Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus.

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0022-538X/78/0027-0560$02.00/0

Rebinding of Transcriptase Components (L and NS Proteins)

to

the

Nucleocapsid Template of Vesicular Stomatitis Virus

MARGARET G. MELLON AND SUZANNE U. EMERSON*

University of Virginia Medical School, Department of Microbiology, Charlottesville, Virginia 22901

Received forpublication 7 April 1978

The L and NS proteins of vesicular stomatitis virions (New Jersey serotype) were solubilized with Triton X-100 and high-salt buffer and recombined with purified nucleocapsids under conditions similar to those used to reconstitute transcriptase activityinvitro. Thenucleocapsid-boundL andNS proteins were separated from unbound proteinsonaglycerolgradient. Therebinding of L and NS proteins mimics the invivo binding in that at saturation the ratio of L and NS moleculestoNmoleculesisapproximately the same as observed in the intact

virion. L andNSproteins were separated and added back independently and in

combinationtothetemplate.Thepurified NS proteinbound to thetemplate in the absenceof Lprotein. However,the Lproteinbinding appearedtodepend on

the presence of NS protein. The presence of

Mg2"

and nucleotides, which is

required for transcription, was not necessary for the rebinding of L and NS proteins.

Most, ifnotall,rhabdoviruses carryan

RNA-dependent RNApolymerase in the virion (14).

In several rhabdoviruses, including the

well-characterizedvesicular stomatitis(VS) virus,the

polymeraseactivity may be dissociated from the ribonucleocapsidtemplateand thetranscription

systemthen reconstitutedbyrecombination of

the separated components (7, 5). Under this reconstitution system, three of the five viral

proteins have been identified as necessary for

transcription (8, 9, 11). These are the major

structural protein Nand the minorproteins L

andNS;thetwomembrane-associatedproteins,

G andM,areapparentlynotrequired (7,6). The

N protein is probably required as an integral

structural component of thetemplate; onlythe

nucleocapsid consistingof Nprotein tightly

com-plexedtotheviralRNAserves as atemplatefor transcription (7, 11).Deproteinizedviral RNA is

not transcribed in the in vitro system. The L

and NSproteins,which have beenpurifiedas a

complex, active in in vitro transcription (11), almostcertainly constitute thetranscriptase

en-zyme. However, mRNAsynthesis catalyzed by

the transcriptase is a complex process. The

mRNA's transcribed in thereconstituted

tran-scription systemarecappedandmethylatedat

their 5' endsandpolyadenylatedattheir 3' ends

(1). Thecappingandpolyadenylating activities

whichmodify the mRNA'sare sotightly coupled

with thetranscriptase activitythat methods for

separating these activities from transcription

haveso fareluded investigators. Thus, the

de-tailedfunctionsofthe L and NSproteinsin the

overall production of mRNA's remain obscure.

As anapproach to understanding the roles of

the L and NS proteins, we have sought

infor-mationontherebindingof thesetwoproteins to

thenucleocapsid of VS virus. In this report we

havedeveloped an assay for the rebinding of L

and NS proteins of VS virus New Jersey to

nucleocapsids and used it to characterize the

bindingcapabilities of the separated L and NS

proteins and to determine the stoichiometry of

and therequirements for the binding reaction.

Since the polymerase rebinds to the template

during transcription, our assay conditions are

basedonthose forthe in vitro reconstitution of

transcriptase activity (7).

MATERIALS AND METHODS

Viruses andcells. TheNewJerseyserotypeofVS

virus wasoriginally obtainedfrom the U.S. Depart-mentofAgriculture, Beltsville,Md., by R. R. Wagner and wasrecentlyclassifiedasthe Concansubtype (13). The viruswasgrownat31°Cinconfluent monolayers of BHK-21 cells in 150-mm2 Corning tissue culture flasks with Dulbecco modified Eagle medium, high glucose, and 10% tryptose broth. Virus was radiola-beledby including150juCiof[3S]methionineperml,

100,uCi of3H-labeledL-aminoacid mixture perml,or

50,Ciof[2H]leucineper ml in theinfectingmedium.

Cultureswereinfected at0.5 to 2 PFU percell, and virions wereharvested at16to18hpostinfection. The

overlaymediumwasclarifiedbya10-min

centrifuga-tionat1,000xg,and the virionswerepelleted through

alayerof 50% glycerolonto apadof Flouroinertby centrifugationat80,000xg for90min. The virus band wasremoved from the surface of the Flourinertpad

andlayeredontoapreformedlinear0to40%sucrose 560

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gradient containing 1 MNaCl and 1mM EDTA (7,

10). The band ofB virions wascollected from the

gradient andconcentratedbycentrifugationat80,000 x g for 90 min. The virus was then centrifuged to

equilibriumovernightonapreformedlinear 10 to40%

sucrose gradient containing 1 M NaCl and 1 mM EDTAandconcentratedagain bycentrifugation.The finalpellet,the harvest of 15bottlesof tissue culture cells (5 to 10mg),wasresuspendedin 1 to 2 ml of 0.01 MTris-hydrochloride (pH7.4) plus15%glyceroland stored at-18°C untilneeded.

Solubilization of viralproteins for rebinding.

Viral proteins for rebinding were labeled with

[3S]-methionine or a 3H-labeled mixture of amino acids. The virus harvest fromapproximately five bottlesof BHK-21cells (-3mgofpurifiedvirus in 0.3ml)was mixed withanequal volume of 2x Triton-high-salt solubilizer(2xHSS=18.7%glycerol,1.44MNaCl,1.2 x 10-3Mdithiothreitol,3.74%Triton X-100, and10-2

M Tris-hydrochloride, pH 7.4) (7). The solubilized virions were incubated at31°C for 30min.The

nu-cleocapsidtemplatewaspelletedfrom thesolutionby centrifugation at 125,000 x gfor 2 h, leaving the

supernatantfractioncontainingthesolubilizedG, M, L, andNS viralproteins. Viralproteinswerealways solubilized immediately before their use in binding experiments.

Templatepurification.Tocompletelyremovethe

tightly adhering NS protein, very dilute samples of virus were used to prepare anucleocapsid template.A

15-bottleharvestofpurifiedvirus(5to10mg) labeled

with[3H]leucineor[3S]methioninewasdilutedto43

ml with 0.01 MTris-hydrochloride, pH7.4.The virions weredisrupted by mixingwith anequal volumeof 2x

HSS (seeabove).Thenucleocapsidswerecentrifuged

onto a pad of renografin at 90,000 x governight,

collectedfrom the surface of thepad,andthen

chro-matographed on anagarose (Bio-GelA-5M) column equilibratedwith 10%glycerol,1MNaCl,0.1%Triton,

10-4Mdithiothreitol, and 0.01 MTris-hydrochloride,

pH 7.4. Approximately2-ml fractionswerecollected

fromthe column. The fractionscontainingthelabeled

nucleocapsids, which eluted with the void volume,

werepooled andcentrifugedthroughalayerof 20%

glycerolonto a100%glycerol padbycentrifugationat 125,000 x g. Thenucleocapsidswere removedalong

with the 100%glycerolpadand storedat-18°Cuntil used.

Binding assays.Details of eachbindingassayare

givenin thefigure legends.The standard assay, based ontheoptimalconditions fortranscription,was setup

accordingtothefollowingscheme (7). Since the salt

optimumfortranscription is -0.15 MNaCl,the viral

proteins, solubilizedin 0.72 MNaCl,werediluted1:4 into thestandardassay mixture togiveafinal NaCl concentration of0.144M(80

id

ofsolubilized proteins

ina0.4-ml final reactionvolume).Theprereactionmix (1.4x10-3MATP,GTP, and CTP; 8x10-3

MMg2m

; 1.2x 10-3Mdithiothreitol; and 5x 102MTris, pH 7.4) was two times the desired concentration of nu-cleotides and

Mg2e,

and 0.2mlof theprereactionmix wasincluded in the 0.4-ml final volume. Various vol-umesofpurifiedtemplate (in100%glycerol) and0.01 MTris-hydrochloride, pH7.4, made up the remaining 0.12ml of the final reaction volume.

Glycerol gradients. Linear 15 to 35% glycerol

gradients (5ml)wereformedby usinga custom gra-dient maker. The 15 and 35%glycerol solutionswere made bydiluting either 3 or 7 ml ofglycerolto 20ml with glycerol gradient mix (7 x 10' M ATP, CTP, andGTP, 4 x 10-3 Mmagnesium acetate, 0.144 M NaCl, 2%Triton, and 6 x10-4M DTT in 0.01 MTris, pH 7.4). ATP, CTP, andGTP were included in gra-dientsonly in early experiments. Once it was deter-mined thatnucleotideswerenotrequired for binding, they were omitted from the gradients.

SDS-polyacrylamide gel electrophoresis. Pro-tein sampleswere mixed with 100,ug ofhemoglobin

carrierand 1 volume of 40%trichloroaceticacid and centrifuged at 2,000 x g to pellet the precipitated

protein.Thepellet was washed one or twotimeswith acetoneand dissolved in asolution of 0.01 M

Tris-hydrochloride with 8 M urea, 1% sodium dodecyl

sulfate (SDS),and 1%mercaptoethanol.Afterboiling

for 2min,thesamplesweresubjectedto electropho-resis on 7.5% SDS-phosphate acrylamide gels. Gel

sliceswereincubated with 0.5 ml ofNuclear-Chicago solubilizer(NCS)for 2 h at 50°C and counted in LSC

Complete toluene-based scintillationfluid.

Preparation andrecombination ofthe Land NSprotein fractions. The preparation of L and NS

proteins from VS New Jersey serotype virus is a modification of thepublishedprocedure using VS In-diana serotypevirus(8, 9).Approximately 1 ml (-10 mg) of VS NewJerseyviruslabeledwith [3S]methi-onine wasdilutedto 10ml and mixed with an equal

volume of 2x HSS (see above). The nucleocapsids were removedbycentrifugationfor 2 hat 125,000 x g. The supernatant wasmixedwith 5 to 10 ml of

phos-phocellulose previously equilibratedwith Tris, pH 7.2,

and thendialyzed overnight against column buffer(75 ml ofglycerol,3ml of 20% Triton, 9 mg of DTT, and 222 ml of 5 x 10-2 M Tris-hydrochloride, pH 7.2)

containing0.1MNaCl. The phosphocelluloseand 3S-labeledsupernatantprotein mixturewas then trans-ferred to a 12-mlcolumn, drained,and washed with more0.1M NaCl columnbuffer;theflow-throughwas saved and used later. Thephosphocellulose column waselutedwith 0.5 M NaClcolumn buffer, andthe

peakfractionscontainingthe3S-labeled proteinswere calledthe L-protein fraction. Becauseof the instability

ofthe L protein, the L-protein fractionwas always

usedimmediately.

Thereservedflow-throughfrom the

phosphocellu-losecolumn, containingprimarilyNS andGproteins, wasappliedto a 4- to6-mlDEAE-cellulose column,

and thecolumnwaswashed with 0.1 M NaCl column buffer. The NS proteinwasthen eluted with 0.4 M NaCl in column buffer. Tocompletelyremove L

pro-tein,it wasnecessary todialyzetheNS-protein frac-tionovernight against 0.1 M NaCl column buffer and toprocess the NS proteinthrough anothercycle of

phosphocellulose and DEAE-cellulose chromatogra-phy. Purified NS protein was stored at-180C.

When used inbindingassays, the35S-labeledL-and

NS-proteinfractions were recombinedwith3H-labeled

purified template. The binding assay mixture with both L andNSproteinscontained0.06ml ofL-protein fraction, 0.08ml ofNS-protein fraction,0.025 ml of

template,0.035ml of0.01MTris,pH7.4, and0.2ml

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562 MELLON AND EMERSON

ofprereaction mix(see above), pH7.4. When L- or

NS-protein fractionwasaddedseparately,either 0.08

ml of 0.4 M NaClcolumn wash or0.06ml of0.5M NaCl columnwash, respectively, wasaddedto main-tain thepropersaltconcentration. Thebinding reac-tions wereincubatedat31°Cfor30min.The

template-boundproteinsineachpreparationwereisolatedon

glycerol gradients and analyzed on

SDS-polyacryl-amidegels.

Stoichiometry ofLandNSproteins boundto

template.The relativedistribution of label inL, NS,

and N proteins was obtained from

SDS-polyacryl-amide gels. Smallsamples of intact viruswere dis-solvedingelsamplebuffer andapplied directlytothe

gel.Templateand reboundproteinswereisolatedon

glycerol gradients, trichloroacetic acid precipitated,

and thenanalyzedongels. In the casewhere intact virionswerecomparedtothedissociated and

imme-diately recombined supernatant and pellet fraction,

the virus was labeled with a 3H-labeled mixture of

amino acidstouniformlylabel all viralproteins.The relative amount of radioactivity in L, NS, and N proteinswasdetermined and dividedbythemolecular weightsforL,NS,and Nproteins(190,000,40,000,and 50,000,respectively)togivemolecular ratios. The

mo-lecular ratio for N was divided into 2,000, and the molecular ratiosfor L andNSproteinsweremultiplied

by this conversion factor. The final valuesrepresent the number of L andNS moleculesper2,000N mole-culesonthegel.

Whenpurifiedtemplate andsolubilized viral pro-teinswererecombined,thetemplatewaslabeledwith

[35S]methionine, and the solubilized viral proteins

werelabeled witha3H-labeledmixtureof aminoacids. In thiscasethespecificactivities of the3H-solubilized proteins and the3S-labeledNproteinwerecalculated based on a Lowry protein assay, and the specific

activities of the three proteins were then used to

determine thestoichiometryof L and NSproteinsper

2,000Nmolecules.

Chemicals. [3H]leucineand[3S]methioninewere

obtained fromAmersham/Searle, ArlingtonHeights,

Ill.,and the3H-labeledL-aminoacid mixturewasfrom

NewEnglandNuclearCorp.,Boston,Mass.Whatman

P1l-cellulose and microgranular DEAE-cellulose

DE52 were purchased from Whatman Biochemicals

(Maidstone, England).Renografinwasobtained from

E.R.Squibb &Sons, Princeton,N.J.,and Flouroinert was from Instrumentation Specialties Co., Lincoln,

Neb. LSCCompletescintillation fluidwasfrom York-townResearch,Hackensack,N.J.Agarose(Bio-Gel A-5M), acrylamide, and N,N-methylenebisacrylamide

wereobtained from Bio-RadLaboratories,Richmond,

Calif.

RESULTS

Solubilizedviral proteinsrebound to the template. The L and NS proteins were

solubi-lized along withthe M and Gproteins by treating

whole 35S-labeled virus with Triton X-100 and

high salt (0.72 M NaCl) andpelleting out the

nucleocapsids. This supernatant fraction was used immediately in binding assays. Purified template, consisting ofonlythe N protein and

viral RNA,waspreparedfrom anotherbatch of

3H-labeled virus by standard methods (see

Ma-terialsandMethods).

The purified 3H-labeled template was

recom-binedwith the3S-labeled supernatant proteins

under transcription assay conditions. Final

con-centrationsofMg2+, NaCl, ATP, GTP, and CTP

were optimal for transcription, but UTP was

omittedtopreventtranscription from occurring

(8). Thereaction was incubated at 31°C for 30

min, and then the entire 0.4-mlreactionmixture

was layered on a 15 to 35% glycerol gradient

made up in a solution similar to the reaction

mixture. Figure 1 shows the distribution of

'S

and 3Hlabel inthe glycerol gradient. The

tem-plate along withbound 3S-labeled proteins

sed-imented to the middle of the gradient. The

un-boundproteins remained at the top. Aggregated

template (30 to 50% of total)and proteins(-10%

oftotal) sedimented through the gradient and

pelleted. When supernatant proteins were

ap-plied to thegradient in the absence of template,

no proteins were found in the middle of the

300 200 100

4-V

2 4 6 10

GRADIENT FRACTION

FIG. 1. Glycerol gradient of the binding reaction mixture. High-salt-solubilized VS New Jersey viral proteins labeledwith[3SJmethionine andtemplate labeled with[3H]leucinewereprepared as described in the text. The binding reaction mixture contained 80ulof the solubilizedmS-labeledviralproteins, 70

,ulof template,50,tlof0.1MTris,pH 7.4, and 0.2 ml

ofprereaction mixture (1.4 x 103 MATP, CTP, and

GTP, 1.2x 103 M dithiothreitol, and5 x 102 M

This-hydrochloride,pH7.4).After a30-min

incuba-tionat31°C, the entire0.4-mlreaction mixture was

layeredona 15 to35% glycerol gradient (see text) and

centrifugedat125,000 x g for I h at 4°C. Portions (50

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1d) of0.5-ml gradient fractions were counted and

plotted. Thedirection of sedimentation is from right toleft.

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gradient(datanotshown).

To determine which of the four solubilized proteins had boundtothetemplate,the

gradient

fractions containingthecosedimenting S- and 3H-labeled proteins were precipitated with

tri-chloroacetic acid andanalyzedon an

SDS-poly-acrylamide gel. Despite the fact that M and G proteins were present in much greater abun-dancethanthe LandNS proteins inthe solu-bilized supernatant fraction, only traces of M and Gproteinswerefound

cosedimenting

with template (Fig. 2).Essentially onlythe L and NS proteins reboundtotemplate.

Invitro

rebinding

of L and NS proteins mimicked the stoichiometry of intact

vin-ons. Inview of the marked

tendency

of the L

protein toaggregate invitro,it is

important

to

show that the observed cosedimentation of L and NS proteins with template represents a rebinding ofLandNSproteinsto

specific

sites on the template. If the L and NS

proteins

are

rebinding to

specific

sites on the

template,

it 32k

281-

241-x

C4

E

U,1

201-16

[-12H

8

4

AND

should be possible to saturate the available sites

on thetemplate toapproximatelythesame

ex-tent asinundisruptedvirions.

Aseries of binding reactions was set up with

constant amounts of 3S-labeled supernatant proteinsand decreasingamounts of3H-labeled template, in effect increasing the concentration

of Land NSproteins relative to the template.

Each ofthe reactionmixtures was applied to a

glycerolgradient, and the template-bound

pro-teins were analyzed on SDS-polyacrylamide

gels.The amountsof L and NS proteins bound

were normalized to the N protein on the gel.

The amounts ofboundL and NS proteins

in-creasedlinearly with increasing relative

concen-trationsof LandNSproteins and then reached

a plateau, showing that sites on the template

canbesaturatedwith L and NS proteins (Fig.

3).

Further evidence that the binding is

signifi-cant wasobtainedbycomparingthe number of

Land NS proteinsreboundtothetemplateand

N

NS

_ 10 20 30 40 50 +

GEL FRACTION

FIG. 2. SDS-polyacrylamidegelelectrophoresisof template-boundproteins.Fractions4,5, and6fromthe

gradientrepresented inFig.)werepooled,and theproteinswereanalyzedonSDS-phosphatepolyacrylamide

gels.Thetemplatewaslabeled with3H,and the solubilizedsupernatantproteinswerelabeled with 'S. No

!2M

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MELLON AND EMERSON

cn

5.

-n

2

0

3

2

1

FIG. 3. Bindi A seriesofbind

asdescribed in

mlof theprerea

lized viralprote ,ul) of 3H-label hydrochloride, bring the total boundfromunl

gradients, theI the rebound L SDS-polyacryla

orNSproteins

labeled Nprot plotted against lizedsupernata ratiowasdeter each reactiona gradients.

thenumbero

the intact vir

ecules, arbitrarily expressed per 2,000N

mole-cules,weredetermined from eachgelasdetailed

inMaterials and Methods. Wherethetemplate

0 was not

saturated,

the number of L and NS

}rl/N molecules rebound per 2,000 N molecules was

0 less than the number of L and NSproteinsfound

o0 inthe intact virion (Table 1).

However,inthecasewhere the templatewas

-v--NS/N saturated, there was excellent agreement

be-,,+,,A

aNS/N

tween the numbers of L and NS molecules

bound in vitro and in vivo (Table 1). In this

experimentLandNS proteinswerereboundto

purified template and the concentration of L

20 40 60 80 and NS proteins was shown to be saturating.

The

amount

of L andNS

proteins

reboundper

INITIAL RATIO35S/3H 2,000 N molecules in this experiment

ap-proachedclosely thenumber determined forthe

of LandNSproteinstotemplate intactvirion. These resultssuggestthatthe

re-Fingassayswassetupandprocessed binding is specific inthat saturationthe

Fig. and 2. Each tube contained 0.2

actionmixture,80Alofthe

XS-solubi-

binding

of L and NS

proteins mimicked

the

?ins, anddecreasingvolumes(80to10 bindingobservedintheintact virion.

ed template. Sufficient 0.01 M Tris- NSboundtotemplate in the absenceofL

pH 7.4, was added to each tube to protein. It wasofinterest to examine the sep-volumeto0.4ml.After separationof arate binding capabilitiesof the Land NS

pro-found'S-labeledproteinsonglycerol teins. The NS proteincanbe purifiedfrom the

pooledgradientfractions containing mixture of solubilized viral proteinsbyseveral and NSproteins were analyzed on cycles of column chromatography(seeMaterials

cmidegels. Thecountsof'Sin the L andMethods). A series of bindingassayswasset

werenormalizedtothecountsof 3H- upwithincreasingconcentrations ofNSprotein

'ein on each gel. These values were

the initial ratio of

5S-labeled

solubi- and constant amounts of

template.

An

SDS-ntproteins/3H-labeted

template.

Ths

i polyacrylamide gel ofthe purified NS fraction ,minedby countingsmallsamples of showed 95% of the fraction to consist of NS

iixturebefore layeringontheglycerol protein. Purified NSproteinbound to the

tem-plate in the absence of L protein (Fig. 4). Figure

scontained in 5 shows that the binding reaction was linear

rfL andNS proteins with increased protein concentration and that

ion If the number of L and NS NSprotein saturated the template.

proteins rebound to template

in

vitro

greatly

exceeded the number of L and NS proteins

normally carried by the virion, a nonspecific

aggregation might be suspected. On the other

hand,ifundersaturatingconditions the numbers

of L and NS proteinsreboundto thetemplate

wereapproximately equivalentto the virion

val-ues,specific rebindingwould besupported.

Forthese experimentsa3H-labeledaminoacid

mixturewasusedtouniformlylabelallthe viral

proteins.Asampleof viruswasdissociated with

the 2x HSS and separated by centrifugation

intoa supematant and pellet fraction.The sol-ubilized supematant fraction and either 0.1 or

0.05 ml of the pellet fraction which contained

thetemplatewereimmediatelyrecombined

un-der standard binding assay conditions and

ap-pliedtoaglycerol gradient.Thetemplate-bound

proteinswere thenanalyzedon

SDS-polyacryl-amidegels. Undissociatedvirus from the same

preparationwasalsoanalyzedonan

SDS-poly-acrylamide gel.Thenumbers of L and NS

mol-TABLE 1. NumbersofLand NSmoleculesbound

per2,000Nmoleculesa

Rebound Land NSproteins Pro- Mol Intact Subsaturationc

teins virionsh

Satura-0.1Tem-0.05Tem- tiond

plate plate

L 190,000 131 47 76 142

NS 40,000 267 160 224 230

aSee thetextforcalculations.

bIntact virions were solubilized and analyzed di-rectlyonpolyacrylamide gels.

'Samepreparationsasintact virion. The viruswas

solubilized,andsupernatantandpelletfractionswere obtained by centrifugation. Portions (80

pd)

of the

supernatant protein and0.1 or 0.05 ml of thepellet

fractionwereimmediatelyrecombined in the standard

bindingassay.

dPurified template and solubilized proteins

pre-paredfrom other virussampleswererecombined un-dersaturatingconditions inastandardbindingassay.

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201

'Vo

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E

0

OU 10

N

NS

- 10 20 30 40 50 60+

GEL FRACTION

FIG. 4. SDS-polyacrylamidegel electrophoresisof

template-bound NS protein from a binding assay

containing saturatingamountsof NS protein.

Bind-ingassay conditionsaredescribedin the legendto

Fig.5. 0.3

z

o 0.2 -z

0 z

0.1

_

0

O 0o

0 z

5 10

NS PROTEIN ADDED (cpmx10o3)

FIG. 5. Binding of NS proteintothenucleocapsid template.NSproteinlabeled with3Swaspurified by

chromatographyasdescribed in thetextanddialyzed against glycerolcolumn wash with 0.1 M NaCl. Tem-platelabeled with35Swaspreparedasdescribed in

thetext.Serial dilutionsofthe NSproteinweremade in 0.1 MNaCIcolumn wash. A 0.1-mlamountofNS

protein from each of thedilutions wasaddedto20 ,ul of template,60,1. oflxHSS,20!l1 of Tris,and 0.2 mlofprereactionmix(see text).Thebindingreactions

wereincubatedat31°C for30 min. Thetemplateand

boundproteins wereisolated onglycerol gradients

and analyzed on SDS-polyacrylamide gels. The countsin the NSproteinboundwerenormalizedto

thecounts intheNproteinon thegelsandplotted against the 35Scountsperminute in 20jlofeachof the dilutionsofpurifiedNS addedtotheassay.

Lprotein binding depended onthe

pres-enceof NSprotein.Incontrast tothe binding

capabilityofNSprotein, the Lprotein did not

appeartobindtotemplatein the absence of NS

protein. Forthisexperiment L-andNS-protein

fractions wereprepared by column

chromatog-raphy as described in Materials and Methods.

The L-protein fraction was free of NS protein

but contained substantial amounts of M protein. However, since the M protein did not rebind to

the template under these conditions,further

ma-nipulations to

purify

the extremely unstable L

protein were not attempted. The NS fraction

usedwas atleast95% pure.

The L- and NS-protein fractions were

enzy-matically active when combined and tested in transcription assays. However, when tested alone each fraction was inactive, showing that the fractions were free of cross-contamination (datanotshown).

The L and NS fractions were added either

alone or in combination to purified template.

Table 2 shows that whentheL-proteinfraction

wasaddedalone,nobindingtothetemplatewas

seen.However, when Lproteinwasadded in the

presence of NS protein, Lprotein did bind to

the template. Thus, the binding of L protein seemedtobedependentonthe presenceof NS protein. Similaramounts of NSprotein bound to template in the presence or absence of L protein.

Requirements for

binding. Although

the bindingassaywas

originally developed by using

conditionsoptimalfortranscription, further

ex-perimentsweredoneto seetowhatextentthese conditionswere

actually required.

Table 3

com-paresbindingof L and NSproteins under

satu-ratingconditions in the presence and absence of

Mg2e

and nucleotides. The omission of

Mg2e

and

nucleotides did not

significantly

affect the

amountof

binding

of the L andNS

proteins

to

thetemplate. Thus,

Mg2e

andnucleotideswere

notrequiredforbindingLand NS

proteins.

Another experiment was done to determine

whetherthebinding reaction wouldoccur atlow

temperature. After mixingthe components

un-dersaturating

conditions,

the

binding

reaction

wasleft in the ice bucket insteadof

being

incu-bated at

310C.

Similar amounts of L and NS

[image:6.501.55.215.59.218.2]

proteins bound at4 and31°C

(Table

3).

Thus,

TABLE 2. Binding ofLandNS proteins separately and incombination to purified template

Proteins bound(35S/3H)b

Fractions added'

L/N NS/N

L <0.005c

NS 0.232

L+NS 0.238 0.249

a35S-labeledL and NSfractionswereprepared and

recombined with3H-labeledpurified template as de-scribed in the text.

bTemplate-boundproteins isolatedonglycerol gra-dientswereanalyzedonSDS-polyacrylamide gels.

'NoLproteinwasdetected on thegel.

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TABLE 3. RequirementsforthebindingofL and NSproteins

Proteins bound Incubation

(35S/3b

Reaction mixture temp0a H)

(0C)

L/N

NS/N

Completec 31 2.41 0.80

-Mg2e,

ATP, GTP, 31 2.22 0.78

CTP

Complete 31 2.59 1.08

Complete 4 2.54 0.86

aAfter incubation at the indicated temperature,

all

gradientswereloaded inacold room andcentrifuged at40C.

bTemplate-bound proteins isolated onglycerol gra-dientswereanalyzedonSDS-polyacrylamide gels.

c35S-labeledsolubilizedviral proteins were prepared

asdescribed in the textand recombinedunder stan-dard assay conditions. All glycerol gradients corre-spond toprereactionmixtures withrespect to nucleo-tides andMg2+.

unlike transcription, the binding reaction

oc-curred at40C.

DISCUSSION

Thereconstitutionoftranscriptase activity by

recombination of template and soluble L and

NSproteins impliesthat theseproteinsrebind totemplatein afunctionally significantmanner. Therefore, the assay to detect this binding is

baseddirectlyontheconditions for

reconstitu-tion of transcriptase activity in vitro (7). The

greatest difficulty in developing an assay for

rebinding is the denaturation and aggregation of

the reaction components, especiallythe L

pro-tein. The assayreported here circumvents this

problem by isolating the nucleocapsid with

bound proteins on glycerol gradients through

whichaggregated materialspellet. Theamount

of bound L and NS proteins detected by the

assay saturates with approximately the same

number of L and NS molecules per 2,000 N

molecules as are present in the intact virion,

indicating that the binding detected is a specific

rebinding, similar tothat whichoccursin vivo.

Inaddition, theseresults suggest that the sites

onthetemplate available for binding L and NS

proteinsareprobablyfully occupiedinthe

pack-agedvirion.

In determining the numbers of L and NS

molecules bound in the intact virion, it was

alwaysnoted that the numbers ofNS molecules

weresignificantlygreater than the numbersof L

molecules. Of course, such calculations depend

on thestillunsatisfactory molecular weight

as-signments for L and NS proteins. The values

listed in Table 1 (131 molecules of L and 267

molecules of NS per 2,000 N molecules) were

calculated by using 190,000 and 40,000 as the

J. VIROL.

molecular weights for L and NS proteins and

result in an NS:L stoichiometric ratio of 2:1.

This ratiocanbecomparedtothe NS:L ratio of

1:1 reported for intact virions (and the in vitro

purified polymerase) by Naito and Ishihama

(11). These authors also used VS New Jersey

virusand molecular weights of 40,000 to 45,000

for NS protein and 190,000 for L protein.

Al-thoughreliable stoichiometric ratios await

bet-termolecular-weight assignments,especially for

NS protein, our data suggest some caution is

necessary in accepting the 1:1 stoichiometric

ratio forNS to L protein in the intact virion.

When NS and L proteins aresolubilizedfrom

thevirus, it isimpossibletodetermine to what

extenttheyexist insolutionaseitherindividual

proteins or asL:NScomplexes. It is clear that

when using the solubilized viral proteins as a

sourceof L andNS proteins, both L and NS are

bound to template, but analyzing the binding

from such acomplex,and as yetuncharacterized,

mixtureisdifficult. Our experimentsadding NS

and Lproteins separatelytothetemplatearea

start insortingoutthebindingcapabilitiesof L

andNS proteins. The results with the purified

NSprotein show that NS proteinbinds to the

template directly in the absence of L protein,

i.e., thetemplatemusthaveanaccessible

bind-ing site forNS protein.However, thebindingof

Lproteinappearstobedependentonthe

pres-enceof NSprotein.Since the inherentinstability

of Lproteinisaccentuatedby the manipulations

necessary to separate Lproteinfrom NSprotein,

this observation maysimply reflectthe

stabili-zation of L protein by NS protein. However,

since Land NSproteinscanbindtoeach other

(11), itispossiblethat Lproteinmaybind

indi-rectlytotemplatevia NSprotein.Alternatively, the NS protein may modify the template in

some way (e.g., exposing sites on the RNA)

whichallowsLproteintobinddirectly to

tem-plate.

Ourdataonthe numbers of bound L andNS

molecules have been arbitrarily calculated per

2,000 Nmolecules,notper virion.However,since

reported values for the numbers of N protein

per virion range from 1,000to2,000 (6, 12), we

would roughly estimate 65 or 130 L molecules

and134 or 267 NS molecules per virion. These

numbersmay becomparedtotheprobable

num-ber of promoter sites pergenome.Asingle

pro-moter site per genome is consistent with the

sequential transcription of VS virus genes

de-duced from UV-inactivation studies(4, 2,3),but,

atmost, the VS virus genome couldreasonably

beexpectedto have fiveor sixpromotersites,

onefor eachgene. Since wedetectmanymore

Land NS molecules

packaged

per virion than

could be boundtotheone tosixpromotersites,

http://jvi.asm.org/

(8)

it is likely that the nucleocapsid has another

class ofbindingsites for thepolymerase.These

may be"reserve" sites, whichallow the virion to

package an extra supply ofpolymerase

mole-cules.Experiments in which thetranscriptionof

solubilized whole viruscanbe stimulatedatleast

threefold by the addition of extrapurified

tem-plate (unpublished data, S.U.E.) confirm the

suggestion thatVS virus packages more

polym-erase than can be used.

The two classes ofbindingsitesmay bequite

different. The promoterbindingsitesare

prob-ably restrictedtospecificsequences of RNAat

one orafewsitesonthe genome. The reserve

binding sites may be determinedbymore

gen-eral interactions with RNA and/or N protein

and may be distributed allalongthe

nucleocap-sid. Since our reactionswere done under

tran-scriptase conditions, rebinding to both sites probably occurs, but the characteristics of the

reaction are dominatedbybindingtothemore

numerous reserve sites. Further experiments,

including the binding ofpolymerase to naked

genomicRNA,areneededtofullycharacterize

the two types ofbinding.

ACKNOWLEGMENTS

This resech wassupported by PublicHealth Service grants AI-11722 (S.U.E.) and AI-05292 (M.G.M.) from the NationalInstituteofAllergyand Infectious Diseases. S.U.E. is therecipient of Public Health Service Research Career DevelopmentAward AI-00013 from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED

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2.Abraham, G., and A. K. Banerjee. 1976. Sequential transiptionof thegenes of vesicular stomatitis virus. Proc.Natl.Acad. Sci. U.S.A.73:1504-1508.

3. Ball,L A. 1977. Transcriptionalmapping of vesicular stomatitis virus in vivo.J.Virol. 21:411-414.

4. Ball, iA.,and C. N. White. 1976. Order oftransciption of genesofvesicular stomatitis virus. Proc.Natl. Acad. Sci. U.S.A.73:442-446.

5. Bishop, D. H.L,S. U. Emerson, and A. Flamand. 1974. Reconstitution of infectivity and transcriptase activity of homologous andheterologousviruses: vesic-ular stomatitis(Indianaserotype), Chandipura, vesicu-lar stomatitis (New Jersey serotype), and Cocal Viruses. J.Virol. 14:139-144.

6. Bishop, D.H. L, and P. Roy. 1972. Dissociation of vesicular stomatitisvirus and relation of the virion proteins to the viraltranscriptase.J.Virol.10:234-243. 7. Emerson, S. U., and R. R. Wagner. 1972. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions.J. Virol. 10:297-309.

8.Emerson, S. U., andR. R.Wagner. 1973. Lprotein requirement for in vitro RNA synthesis by vesicular stomatitis virus. J.Virol. 12:1325-1335.

9.Emerson, S.U.,and Y.-H.Yu.1975.Both NS and L proteins arerequired for in vitro RNA synthesis by vesicular stomatitisvirus. J. Virol. 15:1348-1356. 10.Hunt,D.M.,S. U.Emerson,and R.R.Wagner.1976.

RNA-temperature-sensitivemutantsofvesicular sto-matitis virus:L-protein thermosensitivityaccounts for transcriptaserestriction of group I mutants. J. Virol. 18:596-603.

11. Naito,S.,and A.Ishihama.1976.Function and structure of RNApolymerasefromvesicularstomatitis virus. J. Biol. Chem.251:4307-4314.

12.Nakai, T.,and A. F.Howatson.1968.The fine structure of vesicular stomatitisvirus.Virology35:268-281. 13. Reichman4nM E.,W. KLSchnitzleln,D.H.L.Bishop,

R. A.Lazzarini, S. T. Beatrice, and R.R.Wagner. 1978.ClassificationoftheNewJerseyserotype of vesic-ular stomatitis virus into two subtypes. J. Virol. 25:446-449.

14.Wagner,R. R. 1975. Reproduction ofrhabdoviruses, p. 27-30.In H.Fraenkel-Conratand R.R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Publishing Corp.,New York.

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