mostinterestingwith LA672is the fact that thereversetranscriptasedefect resultsonlyina

late defective phenotype; hence, cells infected withstocksof LA672preparedatthepermissive

temperaturedonotexhibitany temperature

sen-sitivityin the initial establishment of infection. The noninfectious particles of these mutants havethesamestructuralpropertiesasinfectious

virus, and all themajorstructural proteinsare

present in normal amounts (4). We have studied twoproblems with these mutants. (i) Does the reversetranscriptasecontaina temperature-sen-sitivelesion which effects the initiationof DNA synthesisasmeasuredbythetRNAprimer bind-ingactivity,and (ii)is the reducedDNA

polym-eraseactivityin virionsproducedatthe

nonper-is.sive

temperature (42°C) due to an inactive enzymeor to areducedamountofactivereverse

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FIG. 3. Inactivation oftrp-tRNA binding activity in LA 335 and LA 336. (B, D, and F) Virions were

preincubatedinbindingmixtureathightemperature(450C)for30min, and[3HJtrp-tRNAwasthen added.

(A, C,andE)Bindingwasconductedwithoutpreincubationasdescribed in thetext. (AandB)wtPR-C(330 pug); (CandD)LA335(325pig);(EandF)LA 336(330

pg).

transcriptase assembled into virions?

The observation ofVerma etal. (25) that in vivo proviral DNA synthesis is reduced when infection with LA338is carriedoutatthe

non-permissive temperature, even though in vitro DNA polymerase activity is heat stable, prompted us toinvestigate whether the initia-tion of DNAsynthesis,asmeasuredbythe trp-tRNAbindingactivity,istemperaturesensitive inthis virus. The results shown inFig.4indicate that thetRNAbindingactivityofreverse

tran-scriptase from LA 338virions, grown at either thepermissive(350C)orthenonpermissive tem-perature (420C),isnotthermolabile. Disrupted virionswere preincubated athigh temperature (45°C) or left at

40C

for 30 min, followed by addition of [3H]trp-tRNA and analysis by gel

filtration. The enzyme in LA 338grown at the permissive temperature andlater preincubated

at

450C

canbindasmuch[3H]trp-tRNAasthe same enzyme which was kept at

40C

(Fig. 4A andB). Similarly, trp-tRNA binding to nonin-fectiousvirions,producedat

420C,

is heatstable,

and preincubation at 45°C for 30 min did not

affect thebinding activityof thatreverse

tran-scriptase (Fig. 4CandD). Preincubationof dis-rupted virions at 45°C resulted, however, in a

small decrease in the total amount of DNA

polymerase eluted from the Sephadex column (see alsoTable 1).

To investigatethe biochemical nature of the latereplication lesion in LA338, wequantitated the DNApolymerase and tRNAbinding activi-ties in virions madeatboth35and

420C.

Agiven amount of virionsproduced at

350C

bind four-fold more trp-tRNA as compared with virions madeatthenonpermissive temperature

(420C)

(Fig. 4A andC; Table2). Asimilarly enhanced (three-tofourfold) specificactivity of DNA po-lymeraseisnoticed in virionsproduced at

350C

ascomparedwith those madeat

420C.

The total DNApolymeraseand[3H]trp-tRNAbinding ac-tivities eluted from the columns (Table2)were calculatedfromtheresults showninFig.4Aand Cbyintegratingtheareaunder the voidvolume peaks.

LA 343 wasalsoanalyzedforitsreverse tran-scriptase properties (Fig. 5). The virions pro-ducedatthenonpermissivetemperature

(420C)

show a fourfold-lower specific activity for trp-tRNA binding compared with virionsgrown at

350C,

and the DNApolymerase specific activity

in the noninfectious virus was reduced by a factor of2.5.Wild-type PR-C producedateither

42 or

350C

areboth infectious and havespecific activities for tRNAbindingand DNA

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FIG. 4. trp-tRNA binding and DNA polymerase activities in LA 338. Binding reactions were

con-ductedasdescribed in thetext.(A)LA338,grownat 35°C (330pg), and(B) thesamevirions were

prein-cubated in thebindingmixturefor30min at45°C before[3Hltrp-tRNA wasadded. (C) LA 338grown

at42°C (330pg)and(D)thesamevirionswere

prein-cubated, for30 min at45°C, before ['HJtrp-tRNA

wasadded.

asesimilartothemutantvirus LA 343produced

at 350C (Fig. 5A). The results of these two

independentassaysfortRNAbinding and DNA polymerase suggest that the late

temperature-sensitive lesions in LA 338 and LA 343 affect the amounts ofactive reverse transcriptase in

virions producedatthe nonpermissive temper-ature.

LA672alsoappears tobe defective ina late

stageofreplication in the synthesisorassembly

ofafunctionalreversetranscriptase (4). We have

compared the tRNA bindingaswellastheDNA

polymerase activities in infectious virions

pro-duced at 350C and noninfectious virionsmade at420C. Suchastudy givesusinformationasto

whether the noninfectious virions of LA 672

activities and phenotypesofLA672,LA 338, and LA 343propagated at permissive and nonpermissive

temperatures

DNApo- [' fl

lymer- ]trp- Relative Ei-Virus ase" (en- _binding'tRNA amt of ciencyof

realica-zyme (pmol) antigen'

tionrl

LA338/350C 16 0.64 0.36 1

LA338/420C 5 0.15 0.19 0.08

LA343/350C 23 0.94 0.5 1 LA343/420C 9 0.22 0.14 0.07 LA672/350C 4.0 0.55 0.45 1 LA672/420C 0.7 0.15 0.09 0.0025

aTotal DNApolymeraseactivityeluted from Seph-adex G-100columnswas calculated bysumnmingthe enzyme activity in the void volume(usuallyfractions 13 to 18inFig. 4, 5, and 6).

b[3H]trp-tRNA bound is the sum of radioactivity

co-chromatographedwith theenzyme in the void vol-ume(usuallyfractions 13 to 18). Virion protein input wasaround 330,ug (see legends to Fig. 4, 5, and 6).

'Relative amount of reverse transcriptaseantigen in virions wasderived from amount (micrograms) of viralprotein needed to restore 20 or 30% of the DNA polymerase activity in the immunoassay (Fig. 7). At 20 to30% of enzyme activity the competition immu-noassay is linear, and therefore the amount of

com-petingvirus needed torestore thisactivity is inversely linear to the virus content of reverse transcriptase

antigen.The amount ofantigeninwtPR-Aor PR-C wasadjustedto 1unit, and the relative amounts of ts mutants wereaccordingly corrected.

dEfficiency ofmutant replication was calculated from the titers of infectious virus released to the medium at 35 and 42°C. The focus assay wascarried

outunder standard conditions (2, 3).

contain enzyme protein which has lost DNA polymerase activity but still retains its tRNA

binding activity. The results illustrated in Fig.

6Aindicate thatinfectious virusproducedatthe

permissivetemperature (35°C) containsa high

tRNA binding activity. An equal amount

(mi-crograms of protein) of noninfectious virions

(produced at 42°C) binds threefold less trp-tRNA but exhibits a sixfold reduction in DNA

polymerase activity as compared with LA 672 producedat35°C(Fig.6; Table2). The standard LA 672virions usedinthisstudywereharvested every 24 h and then purified. To exclude the possibility that the virions produced at 42°C were inactivated due to the high temperature afterreleasefromcells,wealsoinvestigatedthe reverse transcriptaseinrapidly harvested (3h)

virus. A rapidly harvestedvirus grown at42°C

(Fig.6C)behavessimilarlytothe24-h-harvested virus (Fig. 6B), and its polymerase and tRNA binding activities are both reduced compared

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FIG. 5. trp-tRNA binding and DNA polymerase activities in LA 343. Binding of[3HJtrp-tRNA was carriedout asdescribed in thetext. (A)PR-C grown at420C(300pg);(B)LA343grownat35°C (325ug);

(C) LA 343 grown at420C (327 pg).

with virusgrownatthepermissivetemperature,

35°C (Fig.6A). Allof these results indicate that

qualitatively the lesion in LA 672 is similarto

the late temperature-sensitive function in LA 338and LA343.

Quantitationofreverse

transcriptase

an-tigen in

temperature-sensitive

ASV

mu-tants.Thefmdingof reducedDNApolymerase

andtrp-tRNAbindingactivities in ASVmutants

LA 338, LA 343, and LA 672 grown at the nonpermissive temperature could beexplained

in either oftwoways: (i) packaging ofenzyme into virions is a temperature-sensitive process, and virions produced at 420C contain less

re-verse transcriptase protein;or (ii) enzyme syn-thesized in thecellsathigh temperatureis de-fective, and,as aresult, virionscontain normal amountsofenzymeproteinwithlowenzymatic activities. To differentiate between these two

possibilities, wemeasured the relative amounts

of reverse transcriptase antigen in virions by using a competition immunoassay (15).

Deter-gent-disrupted virions are reacted with NEM,

which inhibits in a nonreversible fashion the DNApolymeraseactivitybutdoesnotaffect the

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10 20 30 40

FRACTION NUMBER

FIG. 6. trp-tRNA binding and DNA polymerase activities in LA 672. Conditionsfor binding and DNA polymeraseactivityaredescribed in thetext.(A) LA

672,grownat35°C,harvestedat24-h intervals(345

pg); (B) LA 672,grown at42°C, harvested at 24-h intervals (345pg); (C) LA 672,grownat42°C, har-vestedevery3 h(360 pg).

antigenic properties of thereversetranscriptase,

andexcessNEMisdestroyed bydithiothreitol.

Increasingamountsof theNEM-treatedvirions

arethen reacted withalimitedamountof

anti-reverse transcriptase IgG. The ability of the

mutantvirusestoabsorb the monospecific IgG

canthenbeestimated by adding purified active reversetranscriptase toreactwith the free IgG, and finally the residual reverse transcriptase

activity is estimated with poly(A) oligo(dT) as

template primer for DNA synthesis (Fig. 7). Virions whichcontainalargeamountofreverse

transcriptase antigen will absorbmore

antibod-ies, and as a result less ofthe purifiedreverse

transcriptase willbeinactivated inthe final

re-action. The neutralization inhibition method used hereprovedtobe asaccurateasthe

stan-dard competition radioimmunoassay technique

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FIG. 7. Neutralization inhibitionofDNApolymerasein RSVtsmutants. The reactionswereconductedin

foursteps. (i)DNApolymeraseactivity in virionswas inactivated by incubationwith 2 mMNEM, 50mM

Tris-hydrochloride (pH7.5), 1.0 mgofbovine serumalbuminperml, and1.0% TritonX-100.After15mm at 37"C, the tubes were transferred to40C and dithiothreitol (7mM) was addedto destroyexcessNEM. (ii) Increasingamountsofvirus-NEMwerereactedfor20minat370Cwithanti-reversetranscriptaseIgG (3 pg)

in mixtures (45 gil) containing 10mM Tris-hydrochloride (pH 8.0), ffXJ mMKCI, 0.5 mg ofbovine serum albumin per ml, 10%glycerol, and 0.1% Triton X-100. (iii) ActivepurifiedAMvVreverse transcriptase, 1.5

units/S

[LI,

was added, and incubation was continuedforanother 20 min at370C. (iv) The residual DNA

polymerase activitywasdeterminedby addingamixture(50Md) ofall thecomponentsneededfor assaying

DNApolymerase activity (see text), andthe incubation time was20min. Under the above conditions, the

enzymaticactivitywaslinearforatleast 40min. Thepercentageofresidual DNA polymerase activity was calculatedbycomparisonwith control reactions in duplicate tubes that contained no immune IgG (taken as 100%).

for reverse transcriptase (15). Thus, torestore 20% of theprobe DNA polymerase activity, 55

,ug

of LA 672 virions grown at 42°C, 11 ,tg of virionsgrown at350C,and5

,ug

ofwtPR-A virus were needed (Fig. 7A). These results indicate that LA672virusgrownat420C contains about fivefold less enzyme protein as compared with thesamevirusgrownatthepermissive temper-ature(350C).LA338and LA343 mutantsshow a similarlack, and virionsproducedatthe

non-permissivetemperaturecontaintwo-tofourfold lessreversetranscriptaseascompared with virus madeat35°Candsomesixfold lessenzymethan thewtPR-C (Fig. 7BandC;Table2).

All of these results indicate that the three conditional mutantsanalyzed, LA 672, LA 338, andLA 343,contain reducedamountsofreverse

transcriptase protein per total virus proteins

whenharvested atthenonpermissive tempera-ture.Itisinterestingtonote,however,thateven

the infectious virus mutants produced at the permissivetemperaturealways contain less

en-zyme protein than the wild-type parents. Whereas the reduced incorporation of reverse

transcriptaseinto virions canexplainlower DNA polymerase and trp-tRNA binding for LA 338 and LA 343 grown at

420C,

LA 672 grown at

420C

probablyincorporatessignificantamounts

ofdamagedenzyme,since the trp-tRNA binding andDNApolymerase activities for these virions grownat

420C

are notparallel.

DISCUSSION

Inthepresentstudywe describe the interac-tion betweenthe tRNAprimerfor DNA

synthe-sisand thereverse transcriptase of five temper-ature-sensitive mutants of ASV. The finding that trp-tRNA binding activityin LA 335 and LA336 is thermolabile provides the best proof that this function is virus coded like the DNA polymerase and the RNaseHactivities (24, 25). The active sites for these three activities are apparentlyapartof thesamegeneproduct since they are coordinately affected by single muta-tions. Biochemical studies, however, show only littleoverlappingof the three active sites. Thus, each of the three activities can be separately inhibited by different chemical reagents (Go-recki andPanet, manuscriptinpreparation).

Our findings with the late replication-defec-tive mutants indicate that in terms of DNA polymeraseandtrp-tRNAbinding, reverse

tran-scAiptaseis either not atall (LA 343 and LA 672) or only slightly (LA 338) thermolabile. In fact, the lesions responsible for the phenotype

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442 PANET, WEIL, AND FRIIS

served need not necessarily be in the genetic

elementcodingfor theviralreversetranscriptase

(pol) butcould be locatedin anunknown func-tion essential for correct virus assembly. More

probably, however, defective reverse transcrip-tasemoleculesarethemselvesimpededinsome kind of self-assembly process, and the actual defectsarecausedbylesions inpol. Thefindings

presented inFig.7andTable 2, inany case, go alongwayinexplainingthe lack ofinfectivityof virusisolated from cultures maintainedat42°C.

Certaincomplications remain, however,if the late defective mutants are to be explained

en-tirelyonthe basis of lack ofreversetranscriptase incorporation. First,there is theinterestingfact thatwhereasspecificactivityof DNA

polymer-aseobserved with LA672isolated from cultures at42°C was reduced sixfold(andmore insome experiments), trp-tRNA binding by the same viruspreparationswasless reduced. Thisfinding

was consistently observed in repeated experi-ments.Hence,for LA672,the fractional

comple-mentofreversetranscriptase,whichdoes incor-porateintovirionsgrown at42°C,seems tohave retained more trp-tRNA binding activity than DNA polymerase activity. This would point

quite definitely to a pol lesion. Furthermore,

analyses of recombinants made between the avian leukosisvirus, RAV-6, and LA672 (Joho, Stoll, Friis, andWeissmann,unpublished obser-vations) showed that T, RNase-resistant

oligo-nucleotidesdirectlyassociatedwith the temper-ature-sensitive phenotype were those which mapped in theregion defined ascodingforpol. Likewise, LA 338 can be considered a likely

mutant inpol. A

significantly

shorter half-life for inactivation ofthereversetranscriptasewas

noted with thismutant (Table 1),regardlessof whether virus preparationshad beengrown at 35 or 42°C. Since this mutant shows an addi-tionalveryleaky, early, transientdefect (27), it may well be speculated that there is a slight

(leaky) early pol-related defective phenotype.

Evidence for this earlydefect can be found in thethermolabilityreferredtoabove and in sim-ilar recent experiments showing temperature-sensitive behavior of LA 338 in terms of both DNApolymeraseand RNaseHactivities

(Moll-ing andFriis,manuscriptinpreparation). The amountofenzyme innoninfectious viri-onsofLA 338, LA343, and LA 672 produced at the nonpermissive temperature is three- to six-foldlower thanin the samevirusesproducedat the permissivetemperature. The amount of re-verse transcriptase per wtASV virion was cal-culated by radioimmunoassay to be 30 to 70 molecules (15). Asixfold reduction would leave 5 to 12 enzyme molecules in mutant virions

produced atthe nonpermissive temperature. It isinteresting thatthereductionininfectivity of mutantviruspreparationsgrown atthe nonper-missive temperature is 100- to 500-fold. This disparity between loss ofenzyme and reduced infectivitymaybeseen asevidence that infectiv-ity requires more thanjust a few molecules of reverse transcriptase per virion, or it may be interpretedtosuggestthat, indeed, LA 338,LA 343, and LA 672 incorporate notjust reduced amounts ofreverse transcriptase, but also con-tain molecules of lowerspecific activity.

ACKNOWLEDGMENTS

Theencouragement ofN.Goldblum isgratefully acknowl-edged. This workwasinitiated whileoneofus(A.P.) was the recipient ofanEMBO short-termfellowship.

This workwassupported bytheStiftung Volkswagenwerk and the Israel Academy ofSciences, and in part by the Sonderforschungsbereich47(Virologie)ofthe Deutsche For-schungsgemeinschaft.

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1976.Temperature sensitive mutants of avian sarcoma

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