Effect of Temperature-Sensitive Mutation on Activity of the RNA Transcriptase of Vesicular Stomatitis Virus New Jersey

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0022-538X/79/06-0692/09$02.00/0

Effect of

Temperature-Sensitive

Mutation on

Activity

of

the

RNA

Transcriptase

of

Vesicular Stomatitis Virus

New Jersey

J. F. SZILAGYI* AND C. R. PRINGLE

Medical Research CouncilVirologyUnit,Instituteof Virology, University of Glasgow, Glasgow Gll 5JR, Scotland

Received forpublication23January 1979

The virion-associated RNA transcriptase activity ofvesicularstomatitis virus New Jersey temperature-sensitive (ts) mutants was assayed in vitro at the permissive (3100) andrestrictive (3900) temperatures. RNA synthesis at390C

bytheRNA-negativets Al andtheRNA-positive tsCl andtsDl mutants was

similar to that ofwild-typevirus. The RNA-negativetsBlsynthesized only small amountsof RNA in vitro at390C.The three mutants ofcomplementationgroup E were dissimilar in the amounts of RNA they synthesized at 390C: ts El synthesized very little RNA, ts E2 synthesized moderate amounts, and RNA synthesis by ts E3 was not inhibited. The two mutants of group F were also

dissimilar, since ts Fl synthesized very little RNA at 390C, whereas ts F2

synthesized asmuch RNA aswild-typevirus. The revertant clones ts Bl/Rl, ts El/Rl, and tsFl/Rlsynthesized RNA at

390C

in amounts comparable to

wild-typevirus, indicatingthat the heatsensitivity of thetranscriptaseactivity of the

mutants ts Bl, ts El, and ts Fl was associated with temperature sensitivity.

Similarheat sensitivities were observed when transcribing nucleoprotein

com-plexeswereused in the assays,showingthat the mutatedpolypeptideswerepart

of the viral core. The heatstability of the mutant ts Bl was similar to that of wild-type virus, and in vitro RNA synthesis wasfullyrestored when the temper-aturewasloweredto 310Cafter 30 min ofpreincubation at390C, showing that the inhibition was due to reversible configurational change of the mutated

polypeptide.When virions ofthemutant tsElwereheated for5hat

390C,

their

infectivity and transcriptase activity were as stable as those of the wild-type virus, whereastranscriptaseactivitybecame very heat labile afterdisruption of the viral coatwith a neutraldetergent. This suggests an interaction between the mutated

polypeptideanda coatpolypeptidewhich stabilizes theactivityof the

transcrip-tase. The RNAtranscriptase activity of themutant tsFl was also heatlabile,

althoughto alesserextentthanthat of tsEl.Thus, the defects intranscriptase

activityof groupsB,E, and F suggest thatall threepolypeptidesofthe virus core,

polypeptides L, N, and NS, are involved in the transcription. In addition, we

postulatethat themutated gene products of groups E and Faremultifunctional,

being requiredboth intranscriptionandreplication,and that the geneproductof

group E mayalso be involved insomelate stage of virusdevelopment. Temperature-sensitive (ts) mutants of

vesic-ular stomatitis virus (VSV) Indiana have been classified into six non-overlapping

complemen-tation groups (3, 4, 13, 19). At the restrictive temperature

(390C),

mutants of

complementa-tion groups I and IV do not synthesizevirion

RNA, whereasmutantsofgroups IIIandV do (15). GroupHisuniqueinthat it contains mu-tants of both RNA-negativeand RNA-positive phenotypes (15). Theviriontranscriptase

activ-ity of several tsmutants was comparedin vitro

at 31 and

390C

by Szilagyi and Pringle, who

found thatmutants ofgroupsII, III,and IVall synthesized RNA at

390C,

indicating that in

these groups the RNA transcriptase was not

affectedby the mutation (20). However, several

mutantsin group I did notsynthesize RNA in

vitroat390Cbecause of the heatlability oftheir

transcriptase (20).Studies ofprimaryRNA

tran-scriptionininfected cellsconfirmed thatin

com-plementation group Ithe mutation altered the

activity of the RNAtranscriptase (18).

Dissocia-tion and reconstitution experiments indicated

thatpolypeptideL wasthe siteofthe mutational lesion(2, 5, 6).Recentlyweexaminedmutant ts

045 from the RNA-positive complementation

group V (17,24) and mutant ts M602from the

RNA-negativegroupVI(7,8,19)and foundthat

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VOL. 30, 1979

bothmutantssynthesizedRNA in vitro at390C, indicating that the transcriptase activityin these

groupsis not affected by themutation (unpub-lished data).

ts mutants isolated from VSV New Jersey have also been classifiedinto six non-overlap-ping complementationgroups by Pringle etal. (16). Complementation groupsA, B, and F did not synthesize virion RNA at the restrictive temperature(3900),whereasmutantsofgroups

C and D did (16). Complementation group E

contained both RNA-negative and RNA-posi-tive mutants, since two of themutants (tsEl and ts E3) did not synthesize virion RNA at

39°C whereas the third mutant (ts E2) did (16). Lesnaw and Reichmann usedone mutantfrom each of the sixcomplementationgroups toshow that ininfected cellstwo of the RNA-negative

mutants, tsAlandtsEl,wereableto carry out

primary transcription but not replication of vir-ionRNA at39.50C (10). The RNA-positive

mu-tants tsCl andtsDl synthesized virion RNAat

the restrictive temperature, whereas mutants from the othertwoRNA-negative

complemen-tationgroups(tsBlandtsFl) exhibited neither transcription nor replication (10). Recently

Les-naw and Dicksonhave carried out in vitro dis-sociation and reconstitution experiments which indicate that the mutational lesion in ts Bl involves the L or NS polypeptides (9).

Conditional temperature-dependent (td CE) mutantshave been isolated which grow both at 31and 390C in BHK cells but only at 310C in secondary chicken embryo cells (14, 21). Some of thetd CE mutantsof VSV New Jersey syn-thesize RNAinvitro at390C similarly to wild-typevirus, while others do not (21). One mutant of the latter category, td CE3, was studied in

somedetail, and it was shown that the activity

ofitstranscriptase was fully and instantaneously reversible bylowering the temperature from 39

to310C (21). Thus, the mode of inhibition of the

transcriptase of this mutant differs from that

observed in some of the group I mutants of VSV

Indiana, where thermal inactivation was

irre-versible (20). Dissociation and reconstitution ex-periments showed that the td CE3 mutation affectedpolypeptide L(22).

Inthiscommunication we examine the

kinet-ics of in vitroRNAsynthesis by purified virions

of the conventional ts mutants of VSV New Jerseytodetermine whether virion transcriptase activity was defective in any of the six

comple-mentationgroups.

MATERIALS AND METHODS

Mutants. The tsmutantsof VSVNewJersey de-scribedby Pringleetal.(16)wereused. We choseone

mutantfrom groupsA, B,and C (ts Al,tsBH,andts C1), the onlymutant of group D (ts D1), allthree mutantsof group E(tsEl,tsE2,andtsE3),and both mutantsof group F (ts Fl andtsF2). We also isolated revertantclones frommutants tsBi,tsEl,andtsFl. These revertants(tsBl/Rl,tsEl/Ri,andtsFl/Ri) were isolated from BS-C-1 monolayersincubatedat 390C.

Growthandpurificationof virus.BHK-21clone 13monolayersin 6to 10Burrler bottleswereinfected withlow multiplicity of themutantsand incubatedat

310C. Growth andpurification followed the method describedby SzilagyiandPringle (20). Thepurified virus wassuspendedin 20 mMTris-hydrochloricacid buffer (pH 8.0), the amount of the protein in this purified preparationwasdeterminedbythe method of Lowry etal. (11), and the concentration ofproteinin eachsuspensionwasadjustedto1.3mg/ml. Infectivity assayswerecarriedoutunder agaroverlay using BS-C-1 cellmonolayers.

Thermalinactivation ofinfectivity.Purified vi-rus preparations (0.005ml)weresuspendedin1mlof Eagle mediumcontaining 10% fetal calfserum and wereincubatedat390Cfor 5 h. The residualinfectivity of thesesuspensionswastitrated at310ConBS-C-1 cell monolayers, andresults areexpressed as a per-centageof the infectivityofidenticalsampleswhich werekept at00Cfor thesamelengthoftime.

TNP preparations. Transcribing nucleoprotein (TNP)complexeswerepreparedfrompurifiedvirions bythe method ofSzilagyiandUryvayev (23) usingthe modifications described bySzilhgyiandPringle (21). Assay of theRNAtranscriptase.Thepreviously described RNAtranscriptaseassays(1, 12, 20, 21, 23) were modified. Incubation mixtures (0.3 ml) were made upat00Cbyaddingthefollowing ingredients: calculated volume of 20 mMTris-hydrochloridebuffer (pH 8.0) tomakethe final volume0.3ml,0.03mlof TNP or purifiedvirus suspension, 0.015 ml of 0.8% Triton-N 101 (added when virus was used but not when TNPwasused),0.015mlof70mM dithiothrei-tol,0.006ml ofSearle "ribonuclease inhibitor" prepa-ration (50units/ml),0.018ml of 32 mMATP,0.0075 ml of1 mMS-adenosyl methionine, and 0.075 ml of "reagent mixture" (containing Tris-hydrochloride buffer, pH8.0,NaCl, actinomycinD,ATP, CTP,GTP, and3H-labeled UTP) (22). The incubation mixtures werethen placed in 31 or390Cwaterbaths for 1 min, and RNA synthesis was started by the addition of 0.0075ml of 220 mMMgCl2. Samples (0.02ml) were taken eitherbefore the addition ofMgCl2 (zero-time sample) or at intervals duringincubation and were placed on Whatman DE-81 paper disks. The disks werewashedatroomtemperaturefivetimeswith5% sodiumpyrophosphate, twice with water, twice with ethanol, and twice withether,andradioactivitywas measured by liquid scintillation spectrophotometry. Thermal inactivation of transcriptase. Two identical incubation mixtures containingallthe ingre-dients exceptMgCl2wereprepared for eachmutant. One incubation mixture of eachpair was incubated at

390C for30minwhile the otherwaskept inan ice-waterbath.Thetranscriptase activity of both mixtures wasassayed at310Cafter the additionofMgCl2, and theamountofRNAsynthesized by the heat-treated

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incubation mixture during 3 h was expressed as a percentage of theamountsynthesized by itscontrol.

RESULTS

Temperature sensitivity of the mutants

of VSV NewJersey. Throughout the

experi-mentsdescribed in thiscommunication,weused purifiedpreparations of the varioustsmutants

of VSV NewJersey. Inpreliminary experiments wenoticed that somepreparationsof mutant ts E1, and to a lesser extent ts D1, appeared to

have ahigh frequency ofrevertantvirus. This

difficultywasovercomebyrecloningthese

mu-tants. The purified preparations of the ts mu-tants used in the experiments exhibited a ts

phenotype,and the revertant clones showed ef-ficiencies ofplatingat39°Csimilartowild-type virus.

Invitro RNAsynthesisby thets mutants. Todetermine the temperaturesensitivityof the

transcriptaseactivityof the varioustsmutants

ofVSV New Jersey, we assayed the virion-as-sociated RNA transcriptase in vitro at 31 and

390Candcomparedthe resultstothose obtained

with wild-type virus. These experiments were

repeatedthreetimes,and the results ofasingle

experiment are shown in Fig. 1. The results

obtained in.the othertwoexperimentswere in verygood agreement with thosegiveninFig.1.

Wild-typevirussynthesized RNAat310C

lin-earlyfor1h,and RNAsynthesiscontinuedat a

reduced ratethroughout the 3 h of the experi-ment. At390Ctheinitialrateof RNAsynthesis wasabouthalf of therate at

310C,

and aplateau

wasreachedbyabout60min, the total amount of RNAproducedbeingabout13 to16% of that synthesized at310C.

Mutant ts Al consistently synthesized more RNAat390Cthan thewild-typevirus. Mutants

tsC1 andtsDlsynthesizedRNAat390C

simi-larlyorslightlybetter thanwild-typevirus.

In the case of ts B1, the mutation almost

certainlyaffected apolypeptideinvolved in the

transcriptionprocess,since thismutant

synthe-sizedverylittle RNA at390C (only 2 to4%of

thatsynthesizedat

310C),

whereas itsrevertant,

tsBl/Rl, synthesizedRNA at

390C

atleastas

wellaswild-type virus.

Likewise, mutant ts El synthesized verysmall

amounts ofRNAat

390C,

whereas theamount

ofRNAsynthesizedat390Cby its revertant,ts

El/Ri,

was comparable to that produced by

wild-typevirus. This indicates that themutated

polypeptideofts Elisinvolvedintranscription.

However, mutant ts E3 synthesized RNA nor-mallyat390C, and tsE2synthesized RNA at a

somewhat reduced rate.

Therewaslimited RNA synthesisat

390C

by

mutant tsFl, about 5% of the amount synthe-sized at310C. Since the revertant clone (tsFl/ Rl) of thismutant synthesized RNA as well as or better than the wild-type virus, it is likely that the mutated polypeptide of ts Fl is also involved inthe transcription process. However, mutant ts F2synthesized RNA at390C similarly towild-type virus.

These experiments were repeated using TNP complexes of the tsmutants. These TNP prep-arations are ribonucleoprotein complexes iso-lated from purified virions (23). They contain only threepolypeptides(L, N, and NS) in close association with the virion RNA and retain the transcriptaseactivity of the virion (23). Results with the TNPs of the ts mutants were very similar to those given in Fig. 1, indicating that the temperature-sensitivepolypeptides of ts Bl, tsEl,andtsFlarepart of theviral core (Table 1).

Thermalstabilityofthetranscriptase

ac-tivity of the ts mutants.The mode of

inhibi-tion of RNAsynthesisat390Cwasinvestigated by examining the heatstabilityofthe

transcrip-taseactivityof thetsmutants.Identical reaction

mixtures, containing all ingredients except MgCl2, were incubated for 30 min at either 39 or 00C, and their residual transcriptase activities were assayed after the addition of

MgOl2

at

310C.

Figure 2 shows thatwild-type virus retained

approximately 40% of itstranscriptase activity

after heattreatment at390C. Similar heat

sta-bilitieswereobserved in thecaseofmutants ts

Al, tsC1, andtsDl (Fig.2).

Sincepreliminary experimentsindicated that

thetranscriptase activityoftsBl washeat

sta-ble, we assayed the heat stability oftwo

addi-tionalpreparations of thismutant. In all three

preparations, the heat stabilities of the

tran-scriptaseactivityweresimilarto oronly slightly

lessthan that ofwild-typevirusorthe revertant clone tsBl/Rl (Fig. 2).Thus, theinhibition of

RNAsynthesisat390C bythemutant tsB1was

probably the result of a reversible

configura-tionalchangeof the mutatedpolypeptide. Thetranscriptase activity of mutant ts El was very heat labile at390C. However, the revertant clone tsEl/Rl,aswell as the mutants ts E2 and

ts E3, had more stable transcriptase activities

thanwild-typevirus(Fig. 2). Thus, theinability

oftsEl tosynthesize RNA in vitro at

390C

is

duetothermal inactivationofthetranscription process.

Mutant ts Fl retained only 10% ofits

tran-scriptaseactivity after heat treatment, showing

thatthe transcriptase activity of thismutant is

slightlyless heat labilethanthat oftsEl. The

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m

0 x

E

cr 0

u UC C)

w

L(

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4r

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0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

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TIME

(HOURS)

FIG. 1. Assayofthe virion-associated RNA transcriptaseofthe ts mutantsofVSVNew Jerseyat 31and 39°C.Thevirion-associatedRNAtranscriptaseinpurifiedpreparationsofthets mutants wasassayedeither at31°C(@)or at39°C(0) asdescribed in the text.

transcriptase activity ofts F2 wasconsistently

more heatstable than that ofwild-type virus, and thetranscriptase activityoftherevertant ts

Fl/Rlwasthemostheatstable ofallthe clones

tested (Fig. 2). Thus, heat lability ofthe

tran-scriptase activityof the mutant tsFl probably

accountsfor itsinability tosynthesize RNA in vitroat390C.

In vitro temperature-shift

experiments

withts

Bi.

The mode of inhibition of the tran-scriptaseactivity of the mutant ts

Bi

was further studied by temperature-shift experiments, whereareaction mixture was incubated at390C for30minbefore transfer to 31°C (Fig. 3). The

amountof RNA

synthesized

after the

tempera-ture was lowered to 31°C was

approximately

25% of that synthesized by a reaction mixture

thatwas incubatedat310C throughout the

ex-periment. Another reaction mixture

containing

alltheingredients except

MgCO2

wasincubated for 30 min at 39°C, and the residual enzyme

activitywasassayedat31°C after the addition

ofMgCl2.Theamountsof RNAsynthesizedby

thetwopreparationswereverysimilar, showing

that enzyme activity was fully restored after

temperatureshift from39 to

310C.

These results suggestthatareversibleconfigurational change of the mutatedpolypeptideisresponsiblefor the

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696 SZILAGYI AND PRINGLE

TABLE 1. RNAsynthesizedbyTNPpreparationsof the ts mutantsof VSV New Jerseya

Mutant % RNA synthesized at

Wild type ... 16

tsAl ... 26

tsCi ... 20

tsDl ... 12

tsB1 ... 4

ts BR/Ri ... 22

tsEl ... 3

tsE2 .... 8

tsE3 .... 15

ts El/Ri .... 19

tsFl... 3

tsF2 ... 12

tsFl/Rl .... 42

aUsing TNP preparations of thets mutantsof VSV

NewJersey,complementary RNAsweresynthesized at31and390Cunder conditions otherwiseidenticalto thosedescribed inFig. 1. Theamountsof RNA syn-thesizedat390Cafter 3 h of incubationareexpressed

asthe percentage of theamountsof RNAsynthesized

at 310C after thesame length ofincubation bythe sameTNPpreparation,asfollows: percent RNA syn-thesized at390C = (counts per minute after3 hat 39°C/counts per minuteafter3hat3100) x 100.

inhibition of the transcriptase activity of the mutant tsB1.

Heat sensitivity of the infectivity of the

tsmutants.Purifiedpreparationsof thets

mu-tantswereincubated for5hat

390C,

andtheir residualinfectivitywascompared with the infec-tivity of identical suspensions kept at 00C for the samelengthoftime.Inthe case ofwild-type New Jersey, around 5% of the infectivity sur-vived this heattreatment.Considering the vari-ationsinherent in suchexperiments,theresults indicate that the mutants and the revertant clones under investigation showed similar heat stabilities tothewild-typevirus. The only pos-sible exception is tsFl,where only 0.7% of the

infectivity wasrecovered, suggesting that this

mutantmay be heatlabile.

Since it hasbeenshownthat mutant ts El has

avery heat-labile transcriptase activity (Fig. 1 and2), it wassurprising that the proportion of infectious virussurviving the heat treatment at

390C

for 5 h was similar to that ofwild-type virus. For thisreasonwe retested the heat

sta-bilityof theinfectivity of this mutant using two

furtherindependentlygrown and purified prep-arations (Table 2). The resultsconfirmthat the heat stability of the mutant ts El issimilarto that of the wild-type virus, since in both cases 10% oftheinfectivity was recovered after

treat-ment for5 h at

390C.

Webelieve that the low survival in the case oftsEl/Rl is erroneously

low, since in another experiment the heat stabil-ity of this viruswas significantly higher (13.5%). Comparison of the heat lability of the transcriptase activity of disrupted and in-tact virions of mutant ta El. Two further experiments were conducted to determine how mutant ts El retains itsinfectivity after a rela-tively long incubation at 390C, even though the activity of its transcriptase is heat labile.

In thefirst experiment, a purified preparation of the mutant ts E1 was incubated for 5 h at

390C,and then itsresidual transcriptase activity

was assayed at310C. The results showed that, like wild-type virus, mutant ts El retained ap-proximately 8 to 10% of its RNA transcriptase activity. Therefore it appears that, when the undisrupted virions of ts El are heated, both the mutant's infectivity and transcriptase activity areasstableasthose ofwild-typevirus.

Next, the heat stability of the in vitro tran-scriptase activity of ts El was reexamined using aTNPpreparation(Fig. 4). There was very little 1 173RNA synthesis at 310C after an initial incubaRNA synthesis at 310C after an initial incubation of the reaction mixture at 390Cfor

30min.Similarly, almost all transcriptase activ-ity was lost when a control reaction mixture

containing alltheingredients exceptMgCl2was

incubated at 390C for 30 min and then the residual transcriptase activity was assayed at

310C.These results show thattranscriptase

ac-tivityofthe viralcore, unlike theactivityof the

undisrupted virion, is heat labile, presumably

duetothe irreversible denaturation of the

mu-tatedpolypeptide.

DISCUSSION

Inthis paperweattempttoidentifythe

num-ber of viral polypeptides involved in the

tran-scriptionofthe genome of VSV NewJersey by

comparing the in vitro activity of the

virion-associated RNA transcriptase of the tempera-ture-sensitive (ts )mutantsof all six complemen-tation groups. Similar experiments made with the ts mutants of VSV Indiana (20) and the temperature-dependenthostrange (td CE)

mu-tants (21)provided thefirstevidence thatsome mutants possess a temperature-sensitive

tran-scriptase.

The RNA transcriptase of the mutants of

complementationgroups C andDisunlikelyto

beaffectedbythe mutationsincemutants tsCl

and ts D1 synthesized RNA in vitro at 390C

similarlytowild-typevirus.Theseresultsagree

with thefinding that bothprimary transcription

andreplicationof the viralgenomeoccurincells

infected with either of these mutants (10, 16). Since the mutations didnotappeartoaffectany

J.

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Cr)0 ~~~~~00 0

30-X 20

-E

~~~~~3%1-20

80

10

~

1029%

28%

75L 7 S10 1.0

0 1 0 0

< tsEl

PsE

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otE3

100 ttsEl/R

0 6 5.0

w

~~~~~~~~66%

(1)

~~~~~~~~~53%

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0.4/.

H 50~~~~~~~

)0.2

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H tsFl tsF2

20tsFl/R

7.5

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0 0 0

TIME (HOURS)

FIG. 2. Heatstabilityofthe virion-associated RNA transcriptase activity ofthets mutantsduring incu-bationat39°C for30min.Identical reaction mixturescontainingallingredientsexceptMgCl2wereincubated at39°C(0)and0°C(a)for30min,and their residualtranscriptase activitywasassayedat31°C afterthe additionofMgCl2asdescribed in thetext. Themutants werethesame asdescribed inFig.1exceptthattwo

furtherindependentlygrownandpurified preparations ofthemutanttsBlwereadded.

stageof RNAsynthesis in either of thesegroups,

it islikelythat thepolypeptides M and G of the

virus coat areaffected by these mutations. Wun-ner and Pringle found, paradoxically, that,

al-thoughinmutant tsDltheelectrophoretic

mo-bility of both polypeptides Gand Nwas altered, the tsphenotypeof thismutant appeared to be

due to mutation in the Npolypeptide because

reversion oftemperature sensitivity was

accom-panied by reversion of the N polypeptide to

nornal mobility(25).However, the gene

assign-mentof thismutantisunderreinvestigation by extensive biochemical andgenetic analysis.

In vitro RNAsynthesisat39°C bytsAl was even more pronounced than that by wild-type virus. This makes itveryunlikelythat

transcrip-tion in group A isaffectedbythemutation,and

suggests that some function in the RNA

repli-cation is defective. This is in good agreement with the results obtained with infected cells,

where replicationwas restricted whileprimary

transcriptionwasunaffected(10, 16).

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LU wild type tsBl

>- 80

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~~~~25-z t 3,

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TIME (HOURS)

FIG. 3. Reversibility of the activity ofthe RNA

transcriptase ofthemutant tsBJ. Thetranscriptase activity of purified preparations ofwild-type VSV NewJerseyand themutant tsBJwasassayedeither

at31°C(U)or390C(0).Afurtherreaction mixture was incubated at39tC for30mi and then

trans-ferred to 31'C (0). As control, a reaction mixture which contained all theingredientsexcept

MgCl2

was

incubated at390Cfor30min, and then

MgCl2

was

added and the residual transcriptase activity was assayed at 31'C (El). The conditions ofassay are

described in thetext.

TABLE 2. Stability oftheinfectivity ofpurified group E mutantpreparationsduringincubation in

vitroat390Ca PFU/ml

% Sur-Mutants After

0°C

for After

39°C

vivalb

5h for5h

tsEl(2nd 6x 1010 6x 109 10

prepn)

tsEl(3rd 6.3x 10'0 6.3 x109 10 prepn)

tsE2 1.2x 10"1 1.7x10'0 14

tsE3 1.4x 1010 1.2 x109 9

tsEl/Ri 2.7x109 4.1x 107 1.5 a The purified virus suspensionswereincubated for 5h at39°C, and the residualinfectivitywastitratedat 31°C as described in the text. Two independently

grownand purifiedpreparations of themutant tsEl wereused.The preparations of themutantstsE2 and tsE3 andthe revertant tsEl/Rlwere thesame as

describedinFig.1.

'Percentsurvivalisexpressedas(PFU after39°C

for5h/PFU after0°Cfor5h)x 100.

Mutants of the other three RNA-negative complementation groups (B, E, and F) are the mostlikelytobetemperaturesensitive for

tran-scription,and the in vitroexperimentsindicated

0 1 2 3 0 1 2 3

TIME (HOURS)

FIG. 4. Reversibility oftheRNAtranscriptase ac-tivity ofthemutant tsEl.Wild-typeVSVNewJersey

andthemutanttsElwereused. Transcriptase

activ-itywasassayedat31°C(U),at39°C(0),andat31°C

after 30 min ofincubation at 39°C (0). Another reactionmixturecontainingalltheingredients except MgCl2 wasincubatedat39°Cfor30min, andthen

MgCl2 was added and the residual transcriptase activitywasassayedat31°C(O). Conditionsof

en-zyme assayaredescribed in thetext.

that therepresentativemutantsofthesegroups, mutant ts B1, ts E1, and ts F1, did possess

temperature-sensitive transcriptase activity.

Therefore, the investigation was extended to

includeallthreemutantsofgroupE andboth of groupF.

Therewasverylittle RNAsynthesisat39°C

inthecaseofmutanttsBi,whereasRNA

syn-thesiswasnotreducedin thecaseofthe revert-antclonetsBl/Rl,strongly indicating thatthe mutated gene product is involved in the

tran-scriptionprocess.Thisis ingoodagreementwith

the resultsobtainedwithtsBl-infectedcells(10,

16). Temperature-shift experiments withts Bi

showed that inhibition is a reversible process

presumablybrought about bya configurational

change of the mutated polypeptide. Thus, the

mode ofinhibitionin thismutantisverysimilar

tothat of thetemperature-dependenthostrange mutanttdCE3 (21,22).

Mutantsof complementationgroupEshowed differencesin the amounts ofRNAthey

synthe-sized invitroattherestrictivetemperature.The

involvementof the mutatedpolypeptideof tsEl in the transcriptionprocessis suggestedby the

fact that this mutant synthesized very little RNAat39°C,whereas its revertant clonetsEl/ Rl synthesized normal amounts of RNA at

39°C. On the otherhand, mutants tsE2 and ts

E3 bothsynthesized RNA at39°C. Since

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cationof viralRNAis observed at the restricted temperatureincellsinfected withtsE2(16), the mutated polypeptide in group E is almost

cer-tainly multifunctional. Thus, experiments

in-volving ts Elindicate that the polypeptide takes part intranscription, whereas those involving ts E3 suggestthat thepolypeptideisalso involved in replication, and thoseinvolving ts E2 indicate that thepolypeptide isdefectiveeither in some later stage ofreplicationorin somestageof virus

development following replication.

Alterna-tively,itispossible thatthetranscription

prod-uctssynthesizedat

390C

bytsE2and ts E3 are

notfunctional.

The mode of inhibition of the transcriptase

activity ofts El is irreversible thermal

inacti-vation of the mutated polypeptide. However, whenundisrupted virusparticles aresubjected

to heat, both the infectivity and transcriptase

activityof this mutant and ofwild-typevirus are

verysimilar.This maybe theresult of an

inter-action between the mutated polypeptide and

either the M or,lesslikely, the Gpolypeptide,

resulting in the suppression of the heatlability of the mutated polypeptide. Like interactions,

possibly involving somehost polypeptide, may

accountfor the primary transcription that takes place incellsinfectedwith tsEl (10).

The two group F mutants are alsodissimilar inthe amount ofRNA they synthesize in vitro

at

390C.

One of them, ts Fl, synthesizes very

little RNA at

390C,

and the other, ts F2,

syn-thesizes nornal amounts of RNA at this

tem-perature. Since the revertant clone, ts Fl/Rl,

synthesizesRNA at

390C,

weconclude that the

mutatedpolypeptideoftsFlis also involved in

the transcription process. This conclusion

ac-cords well with the finding that

primary

tran-scription in infected cells was inhibited at the restrictive temperature (10). The heat lability of the mutatedpolypeptide appears to be respon-sible for the inhibition of transcriptase activity. The fact that tsFl and ts F2 synthesized

differ-ent amountsof RNA in vitroat390C suggests that the polypeptide is

multifunctional,

being involved in both transcription and replication.

However,it is alsopossible that the RNA species

synthesizedat390C bytsF2 are notfunctional.

Thus invitro experiments indicate that mu-tation affected transcription in group B, repli-cation in group A, and both transcription and

replication in groups E and F. The mutated

polypeptideingroupE may also be involved in

somelatestage of virus development. Itis hardly surprising that some of the polypeptides should be multifunctional, since there are only five of them, although some host polypeptide may also beinvolvedinsome stages of viral RNA

synthe-S1S.

Becausetherearesixcomplementationgroups

andonly fiveviralpolypeptides,it isdifficultto

make gene assignments. It ispossiblethat VSV codes for a sixth, so far unrecognized poly-peptide,althoughthepossibilitythat one of the six groups represents an extreme caseof intra-cistroniccomplementationhastobe considered

also.Sincepurifiedcoresisolated frommutants

tsBl, tsEl,andtsFlexhibit thesame temper-ature sensitivity as their virions, it is possible

thatthecorepolypeptides L, N,and NSarethe

sitesof themutationallesion in groupsB, E,and

F.

We propose that the mutated polypeptide in group B is polypeptide L, since the mode of inhibition in mutant ts B1 issimilartothat in td CE3, wherepolypeptideLwasshown to be the mutatedpolypeptide(21, 22). Thispossibilityis

strengthenedbya recentreport ofdissociation

and reconstitution experiments in which it was

foundthat the mutatedpolypeptideoftsBl was

either L orpossibly NS (9). If in group B the mutatedpolypeptide is L, then polypeptides N

and NSare the likelyones in groups E and F.

ACKNOWLEDGMENTS

We thank J. H.Subak-Sharpefor criticalreadingof the manuscript.We also thank C.Cunningham and P. Malloy for their excellent technical assistance, and J. T. Poyner for helping with the preparation of the manuscript.

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