Assignment of the temperature-sensitive lesion in the replication mutant A1 of vesicular stomatitis virus to the N gene.

Copyright C) 1985, American Society for Microbiology

Assignment

of

the Temperature-Sensitive Lesion in the Replicatio-n

Mutant

Al of Vesicular

Stomatitis

Virus

to

the

N

Gene

M. DAVIDMARKS,t JENNIFERKENNEDY-MORROW,t ANDJUDITH A. LESNAW* SchoolofBiological Sciences, University of Kentucky, Lexington, Kentucky 40506

Received 6 June1984/Accepted24September1984

The replication defect in the temperature-sensitive mutant Al of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis viruswasconfirmedby the absence ofintracellular nucleocapsidsin infected cells incubated at the restrictivetemperature. After preamplification, the relative yield of the Al N protein accumulatedintracellularly after 1 h of incubationattherestrictivetemperaturewasdecreasedby50% that of thewild-typeorrevertantAl Nprotein. This differencewasnotasapparentinpulse-chase experiments. The functional lesion in Al was correlated with a structural alteration in the N protein on the basis of the thermolabilityof thetemplateactivityof the Al Nprotein-RNA complex in in vitro transcription reactions and thecovariance of thisphenotypewith thetemperature-sensitivephenotype inaspontaneousAl revertant.This correlation wasconsistent with adirect role of the Nprotein in replication and allowed the assignment of the Ngeneto complementation group A.

The set of 48 temperature-sensitive (ts) mutants isolated

from the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus (VSV) has been classified into six

nonoverlapping complementation groups (designated A

through F), each of which presumably represents a

func-tional unit on the viral genome (19). Because only five VSV geneproducts (L,G,M,NS, and N; all structural) have been

identified (23), the existence of six complementation groups

in the New Jersey collection has been of considerable

interest. We, as well as others, have been studying these

mutants with the objective of identifying viral functions

involved inRNA synthesis and correlatingthem with viral

proteins. RNA synthesis in VSV involves two distinct

pathways: transcription (the sequential synthesis of a

47-base"leader" sequenceencodedattheprecise3' endof the genome, followed by the capped, methylated, and

polyad-enylated monocistronicmRNAsfortheN,NS, M,G,and L

proteins) and replication (the synthesis ofa genome-length

positivestrand which, complexed withN protein, serves as

atemplate for the synthesis ofnew genomic RNA) (1). Of the six New Jersey complementation groups, four contain members whichdisplay an RNA-negative phenotypeat the restrictive temperature in vivo (19). Analyses of viral RNA

species synthesized in vivo at the restrictive temperature

suggested that mutants Al and El (the prototypes of

com-plementation groups A and E, respectively) were defective

inreplication,that mutant

Bi

(the prototype of

complemen-tationgroup B) wasdefective in primarytranscription, and

thatmutant Fl (theprototype ofcomplementation group F)

wasdefective in both transcriptionandreplication (12). The

transcription

defects

were confirmed by in vitro studies (2,

10, 21).

In vitro reconstitution studies demonstrated that three of theviralproteins, N(the nucleoprotein) and L and NS(the two subunits of the viral encapsidated transcriptase), were necessary and sufficient for transcription (5). Presumably,

* Correspondingauthor.

tPresentaddress: Department of Plant Pathology, Purdue Uni-versity,West Lafayette, IN 47907.

tPresent address: Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY40536.

these three proteins were also involved in the replication

pathway;therefore, thefour complementationgroups(A, B,

E, andF)displaying defects in RNAsynthesis were believed torepresentthe L, N, andNS genes. The extra complemen-tation group was postulated to represent a sixth, nonstruc-tural, virus-encoded protein involved in thereplication path-way (12).

Assignments have now been made for three of the four RNA-negative complementation groups. On the basis of in

vitro reconstitution experiments assayed in vivo, the ts

lesions in transcription mutants Bi and Fl have been as-signed to the L gene (2), and on the basis ofthe aberrant

electrophoretic mobility andpeptide map oftheNS protein

of thereplicationmutantElwith respecttothoseof the wild type(WT) and revertants, the NS gene has beenassignedto the E group (4, 11). The discovery that two of the four

RNA-negative groupsrepresented theLgeneeliminatedthe

necessity of invoking a yet-undetected sixth viral gene

product, and it seemedlikelythatcomplementationgroupA represented the N gene. Nevertheless, as a result of the initial failure to detect alterations in the Al N protein by

polyacrylamide gel electrophoresis and peptide mapping

(11), coupled with the high levels ofpolymerase activity (in excess of those in WT) in Al virions and transcribing

nucleocapsids assayedin vitroattherestrictive temperature (21), no assignment was made, and the possibility that

complementationgroupArepresentedasixth,nonstructural

protein was left open.

Inthe present communication,wepresentdatawhichnow

permit the assignment of the N gene and confirm the

assignment of a replication function to complementation

group A.

MATERIALSAND METHODS

Cell line and virus stocks. BHK-21 C13 cellswere propa-gated as monolayers as described previously (11), except thatthegrowth mediumwassupplementedwith2mM

L-glu-tamine. Mutants Al andEl and theWT (New Jersey sero-type, Hazelhurstsubtype)fromwhichtheyhadbeenderived (19) weregenerously provided by Craig Pringle. The spon-taneous revertants of El and Al were isolated as plaques

44

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that appeared atthe restrictivetemperature duringthe

titra-tion ofmutantenrichments.

In vivo viral nucleocapsid synthesis. Duplicate monolayers

were infected and incubated at 31 or39.5°C in thepresence

of [5,6-3H]uridine (specific activity, 35 to 50 Ci/mmol;New

England NuclearCorp., Boston, Mass.) andactinomycinD

(Calbiochem-Behring, LaJolla, Calif.). After6 h of

incuba-tion, the contents of each flask (including the overlaying

medium) were frozen, thawed, and separated by low-speed

centrifugation into supernatant (unbound-virion) and pellet

(bound-nucleocapsid) fractions. Virionspresent in the

super-natant (unbound fractions) were pelleted and analyzed by

density gradient centrifugation. Fractions were collected

and analyzed for radioactivity by liquid scintillation spec-troscopy. Viral nucleocapsids wereextractedfromthe

low-speed pellet (bound fractions) with 1% Nonidet P-40-2 M

urea and analyzed by sucrose density centrifugation.

Frac-tions were collected and analyzed as described above.

Additional details of the procedure are presented in

refer-ence6.

Intracellular viral protein synthesis and stability.Replicate

monolayers of5 x 106 cellswereinfectedat amultiplicity of

infection of25, and theinfection mixtureswerepreamplified

by incubationat31°C for3 h in the presenceofactinomycin

D(5,ug/ml). For thedetermination ofyields ofviralproteins

accumulated intracellularly, the medium was replacedwith

mediumcontaining10,uCi of

[35S]methionine

(specific

activ-ity, 600 to 1,400 Ci/mmol; Amersham Corp., Arlington

Heights, Ill.) and 0.01 mM unlabeled methionine (0.1 the

normalconcentration) per monolayer. Incubation wasthen

continued for 1h at either31 or

39.8°C.

Forthe

determina-tion of the stability of pulse-labeled viral proteins, the

medium was replaced with medium containing 10 ,uCi of

[35S]methionine

per monolayer, and the monolayers were

incubatedfor10 min(pulse) at theappropriate temperature

(31 or 39.8°C). A 10-fold excess of unlabeled methionine

(final concentration, 1 mM) was then added, and the

infec-tion mixtureswereincubatedatthe

labeling

temperaturefor

various periods of time (chase). Extracellular (overlaying

medium) and intracellular (monolayer) fractions were then

disrupted with sodium dodecyl sulfate (SDS) and analyzed

by SDS-polyacrylamide gel electrophoresis in gels

contain-ing 10%polyacrylamide and 0.13 or 0.5% bisacrylamide in

thediscontinuous buffersystem(9). Aspreviously reported

(4, 11),the El NS

protein

is anelectrophoretic variant. To

display the WT and El NS proteins on a single gel, we

increased the

bisacrylamide

concentration to 0.5%.

Addi-tional details of thelabeling protocols, electrophoresis, and

fluorography can be found in reference 6. The data were

quantitated by determiningtheamountofradioactivity

con-tainedin eachoftheviralproteinbandsexcisedfromthegel

by liquid scintillation spectroscopy asdescribedpreviously

(6).

In vitro functional analyses. WT, Al, and revertant Al

virionswerepurifiedbydifferential and rate sucrose density

gradient centrifugationsfrominfections incubated for48 h at

31°C, and transcribing cores containing the L, NS, and N

proteins and RNA were prepared as described previously

(10). The procedure for the preparation of enzyme (L and

NS) and template (N and RNA) fractions (10) has been

modified. Transcribing cores at a protein concentration (14)

of 2 mg/ml in 0.1 M column buffer (0.1 M

NaCl,

25%

glycerol, 10 mM Tris-hydrochloride [pH

8.01)

were treated

withanequal volumeof 2x disruption buffer (4 M NaCl, 2% Nonidet P-40. 10 mM

Tris-hydrochloride

[pH 8.0], 2 mM

dithiothreitol) for 2 h at

0°C.

Under theseconditions, Land

NS proteins were released, and the templates became

floc-culated and could be removed by low-speed centrifugation

(11,100 xg,40C,30min). Thesupernatant(enzyme) fraction

wasfreed ofresidual templates byhigh-speedcentrifugation

(176,700 x g, 4°C, 3 h) and stored at -700C. Immediately

before use, the enzyme fractionsweredialyed against0.3 M

columnbuffer. ItwasnecessarytomaintainaNaCI

concen-tration above1 M during storage at -70°C toprevent the L

protein from

aggregating.

Template fractions obtained

dur-ingthelow-speedcentrifugation described abovewerefreed

of residualpolymeraseactivity by

being

dilutedto0.2

mg/ml

in 0.1 M column buffer and retreated withanequalvolumeof

2x disruption buffer for2 h at0°C.Thetemplateswerethen

collected by low-speed

centrifugation

as described

above,

resuspended in0.1 Mcolumn buffer, adjustedto0.5 mg/ml

(on thebasis ofprotein determinations),andstoredat-70°C

until used. Theproteincompositions ofthevarious fractions

weredeterminedbySDS-polyacrylamide gel

electrophoresis

in the discontinuous Tris buffer system (9) as described

previously (10). The residual polymerase

activity

ofthese

template preparations was 10% or less than the

activity

obtained afterreconstitution withthe enzyme fraction.

The thermostabilities ofthese fractions were determined

byheating thematvarioustemperaturesorfor various times

at agiven temperature before reconstitution

(in

the case of

enzyme and template fractions) with the

appropriate

un-heated WTfraction. Polymerase

activity

was then

assayed

at the optimal temperature for the reaction

(310C).

The

conditions forthe

transcription

reactionand assayonDEAE

filterpaper diskswere asdescribed

previously

(10).

RESULTS

In vivo viralnucleocapsid synthesis.

Replication

defects in

mutantsAl and

El,

the prototype mutants of

complemen-tation groups A and E ofthe New Jersey serotype

(Hazel-hurst subtype) of VSV (19), were suggested by the absence

of detectable

genome-sized

viral RNA in infected cells

incubated at the restrictive temperature (12). Because the

level of

genome-sized

RNA in WT VSV infections is very

low in relationtothelevelofmessage-sized RNA, analyses

of intracellular viral

nucleocapsids

have beenused to

verify

replication defects suggested by the absence of

genome-sized RNA in mutant-infected cells

(22).

Although

this

approach was successful in thecase ofanalyses of Indiana

serotype mutants, the failure ofthe

commonly

used

freeze-thaw

technique

(22) toreleaseviral

nucleocapsids

fromWT

New Jersey infections precluded the

application

of this

approachtothe NewJersey serotype mutants. A

technique

fortheisolation of intracellularNew

Jersey

serotype

nucleo-capsids

which is based upon the extraction of

freeze-thaw-disrupted

cells with Nonidet P-40 and urea was

re-cently

developed

(6); we used this

technique

to

investigate

mutants Al and El. The absence of detectable

nucleocap-sidsinAl-andEl-infected cells incubated attherestrictive

temperature confirmed the

replication

defects in both mu-tants (Fig. 1).

Intracellular viral protein synthesisand stability.

Knipe

et al. (8) have shown that certain altered viral

proteins

are

rapidly degraded at the restrictive temperature in vivo.

Therefore,

in anattemptto determine thealtered

protein

in

Al,

weexamined theyieldsof viralproteins synthesizedand accumulated in Al-infected cells after 1 h ofincubation at the restrictive temperature, after preamplification. Visual observation of thefluorograms of

SDS-polyacrylamide gels

ofAl and control

samples suggested

adecrease in the

yield

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5

p

4

0

1,

0r-16

14

162

'10

X Q

u

6r

UNBOUND

10

20

30 40

10

20 30 40

10 20 30 40

FRACTION

NUMBER

BOUND

1-F

0

10

20 30

40

10

20

30

40

10

20

30 40

FRACTION

NUMBER

FIG. 1. Sucrose density gradient analyses of[3H]uridine-labeled virions (unbound viral particles [A]) and nucleocapsids (bound viral particles [B]) synthesized in cells infected with WT (a), mutant El (b), or mutantAl (c) virus at 31°C

(O*-

) or 39.5°C

(0----

-0). Fraction1 correspondstothebottomof the gradient.

ofthe N protein relative to those of theother Al proteins

andcontrolproteins(Fig. 2).

Quantitationofthe datashowninFig.2confirmedthatthe

yield oftheAl Nprotein accumulated during a 1-h labeling

period at the restrictive temperature after preamplification

wasreducedby50%with respect toAl infections incubated

atthe permissive temperature and with respect to control

infections (WT, revertant Al, El and revertant El)

incu-batedat either temperature(Table 1). Intheseexperiments,

the overlaying medium (extracellular fractions containing

mature virions and shed proteins) was removed, and the

ittracetlularand extracellularfractionswereanalyzed

sepa-rately. Thisprocedureprovidedacontrol forpossible

differ-ewesinthe stabilitiesofintracellularviralproteins and viral

proteias associated with mature virions released into the

extracellular fraction in control infections. Moreover, it ensured thatany decrease in theyield of an intracellular viral

proteinwas nottheresultof increasedshedding(13). In the

caseofthe Alinfections incubatedattherestrictive temper-ature, no Nprotein appeared in thecorresponding extrace-Ilular fraction(Fig. 2B).

To ruleout thepossibility that the decrease in the relative accumulation of the Al Nproteinwas asecondaryeffectof theblock inreplication, mutant El, whichis also defective

in replication, was included in the preceding experiment.

Therationale was thatablock in 40S RNAsynthesis would preventnucleocapsid assembly,which inturncould resultin theaccumulation of WT N protein inan abnormal cellular compartment, where it could be degraded. Because El,

which codesfor analteredNS proteinbut a WT Nprotein,

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failsto synthesize40S RNA and therefore cannotassemble nucleocapsids at the restrictive temperature in vivo, it constituted the appropriate control. Indeed, the relative accumulation of the El N protein did not decrease at the restrictivetemperature(Table 1). It is interestingto notethat the relative accumulation of the altered El NS protein did also not decrease at the restrictive temperature (Table 1). This finding is inagreement with the earlierobservation by Knipeetal. (8) that notall alteredproteins aredegraded at the restrictive temperature.

The decrease in the relative accumulation of the Al N protein could have resulted from either its degradation orits reduced synthesis atthe restrictive temperature. We there-fore determined the stabilities ofWT, Al, andrevertantAl intracellular proteins pulse-labeled and chased for various time intervals at the restrictive and permissive (control) temperatures. Fluorograms of the gels are shown in Fig. 3. The data were quantitated and expressed as relative ther-mostabilitiestodetermine whetheradecrease in the relative yield ofa proteinat 39.8°C reflected thermolability of that protein. The relative thermostability of the Al N protein but notthose of the WT andrevertantAl Nproteins decreased slightly asthelength of the chase increased (Table 2).

0IE

L

G N NS

WT Al RAI El REI

V P R P R P R P R P R

-.- eO

V.- V

-MEW

M

0FI

WT Al RAI REI El REl

V PR P R P R P R P R

L _

G _

N w

NS

M _

FIG. 2. SDS-polyacrylamide gel electrophoresisof WT and

mu-tant viral proteinsaccumulated intracellularlyat thepermissive or

restrictivetemperature.After 3 h ofpreamplificationatthe

permis-sive temperature, duplicate monolayers infected with WT, Al,

revertantAl(RAl), El,orrevertantEl(REl)viruswereincubated

for 1 h at31°C (P)or39.8°C (R)inthepresenceof[35S]methionine

and actinomycin D. The proteins in the extracellular fractions

(overlayingmedium[B])and intracellularfractions(disrupted

mono-layers [A])wereelectrophoresed throughSDSgels containing10%

acrylamideand0.5%bisacrylamide. Shownarefluorogramsofthe

driedgels. 14C-labeled marker virion proteins (V) are identifiedto the left of eachgel. Detailsof the infections andelectrophoresisare

[image:4.612.320.565.95.171.2]

givenin the text.

TABLE 1. Temperature dependenceof relative yields of viral proteins accumulated intracellularlya

Relativeaccumulationb of indicated protein

Virus

L G N NS M

WT 86 119 107 84 106

Al 110 130 58 100 126

Revertant Al 86 126 105 85 102

El 89 111 96 96 117

Revertant El 84 117 106 95 100

aBandscorrespondingtothefive viral proteinswere cutfromeachlaneof

the gel containing the intracellular fractions (Fig. 2A) byusingthefluorogram

as atemplate. The.radioactivity in each bandwasdeterminedasdescribedin the text.

bRelative accumulation=(relative yieldat39.8°C/relative yieldat31°C) x

100, where relative yield at31°C =(cpminagiven protein/sumofcpm in all

proteins inthe sample) x 100.

In vitro functional analyses. Because the data suggested

that the N protein was a likely candidate for the gene

assignmentin complementationgroup A,we examined

mu-tant Al with an in vitro functional analysis designed to detect lesions in the three viral proteins (L, NS, and N)

required fortranscription(10). The rationale is based on the

role ofthe N protein-RNA complex as atemplate for both

replication andtranscription.Wereasoned thatalthough the

Al mutant wascapable ofdirecting primarytranscriptionat therestrictive temperature(39.5°C)invivo (12),analteration inthe N protein could affect the relative thermostability of

the N protein-RNA complex at elevated temperatures in vitro. The thermal inactivation could be detected by the

failure of the heat-treated complexes to direct in vitro

polymerization.

In thefirstsetofexperiments, thethermolabilitiesof WT,

Al, and revertant Al transcribing cores (L, NS, N, and RNA), enzymefractions(L andNS), andtemplatefractions

(Nand RNA)werecomparedas afunction oftemperatureof

inactivation. The thermostabilities ofall three transcribing

cores were comparable (Fig. 4A). Similarly, the three

en-zyme preparations were indistinguishable in this assay, in

agreementwith a previousreport (2). Whereas transcribing cores withstood heating for 15 min at 45°C before being

assayed for polymerase activity (Fig. 4A), the enzyme

fractions, when treated for 15 min at 45°C before being

reconstitutedwith the WTtemplate, wereinactive(Fig.4B).

As has been pointed out previously (10), the enzyme is

apparentlystabilized by its interactionwith the template in

transcribingcores. Incontrasttothetranscribing cores, the

ability of the WT template to direct polymerization was

retainedafter 15 minof heatingat60°C(Fig.4C). The greater

thermolabilityof thetranscribingcoresreflectedthatoftheir

least stable component, the enzyme (10). However, in contrast toboth WT andrevertantAltemplatefractions,the

Al template fraction lost50% ofits activity after 15 minof

incubationat50°Candwascompletelyinactivatedby 15 min

of incubationat 60°C (Fig. 4C).

The thermostabilities of these fractions were more

pre-cisely determined in a related set ofexperiments in which

theviralfractions were preheated forincreasingtime inter-vals at a temperature determined from the data in Fig. 4.Al

transcribing cores preheated at 42°C for 0 to 120 min

appeared to be more stable than WT or revertant Al

transcribingcores(Fig.SA).Nodifferences in the stabilities

of the three enzyme fractionsweredetected after0 to45min of incubationat41°C (Fig. SB). In contrast,theAl template

fraction was distinguished from both WT and revertant

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[image:4.612.68.302.308.602.2]

WT Al RAl (E) V WT Al RAI

NS N :'W

o 30 60 0 30 60 0 30 60

MINUTES OF CHASE

0 30 60 0 30 60 0 30 60 MINUTES OF CHASE

Al RAl @ V

~

- L

-fr) V W T L

G

NS

N

M -

_-0 3_-0 60 0 30 60 0 30 60

MINUTES OF CHASE

WT Al RAI

Gso NS

N _

M _

0 30 60 0 30 60 0 30 60 MINUTES OF CHASE

FIG. 3. SDS-polyacrylamidegelelectrophoresis of pulse-labeled intracellularviral proteins chased for various times. Duplicate WT,Al, andrevertantAl(RAl) infections preamplifiedfor 3 hat31°Cin thepresenceof actinomycinDwerepulse-labeledwith [35S]methionine for 10minat31 (A andB)or39.8°C (Cand D). For the chase, excessunlabeled methionine (1 mM)wasadded, and incubationwascontinued

atthelabelingtemperature for 0, 30,or60min. Extracellular fractions (overlaying medium[BandD])andintracellular fractions (disrupted monolayers [A and C])weredissociated with SDSand electrophoresedonpolyacrylamidegels (10%acrylamide-0.13%bisacrylamide) in the

discontinuous Tris buffersystem. Shownarefluorograms ofthedriedgels.'4C-labeledmarkervirion proteins(V)areidentifiedtothe leftof eachgel. Details of thelabeling protocols and electrophoresis aregiveninthetext.

Altemplate fractions by the loss of60% of its activity after 10 minof incubation at52°C (Fig. 5C).

DISCUSSION

Attempts to assign the lesion in the Al mutant to a structuralprotein by in vitro transcription analyses of

tran-TABLE 2. Relativethermostabilities of pulse-labeled intracellular viral proteins as a function of the length of the chasea

Length of Relativethermostabilitybof indicated

Virus chase protein

(min) L G NS N M

WT 0 119 104 100 94 95

30 104 109 99 98 94

60 89 112 97 100 103

Al 0 121 109 99 92 95

30 84 120 113 87 100

60 92 133 102 80 111

RevertantAl 0 131 100 98 93 95

30 98 102 107 100 91

60 92 96 103 108 94

aTheradioactivitycontained in the bands corresponding to thefiveviral

proteins ineach lane of thegelscontainingthe intracellular fractions shown in

Fig.3A andCwasdetermined asdescribedin the text.

b Relativethermostability=(relativeyield at39.8°C/relative yieldat31°C)

x 100, where relative yield = (cpm in a given protein/sum of cpm in all

proteinsinthesample) x 100.

scribingcoreshadpreviously beenunsuccessful(21).

How-ever,wereasoned that because thestability of the transcrib-ing cores reflects that of their least stable component (the enzyme),alesioninthe N

protein

which lowersthestability of the template fraction could be masked by the inherent lability of the enzyme fraction. Indeed, the transcribing

cores as well as the enzyme fractions were completely inactivated by being heated for 15 min at 50°C (Fig. 4). Under theseconditions, the WTand revertantAl template fractions were relatively unaffected, whereas the Al tem-plate fraction lost 50% ofitsactivity (Fig. 4). Onthe basis of theseobservations, werejectedthepossibility that comple-mentationgroup Arepresenteda nonstructuralprotein.

The thermolability of the Al virion template containing the N protein and the genomic RNA coupled with the covariance of this phenotype with the ts phenotype in a

spontaneous revertantof Alallowedustoassignthe Ngene

tocomplementationgroupA.Thelowrelative accumulation of the Al Nproteinatthe restrictivetemperatureinvivowas consistent with this assignment. The small degree of Al N protein degradationdetected in thepulse-chase experiment (Table 2)doesnot accountfor thelowrelative accumulation of the Al N protein observed duringthe 1-h continuous-la-beling experiment (Table 1). We have not resolved this discrepancy. The low relative accumulation may reflect differential synthesis ofWT, revertant Al, and Al N

pro-teins at the restrictive temperature. However, this

discrep-ancy may alternativelyreflect differences in the

experimen-tal conditions used in the twosetsoflabeling experiments.

V

G NS

N

M

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[image:5.612.142.477.69.360.2] [image:5.612.56.296.540.674.2]

A10 WT 300 A1 REV Al

s ~~~420 300

o 20 40 so o 20 40 eo40420

n

4

2X

.

2

500 .500 0

0 20 40 60 0 20 40 60 0 20 40 60

TIME(min)

B s WT - A1 - REV A1 30

400

0~ 0 4060₃₀₀0 020 40 o20 40

X 400

450,500 04 ,50

0 20 40 60 0 20 40 60 0 20 4060

TIME

(min)

C20 WT Al REV Al o

00

Cf) 0 , o

I 10 500

~~~500

600

x ~

~~~~5060

0.~~~~~~~~~~~~~~s

05

0 20 40 60 0 20 40 60 0 2040680

TIME (min)

FIG. 4. Thermal inactivation ofWT, Al, andrevertant Al(REV Al)transcribingcores, enzyme fractions, and templatefractionsas a

function oftemperature. (A)Transcribingcores(5jig)in 50 of 0. 1 M column bufferwereheated for 15minat30,42, 45,or50'Candassayed

forpolymeraseactivityat30°C ina100-,ul reaction mixture.(B) Enzymefractions(25 p.l)in 0.3 Mcolumn bufferwereheated for 15minat

30,40, 45,or50°C,reconstituted with 5 p.gof unheated WT template, andassayedforpolymerase activityat30°Cina100-p.lreaction mixture.

(C)Template fractions (10 p.g)in0.1 Mcolumn bufferwereheated for 15 minat0, 50, or60°C, reconstituted with 25 p.1 ofunheated WT

enzyme,andassayed for polymerase activity at30'Cina200-p.l reaction mixture. Details of thepreparationoftranscribingcores, enzyme

fractions, and template fractions, the polymerase reaction conditions, and the assaying of 20-,ul samples (inthecaseof the 100-,ulreaction

mixtures) and 40-,ul samples (in thecaseof the200-,ul reactionmixtures)aregiveninthetext.

Similarresults were obtained during an analysis of the Dl mutant(6). This mutant also encodes an altered N protein which exhibits low relative accumulation but minimal deg-radation during pulse-chase analysis. This discrepancy ap-pears to be restricted to altered N proteins, as the data obtained from continuous-labeling and pulse-chase experi-mentsareconsistentfor themutantCl Mprotein(6)and the WT Lprotein (6;Tables 1 and2).

The assignment of the N protein to complementation

groupAcompletesthegeneassignmentsof theexistingNew Jersey serotype (Hazelhurst subtype) complementation

groups: Lgene, complementationgroups B and F(2, 10);G

gene, none;Mgene,complementationgroupC(6);NSgene,

complementationgroup E(4, 11); andNgene, complemen-tationgroupA(this paper). Asetoftsmutantsof the New Jerseyserotype(Concansubtype)hasrecentlybeen isolated andclassified intotwo complementation groups: CC/A and CC/B.On the basis ofintersubtypic complementation,these twogroupscorrelated with the HazelhurstsubtypegroupsA and B, respectively. (A. D. Byrd, J. Kennedy-Morrow, M. D. Marks, and J. A. Lesnaw, J. Gen. Virol., inpress). Although nointerserotypic complementation at the level of

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[image:6.612.168.457.71.528.2]

A35

WT Al

3%'

REV

Al

60:

30 0'

₃₀₁

60' 30

O 25 1' 90

20 ' 1

9R'

90

V'C

20-

0o

04

o

o2

0e

2

~~~~~~~~~~~~120"

0.15

120'

10

5

0 20 4060 0 204060 0 204060

TIME

(min)

BIG.

5TiWT Al

REV Al

0

Irl 15

X3 ~ ~ 5'3' 0

0 30'~~~~~~~~~~0 0

1 45'445' 45'

0 204060 0 2040c60 0 20 4060

TIME

(min)

WT

Al of

REV

A 0

0' 10'

6

Cv) ~~10

20'

05

20 30'

30'

3

~~~~~~~~10'

2-20 30'

I

0 20 40 60 0 20 40 60 0 20 4060

tIME

(min)

FIG. 5. Thermal inactivation ofWT, Al, andrevertantAl(REV Al) transcribingcores, enzy'mefractions, andtemplatefractions as a

function of time.(A)Transcribingcores(20p.g)i'n100p.1of 0.1 M columnbufferwereheatedat420Cfor0, 30, 60, 90,or120,min andassayed forpolymerase activityat30°Cin a200-p.lreactionmixture.(B) Enzymefractions (25,ul) in 0.3Mcolumnbufferwere heated at41°C for0, 15, 30,or45min, reconstitutedwith 5p.gof unheated WTtemplate,andassayedforpolymeraseactivityat30°Cin a 100-,ulreactionmixture. (C)Template fractions (5p.g)wereheated at52.5°C for 0, 10, 20,or30min, reconstituted with15 ,ulof unheatedWT enzyme, andassayed forpolymerase activityat30°C in a100-pLIreactionmixture. Additionaldetails are given in the text.

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[image:7.612.150.445.75.647.2]

PFU productionhas been detected between NewJerseyand Indianamutants, thesetwo setsof NewJersey complemen-tationgroups can now be correlated with Indiana comple-mentationgroupsIthroughV(17)onthebasisof theirgene

assignments: complementationgroups B and Fcorrespond to complementation group I (L gene); complementation group C corresponds to complementation group III (M gene); complementationgroupE correspondsto complemen-tation group II (NS gene); and complementation group A

corresponds to complementation group IV (N gene). It is interestingto note that although theextracomplementation

group in the New Jersey mutant collection was originally

considered to represent a sixth viral protein, in factnot all the viral structural proteins are represented; nogroup

cor-respondsto the G protein. Nounique gene assignmentcan

be made formutant Dl, the sole representative of comple-mentation group D (6, 18), and the complementation

ob-served between mutants belonging to complementation

groupsB and F wasapparentlyihtracistronic (2, 19). On the basis of the absence of intracellularnucleocapsids (Fig. 1) andgenome-sized viral RNA(12) and the presence

ofprimary transcription (12) in Al-infected cells incubated at the restrictive temperature, a replication defect can be

assignedtothe Al mutant.Thestructural lesion in the Al N protein, togetherwith thetslesion in Alreplication, implies areplicativerole for the Nprotein.A similarcorrelation has been made in thecaseof the Indianaserotype mutanttsG41 (complementation group IV) (20). These genetic data are consistent with thereplicative role for the N protein postu-lated by Kingsbury (7) andBlumbergetal. (3). Accordingto this model, genome-sized RNA synthesis is coupled to nucleocapsid assembly, and the dependence ofreplication

upon continuous protein synthesis (16, 24) reflects the re-quirement for available N protein. Direct evidence for this model hasrecentlybeenreported byPattonet al. (15), who demonstrated that N protein alone satisfies the requirement forprotein synthesis in acoupled in vitro translation-repli-cation system.

The Nproteinappearstoplayadual role inreplication: (i) complexed with genomic RNA, the N protein serves as a template for replication; and (ii) newly synthesized N

pro-tein is required fornucleocapsid assembly. We have not at present resolved which of these functions isimpairedin the Al mutant. The ability of the Al N protein to function in primary transcription atthe restrictive temperature in vivo (12)suggeststhatthetemplateisnotaffected.However, the templateroles of the N proteinin transcription and replica-tionmaynotbe identical. Moreover, the increased

polymer-ase activity observed in in vitro reactions containing

heat-treated Al transcribingcores (21; Fig. 5), togetherwith the decreased polymerase activity observed in reactions di-rectedby heat-treated Al template fractions(Fig. 4and 5), suggestedapossible alterationintemplate function. Weare currently analyzing the products synthesized in these in vitro reactionsin an attempt toshed lighton thismatter.

ACKNOWLEDGMENTS

We thank M. S.Coleman, S. G. Zimmer, and A. Byrd for valuable

discussions.

Thisworkwassupported by Public Health Servicegrant

R01-AI-13574 from the NationalInstitute ofAllergy and Infectious Diseases

andby Biomedical Researchgrant5-505-07-115-07.

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canyoubepositive?Cell 26:143-144.

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atemperature-sensitivemutantof vesicular stomatitis virus.J. Virol. 39:377-389.

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