Temperature-sensitive mutants of Chandipura virus. II. Phenotypic characteristics of the six complementation groups.

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0022-538X/80/01-0107/08$02.00/0

Temperature-Sensitive

Mutants of

Chandipura Virus

II. Phenotypic

Characteristics of the Six

Complementation Groups

D. A. GADKARItAND C.R.PRINGLE*

Medical Research CouncilVirology Unit,Instituteof Virology. GlasgowGIl 5JR,Scotland

Fifty temperature-sensitive (ts) mutantsof the rhabdovirusChandipuravirus have been classified into six complementation groups designated ChIto ChVI.

GroupChI contains 44 mutants,groupChII contains 2 mutants, and the remaining groupshave 1mutanteach. Mutants ingroupsChI, ChIII,ChIV, and ChVI had

RNA-negative phenotypesinexperiments measuring amplificationofRNA

syn-thesis at restrictive temperature. The two mutants in group ChII had

RNA-positivephenotypes, and the virionswerethermolabile. MutanttsCh851 ofgroup

ChVwasalso RNApositive,and the Mpolypeptideof thismutantappearedto

be unstable in cellsincubated atrestrictive temperature. It is likely, therefore,

thatcomplementationgroups ChIl and ChV represent thegenescodingforthe

twoviralproteinsof the virionenvelope.Noprecise assignment canbe made in

thecaseof the fourRNA-negativegroups,since allthemutantsexaminedshowed

somepolymerase activityinvitroatrestrictivetemperature.Anattempt toobtain

polymerase mutantsby screening for sensitivitytorifampinwasnotsuccessful. Sixtemperature-dependent hostrangemutants (the tdCEphenotype) of

Chan-dipuravirus failedtomultiplyinchickenembryocellsatrestrictivetemperature,

but otherwisetheydifferedintheir hostrangepropertiesfromsimilarmutantsof vesicular stomatitis virus.

Fifty

temperature-sensitive

mutantsof Chan-dipura

virus,

a human rhabdovirus isolated in

India, have been classified into six

complemen-tation groups.

Group

ChI contains 44mutants, group ChII contains two mutants, and groups

ChIII,

ChIV, ChV,

and ChVI have onemutant each. Weak

complementation

wasobserved

be-tweencertain mutantsin group

ChI,

and itwas

possible

to

distinguish

two

subgroups

ChIA and ChIB bytheir interaction with mutant ts

Chl,

an

arbitrarily

chosen prototype for group

ChI

(1).

Wenowdescribe characterization of the

phe-notype of the mutants of groups

ChII, ChIII,

ChIV,

ChV,

and ChVI anda

representative

sam-ple ofthe mutants of

subgroups

ChIAandChIB.

Theproperties examined includedRNA

pheno-typein

vivo,

RNA

polymerase

activity

in

vitro,

thermal

lability,

and intracellular

protein

syn-thesis. Evidence is

presented

which associates

complementation

groupChV with theM

protein

transcriptionalunit of thegenome.

The isolationand hostrange of six

tempera-ture-dependent

host range

(tdCE)

mutants of

Chandipura

virusarealso described.These

mu-tants of

Chandipura

virus failed to

multiply

in chicken

embryo

(CE)

cellsatrestrictive

temper-tPresent address: National Institute ofVirology,Pune 411 001,India.

ature, butotherwise were distinct in their host rangeproperties from previously described tdCE

mutants of the Indiana and New Jersey

sero-types ofvesicular stomatitis virus (VSV) (10).

MATERIALS AND METHODS

Cells. The BS-C-1 line of African green monkey kidneycellswaspropagated inrollerbottlecultures in Eagle minimum essential medium (Glasgow modifi-cation) supplemented with 10% calf serum. BHK-21 clone13cellsweresupplied routinely by the Cytology Department of the Institute of Virology.

Bovine embryo kidney (BEK) cells and hamster embryo (HE)cellswereobtained fromGibco Biocult,

Glasgow. Feline embryo (FEA) cells were obtained from 0. Jarrett, Department of

Veterinary

Pathology, University of Glasgow, and rat embryo (RE) cells were

suppliedby J. Macnab of the Institute of Virology. Mouse embryo (NIH/3T3) cells were obtained from S. A. Aaronson, National Institutes of Health, Be-thesda, U.S.A.

SecondaryCE cell cultures were prepared from eggs obtained fromaleukosis-free flock maintained by the Poultry Research Institute, Edinburgh, Scotland.

Virus.Chandipuraviruswasobtained from F. Mur-phy, Center for Disease Control, Atlanta, Ga., and cloned by three sequential isolations from single plaques on BHK-21 cell monolayers. Isolation and classification of the ts mutants ofChandipura virus have beendescribedinthe preceding paper(1).

The rifampin-sensitive mutant (VSV-29) of VSV Indianaand itswild type (VSV-VJ) were kindly

pro-videdby Ch.Chany, INSERM U-43,H6pitalSt.

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108 GADKARI AND PRINGLE

cent de Paul, Paris, France. The Evans vaccine strain of vaccinia virus was obtained from H. Pennington, Institute of Virology.

Determination of RNAphenotype. BS-C-1 cell monolayers in 30-ml bottles were incubated at 37°C for2hin the presence of 10

fig

of actinomycin D per ml (Cosmegen; Merck Sharp and Dohme, Rahway, N.J.). The cultures were then infected induplicate at a multiplicity of 10 PFU/cell, and the virus was al-lowed to adsorb at +4°C for 30 min. Eagle medium containing10,ugofactinomycinDperml and 10,uCi of [3H]uridine per ml (26 Ci/mmol; Radiochemical Centre, Amersham) was added to each culture bottle, and one set ofcultures was immersed in a water bath at39.5°Cwhile the other set was incubated at 31°C. At 9hafterinfection, 2 ml of a solution of200-yg/ml proteinase K (Boehringen-Mannheim) in 0.01 M Tris-hydrochloride (pH 8.0) containing 0.01 M EDTA, 0.01

MNaCl, and 0.2% (wt/vol) sodium dodecyl sulfate was addedtoeachmonolayer, and the cellsweredigested

at 20°C for 18 h. Trichloroacetic acid-precipitable radioactivity was measured for each sample in an Intertechnique SL30 liquid scintillation counter.

Temperature shift experiments. The effect of temperatureshift onthesynthesis of RNA in ts mu-tant-infectedcellswasdeterminedasfollows. BS-C-1 cells in30-mlbottleswereinfectedat amultiplicity of

10approximately, and the cultureswereincubatedat

31 or39.5°C in 2ml of medium containing 10

jg

of

actinomycin D per ml. After3 h of incubation the culture mediumwasreplaced with2mlofprewarmed (31 or39.5°C) medium containing 10,uCi of[3H]uridine

perml and 10

jig

ofactinomycinD perml.Cultures

previouslyincubated at31°Cwere shiftedto 39.5°C

andincubated for6 h atthat temperature. Cultures

previouslyincubatedat39.5°Cwereshiftedto31°C.

Other cultures were maintained at 31 or 39.5°C throughout the experiment. Allcultures were inocu-lated in duplicate and harvested at9h asdescribed above.

Invitropolymeraseassay. Thepolymerase

ac-tivity ofpurified preparationsofwild-type Chandipura

virusand itsts mutantswasassayedat31 and39°C

under theconditionsestablished for VSV Indiana(11).

Itwasdetermined by experiment, however, that the optimum concentration of Triton N-101 in the reaction mixturewas0.08% forChandipuravirus. The reaction mixtureswerewarmed for1minateither31or390C priortoaddition of220 mM MgCl2. Samplesof20

Al

werewithdrawnimmediatelybefore addition ofMgC12

(thezero time sample) andafter 30, 60,90, and 120

min at 31 or 39°C, transferred to 2.5-cm2 Whatman

DE81(ionic) paper disks, andairdried. The diskswere

then washedfive times in 5% (wt/vol) sodium pyro-phosphateat20°C, followed bytwowashesin deion-izedwater,ethanol,anddiethylether.Residual ether

wasremovedin a stream ofair, andthe diskswere

immersedovernight intoluene-basedscintillantprior

tocounting (12).

Viruspurification.Purified virionswereobtained by inoculating BHK-21 clone 13 cells inrotating

2-literbottlesat amultiplicityof 0.1. The contents of 10

bottleswerepooledand clarifiedby centrifugationat

1,500 rpmfor 20 min at 40Cin aMistralcentrifuge

(M.S.E. Ltd.,London). Virus in thesupernatantwas

sedimented through a 30% (vol/vol) glycerol layer

using a rotor (10 by 100 ml) at 40,000 x g in a

Superspeed65ultracentrifuge (M.S.E. Ltd.,London)

at4°C. The resuspendedpelletwaslayeredon a 15 to

45%(wt/vol) linearsucrosegradient and centrifuged in aBeckman SW27rotor at50,000xgfor60minat

4°C. Theopalescent bandwascollectedby pipette and pelleted by centrifugationat150,000xgfor30minat

4°C in a Beckman SW40 Ti rotor. The pellet was

resuspendedin asmall volume of20mM

Tris-hydro-chloride (pH 8.0).

Determination of heat stability. Virus stocks were diluted 10-fold into phosphate-buffered saline and distributed into thin-walled glassvials. Heat

ex-posurewascarriedoutby totallyimmersing these vials

in a waterbath at 45± 0.1°C. Sampleswere

trans-ferredto anice bathbeforeheat exposure andat

45-minintervals after immersionat45°C.Residual infec-tivitywasassayedonBS-C-1monolayers.

Analysisof intracellularproteins.BHK-21 cells in 30-mm petri disheswereinfectedat amultiplicity of20PFU/cell. The infected cellswerelabeledat 4.5

h after infection in Eagle medium containing

one-tenth the normal concentration of methionine and30

,uCi of [nS]methionine per ml (5 to 7 mCi/mmol;

Radiochemical Centre, Amersham). The cells were

eitherpulse-labeledfor15minorcontinuouslylabeled for3h.

At 7.5 h after infection the medium from each cultureplatewasremoved,0.2ml ofasolubilization mixture (2%[wt/vol] sodium dodecyl sulfate and 5% [vol/vol] 2-mercaptoethanol in0.14M Tris-hydrochlo-ride [pH 6.7]plus 10%[vol/vol]glycerol and bromo-phenol blue)wasaddeddirectlytothemonolayer, and theplateswereleftat20°C for15min.Before electro-phoresis, the samples were heated at 100°C for 5 min. Trichloroaceticacid-insolubleradioactivitywas meas-ured, and the concentration of the samples was ad-justed sothat each track in anysingle gel slab

con-tainedapproximately equalcounts.

Polyacrylamide gel electrophoresis.

Polyacryl-amidegel electrophoresiswascarriedoutin5 to12.5% gradient slab gels accordingto themethod described by Marsdenetal(5).

RESULTS

RNA phenotype. The RNA phenotype of

the mutants was established as described in Materials and Methods. Eight mutants from subgroup ChIA and five mutants from subgroup ChIBwerechosenatrandomtorepresent group

ChI.

Incorporation

of

[3H]uridine

was c1.3%of the wild-type value (Table 1). The group ChI mutants, therefore, were all considered to be

RNA-negativemutants.Thethreemutants

rep-resenting groups ChIII, ChIV, and ChVI also exhibited RNA-negative phenotypes, having levels ofincorporation 0.15% ofwildtype. On the other hand, the two mutants belonging to

group ChII showed goodincorporation, 56 and 73% of wild type, and were designated

RNA-positivemutants.MutanttsCh851of groupChV

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wasalsoanRNA-positivemutantandappeared

to incorporate more [3H]uridine (329%) than

wildtype.

No significant incorporation of [3H]uridine

was observed when mostof the RNA-negative

ts mutant-infected cultures were shifted from

permissivetorestrictive temperatureat3 h after infection. These observations suggested that the input virion proteins,or thosesynthesized

dur-ing the first3hatthe permissivetemperature,

remained nonfunctional atthe restrictive

tem-perature. Cellsinfected with twoRNA-negative

mutants,tsCh2O3 (ChIB) andtsCh472(ChIV), did showsignificant incorporationof[3H]uridine after temperatureup-shift (Table 2).This

incor-poration was not due to presence ofwild-type

virus in the inoculum, nor to reversion during multiplication, since the culturesmaintained at

restrictive temperature throughout the entire

experiment showed no increase in [3H]uridine incorporationbetween 3 and 9 h afterinfection.

These resultssuggestedthat viralpolypeptides synthesized during the first3 h at thepermissive

temperature remained functional after the

in-creaseinincubationtemperature.

TABLE 1. RNAphenotypeandheatstability of19ts mutantsof Chandipuravirus

[3H]uri- Thermal

Comple- dine in- RNA inactiva- Heat

menta- Mutant corpora- pheno- tion

stabil-tion tion(% type 450C ityb

group ofwild (Vt/V0)0

type) a

ChIA tsChi 0.26 - 0.20 R

tsCh9O 0.30 - 0.075 (R)

tsCh363 0.11 - 0.4 R

tsCh449 0.14 - 0.0005 L

tsCh482 0.9 - 0.29 R

tsCh484 0.03 - 0.17 R

tsCh540 0.11 - 0.01 (R)

tsCh867 0.16 - 0.20 R

ChIB tsChl28 0.3 - 0.14 R

tsCh188 0.02 - 0.03 (R)

tsCh203 0.35 - 0.4 R

tsCh315 1.30 - 0.01 (R)

tsCh808 0.25 - 0.0001 L

ChII tsCh4 73.2 + 0.0005 L

tsCh5 55.9 + 0.00001 L

ChIII tsCh465 0.09 - 0.4 R

ChIV tsCh472 0.14 - 0.5 R

ChV tsCh851 329.0 + 0.06 (R) ChVI tsCh319 0.1 - 0.02 (R)

Wild ts+ 100 + 0.30 R

type

aV. the titerbefore andVt the titer after exposureat

450Cfor 4.5 h.

[image:3.504.248.441.79.334.2]

b R, Resistant; (R),partially resistant; L,labile.

TABLE 2. Effectof temperature shifton

intracellular RNAsynthesis

[3H]uridineincorporation (%Y' Group Mutant 3 C 31°C a 39.5°C

39.5 39.5°C 31°C

ChIA tsCh363 0.15 0.09 62.0

tsCh449 0.04 NDb 82.0

tsCh540 0.1 0.16 57.7

tsCh867 0.16 0.12 139.7

ChIB tsChi 0.1 0.1 79.6

tsCh128 0.14 3.5 134.0

tsCh203 0.07 13.1 99.0

tsCh315 0.8 0.16 39.2

tsCh808 0.08 0.08 38.0

ChIl tsCh4 35.4 32.1 221.9

tsCh5 10.5 39.4 96.0

ChIII tsCh465 0.1 0.1 59.5

ChIV tsCh472 0.15 17.7 83.8

ChV tsCh851 37.0 39.6 38.5

ChVI tsCh319 0.1 0.08 39.6

Wild ts+ 12.3 12.5 12.6

type

a[3H]uridineincorporation between3and9hpostinfection aspercentageof the310Clevel.

'ND,

No data.

Inthereciprocal experiment, cultures infected with any of the

RNA-negative

ts mutants

showed

significant

[3H]uridine

incorporation

when incubatedfor 3 hatrestrictive tempera-tureand then labeled after transfertopermissive

temperature for 6 h

(Table

2). These results suggested that the mutant genomes remained

functional

during

the 3-h sequestration at

re-strictivetemperature.

As

expected,

the RNA-positive ts mutants

induced

significant

incorporation

of

[3H]uridine

inboth theup-shift anddown-shiftexperiments

(Table

2).

Heat

stability.

Preliminary experiments showed that themutantscould be differentiated

bestby exposureat45°Cfor4.5h. Table 1

lists

thereduction in

infectivity

for eachmutantand wildtype.Bothmutants

belonging

togroupChII and mutants ts Ch449

(ChIA)

and ts

Ch8O8

(ChIB) appeared to be thermolabile (>2 logio

unitsinactivation). Theremainingmutantswere

fully

orpartiallyheat resistant.

Invitro

polymerase

activity.

The invitro polymerase activity ofpurified preparations of

Chandipuravirusand itsts mutants was deter-mined as described in Materials and Methods.

Wild-type virus and RNA-positive mutants ts

Ch4 (group ChII), ts Ch5 (group ChII), and ts Ch851 (group ChV) showed considerablymore

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in vitropolymerase activity than the remaining mutants atboth 31 and39°C; nevertheless, an

unequivocallyRNA-negative phenotype was not

detected among the remaining mutants (data notshown).

Sincenoneof thets mutantsshowedanyclear

defectinpolymeraseactivity in vitro,anattempt was made to obtain polymerase mutants by

screening clones ofmutagenized Chandipura

vi-rusfor sensitivity to rifampin. Previously, Mo-reau(6)had reportedthe isolation of a

rifampin-sensitive mutant of VSV Indiana which

dis-playedatypical polymerase activityin vitro (7).

Rifampinwasobtained from bothSigma Chem-icals (London) and, as aged andfresh prepara-tions, from G. Tocchini-Valentini, Lancini-Le-petit, Milan, Italy. The antibioticwas dissolved

by adding200mg to 2ml ofdimethylsulfoxide anddilutingto 10 mlwithsterile distilledwater. Thisstock solutionwasdiluted1:100 in solid or liquidincubationmedium to give afinal

concen--M *

*MIt

-.M

.

Ch Ch

I I:I

3 4 5,6

£Ch

78

J. VIROL.

tration of 200 ,ug/ml. A total of 593 clones of

Chandipuravirusgrown in the presence of

100-,ug/ml

5-fluorouracil were tested for ability to

multiply in the presence of 200 ,ug ofrifampin

perml, butnone was sensitive to the drug. All the preparations of rifampin reduced vaccinia virus yields by more than 4log10 units at this

concentration. VSVwas slightly morerifampin

sensitive than Chandipuravirus, but it was not

possibleto clearlydifferentiate the rifampin-sen-sitive mutant (VSV-29) isolated by Moreau from

its wild type

(VSV-VJ)

in BS-C-I cells under these

conditions,

and the attempt to obtain polymerase mutants by this procedure was

abandoned.

Intracellular protein synthesis. BHK-21

cell monolayers were used forradiolabeling of

intracellular polypeptides in preference to

BS-C-1cells, becausethere was less loss of

cells

by

detachmentduringthe labeling period. Figure 1 shows the[3S]methionine-labeledpolypeptides

cb

tIV

9 10

*.. :.. .... :... :.:

" .s.- .

1h1

11 1.2

1*

WT

13 14

3IC 39C

15 16

1N

=rN

NS/A

aM

_ _

4NVP1I

FIG. 1. Polypeptide synthesisin BHK-21 cellsinfectedwithtsmutants tsChl,tsCh4,tsCh465,tsCh472,

andtsCh851,fromcomplementationgroupsChI,ChII, ChIII,ChIV,andChV, respectively.The odd-numbered

tracksarethepolypeptide profiles of samples fromcultures labeledat31°C,and the even-numbered tracks

aresamplesfromcultures labeledat39°C. Monolayers ofBHK-21 cells(orBS-C-1 cellsfortracks 15 and16)

wereinfected at20PFU/cell and labeled between 4 and 7 h with 30,iCi of [35S]methionineper ml. The

cultureswere harvested andproteins were extracted asdescribed in the text. Samples containing equal

amountsofradioactivitywereelectrophoresedon a5to12.5%polyacrylamide gel for3to4hat40mA. The

viralpolypeptidesare labeledL,G,N,NS, andM;Aisactin, NVP1is nonviralpolypeptide 1,and MI isa

mockinfection.Tracks15and 16weretakenfromanothergeltoshow thatNVP1wasalso resolved in

BS-C-1 cellsinfectedwithtsCh851 (groupChV)at39°C.

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CHANDIPURA VIRUS MUTANT PHENOTYPES 111

synthesized in cells infected withwild-typevirus

and mutants ts

Chl,

ts

Ch4,

ts

Ch465,

ts

Ch472,

and ts Ch851, representing complementation groups ChI, Chll, ChIII, ChIV, and ChV,

re-spectively.All the tsmutants,includingtsCh319 of group ChVI (not shown), synthesized less

viral protein at 39°C (even-numbered tracks).

The Lpolypeptide was notresolved in the cul-tures incubated at 390C, except in ts Ch851

(group ChV)-infected cells. The G protein was

resolved intotwobandsin most of the mutant-infected cells at310C. Thesetwobandsprobably correspond to the Gl and G2 polypeptides of

VSV andrepresent two stages in glycosylation

(2; R. M. L.Buller, Ph.D. thesis, University of

Glasgow, Glasgow, Scotland, 1975). The pulse-chaseexperiments illustrated in Fig. 2, 3,and 4

confirmed thatashift inelectrophoretic mobility

1L

4N

NS/A

ofthe G protein occurred duringincubation, as with VSV Indiana (2). The N polypeptide of Chandipura virus migrated just above the cel-lular actin band and was clearly resolved in all the mutant-infected cells. The NS polypeptide

was notresolved, since it migrated in the region of cellular actin. The Mpolypeptidewas present in allthevirus-infectedcell extracts.

A[35S]methionine-labeled polypeptide desig-nated NVP1 (nonviral polypeptide 1) was ob-served in both BHK-21 (track 12) and BS-C-1

(track 16) cells infected with mutant ts Ch851 andincubatedat390C.NVP1 was not observed in tsCh851-infectedcells at310C(tracks 11 and 13), or incells infectedwithwild-type virusand other ts mutants ateither 31 or390C. NVP1was also not resolved incellsinfected witheitherof two spontaneous revertantsof tsCh851 (Fig. 4).

*

;

... ... . N.

C

-'*

''r;}: :i, :: .: .. .. : .:.:

*s.

z z

..X

v o!:

.. .'

::'

....:a

e'.:t

*w,:,,i

...:.

...

..

.::e:#i,r}

;w--

__

44M

_ __

_;w

..._..

*

* .'.

*uNVP1

S

FIG. 2. Thepolypeptides oftsCh851-infectedcells inapulse-chase experiment. BHK-21cellcultureswere

infected withmutant tsCh851at20PFU/cellandincubatedfor4hat31or39°C. The cultureswerelabeled

for15 min with 30 uCiof [35S]methionineperml and then returnedto ordinary medium. Cultures were

harvestedat0, 1,2, and 3hafter labelingasdescribed in thetext.Samples containing equalcountswere

electrophoresed on a 5 to 12.5%polyacrylamide gel. Tracks 3, 4, 5, and6, Cultures incubated at 31°C

throughout and harvested at0, 1, 2, and 3 h, respectively, after labeling; tracks 7, 8, 9, and 10, cultures

maintainedat39°Cthroughout;tracks11, 12,and13, cultures labeledat39°C andsubsequently incubatedat

31°C for 1, 2, and 3h, respectively. Tracks I and 14show markerpolypeptides from partially purifiedts

Ch851,andtrack2showspartially purified wild-type virus. The lettersareidentified in the legendtoFig.1.

si

1£ *

SC

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-4 3C

i}%

3,a

*NVP2

-

*~~~~~NVP

1

FIG. 3. Polypeptide synthesisintsCh851-infected

cells after transfertorestrictive temperatureat4 h

after infection.Theconditionsof infectionand

label-ingwere asdescribed inthelegendtoFig.2.Tracks

2, 3, and 4, Samples from cultures incubated and

labeledat31°Cand chasedfor 0, 1,and 2h,

respec-tively; tracks5, 6,and 7, correspondingcultures

in-cubated at 39°C throughout; tracks 8, 9, and 10,

cultures incubatedat31°Candsubsequentlylabeled

and chasedat39°C for 0, 1,and2 h. Track I shows

thepolypeptides fromamock-infectedculture at3lC.

The lettersareidentifiedin thecaptiontoFig.1.

Thestabilityoftheviralpolypeptides synthe-sized at39°C was examined inpulse-chase

ex-periments (3) asdescribedinthelegendtoFig. 2. Nodegradationof viralpolypeptideswas

de-tected in cells infected with mutants ts Chi (group ChI), ts Ch4 (group ChII), ts Ch465 (group ChIII), ts Ch472 (group ChIV), and ts

Ch319 (group ChVI).IntsCh851-infected cells, however, it was evident that the intensity of

labeling ofthe Mpolypeptidedecreasedduring incubationat390C(Fig. 2, tracks 7to10). This effectwasnotobserved when the cultureswere

incubatedat310Cafterlabelingat390C (Fig. 2, tracks 11 to 13). However, if the cells were

incubatedat310Cand thenlabeledand chased

at39°C,degradationof Mwasobserved(Fig. 3,

tracks8to10).NVP1wasapparent ints Ch851-infected cells,providedthe cultures were

incu-batedat390C duringorafterpulse-labeling (Fig.

2, tracks7to 13,andFig. 3,tracks 5 to10). An

additional polypeptide (NVP2) has been

re-solved in Fig. 3, but the nature of this

poly-peptideor the band between N and M in track 5 isunknown. Neither was observed consistently in other gels.

Aspontaneous revertant (Ch851/R2) of mu-tant ts Ch851 was isolated and compared with the parental mutant in the same experiment (Fig.4). Theinstabilityof the Mpolypeptidein tsCh851-infectedcellsat39°Cwas again appar-ent(tracks5 to7),but incells infectedwith the revertantthere wasless degradationof M (tracks 11 to 13). Densitometertracings of the tracks in

Fig. 4 showed thatduring the chase period

ra-dioactivity in theMpeak declinedto60%of the

initial level forrevertantCh851/R2 and33% for mutant ts Ch851, standardized in each case to

the Npeak. Similarresults were obtained with

anotherrevertantcloneof tsCh851.

Host rangeproperties of ts and tdCE mu-tantsof

Chandipura

virus. Thewild-typeand

conventional ts mutants produced plaques on

CE cells at

310C,

and

only

the wild type

pro-duced plaques at 39°C, i.e., the ts mutants

re-tained their phenotype inavian cells. Mutants

of both VSVIndiana andVSVNew Jersey have

been isolated, however, which were

condition-ally

temperature sensitive in CEcellsand were

designated tdCEmutants(10). Therefore Chan-dipura

wild-type

virus was mutagenized by growth in thepresence of 100,g of 5-fluorouracil per

ml,

and cloneswerescreened for theirability

to form plaques on BS-C-1 and CE cellsat 31

and

390C.

The six tdCEmutantslistedinTable

3 were obtained from 509 isolates, i.e., a fre-quency of 1.2%. No mutants completely

re-strictedonCE cells

(the

hrCE

phenotype)

were obtained. The six tdCE mutants were titrated alsoonmonolayers of hamster (HE),rat (RE),

feline (FEA), bovine (BEK), andmouse

(NIH/

3T3)embryocells andonmouseL929cellsat 31

and

390C

(Table

3). Three ofthe six mutants were temperature sensitive on CE cells

only,

whereasthe other three mutants showedsome

temperaturesensitivity onmammalianembryo

cells.

Indeed, mutanttdCE Ch530 was temper-aturesensitiveinalltypesofcellsexcept

BS-C-1 cells. None of the tdCE mutants

comple-mentedany of the six groups in CE cellsat

390C,

and

consequently

it was not

possible

to

assign

themto

complementation

groups.

However,

for reasons which arestillunclear,little significant complementation was observed between the

conventionaltsmutantsof

Chandipura

mutants

inCE cells (datanotshown).

DISCUSSION

Thephenotypic properties of the ts mutants

ofChandipuravirussuggest that

complementa-J. VIROL.

'm .31

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CHANDIPURA VIRUS MUTANT PHENOTYPES 113

tsaC 851

M * 31C 4

39C

1 2 3 4 5 6 7

Ch 8S1

R/2

m31C 4 *

39C

4

8 9 10 11 12 13 ₁₄M

L

f

'...::...:.:

~~~i6

s v / . > ,.X. M j :*.N

'..:.}.2.§.:N_ A

_. 0 ....

_-

4 M

4

NVP1

FIG. 4. Polypeptidesynthesisin cellsinfectedwith cloneCh851/R2,anon-temperature-sensitiverevertant

oftsCh851. The conditionsof infectionandlabelingwere asdescribed in the

legend

to

Fig.

2,exceptthatthe cultureswereharvestedat0, 1.5,and3hafter labeling. Tracks2, 3,and4, Samples fromcultures

infected

with tsCh851 and incubated throughoutat

31°C;

tracks5, 6, and 7,

corresponding

cultures incubatedat

39°C; tracks 8, 9, and10,culturesinfectedwith therevertantcloneCh851/R2and incubatedthroughoutat

310C;tracks11,12,and13,correspondingcultures incubatedat39°C. Tracks 1 and 14 showpolypeptidesin mock-infectedcellsat31°C.Thelettersareidentifiedin thecaptiontoFig. 1.

tion groups ChII and ChV

correspond

to the

genes

coding

for the two

proteins

ofthe viral

envelope.

Thevirionsofbothmutantsingroup

ChIIwerethermolabile

(Table 1),

andthe

intra-cellularM

polypeptide

oftsCh851

(group

ChV)

wasunstable atrestrictive temperature (Fig. 2,

3, and4).Reversionof the temperature

sensitiv-ity oftsCh851was

accompanied by

anincrease

in stability of the intracellular M

polypeptide

(Fig.4). Mutant ts Ch851 wasalso an overpro-ducer of RNA at restrictive temperature, like the group IIImutantsof VSV Indiana whichare

also considered to be Mprotein mutants (3, 4, 9). It is notpossibletosuggest any gene

assign-ment in the case of the four

RNA-negative

groups.Group

ChI,

which contains88% of thets

mutants ofChandipura virus, may correspond

to group I of VSV Indiana

(containing

77% of mutants), which isconsidered torepresent the

genecodingfor the Lprotein (9). However,none

of thets mutants ofChandipura virus, including thethermolabiletsCh449 ofgroupChIA andts

Ch808

ofgroup

ChIB,

had defective RNA

po-lymeraseactivityinvitroatthe restrictive

tem-perature.

TheapparentmutantspecificityofNVP1

sug-gested that it might be a minor virus-coded

polypeptide which accumulated in conditions wherematurationwasblocked buttranscription

continued unrestricted. However, NVP1 could not be chased into virions by

labeling

at39°C

and incubation at

31°C (data

not

shown).

In

addition, NVP1didnotappeartobederived

by

proteolytic cleavage from any of the five viral

VOL. 33,1980

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TABLE 3. Titers of six tdCE mutantsof Chandipura virus in various cell linesa

Temp of tdCE tdCE tdCE tdCE tdCE tdCE

Cels incubation Ch514 Ch516 Ch525 Ch530 Ch565 Ch859

(OC)

BS-C-1 31 5x 108 5x 108 1.5x 105 1.5x108 1.5x108 2x 108

39 2x 108 108 2.5x 107 107 107 5x 107

CE 31 1.5x107 107 io7 107 5X 106 3x 107

39 104 2X 103 1o2 102 5x103 2X 104

HE 31 1.5x 107 3X106 1.5x 107 107 5X106 1.5x 107

39 3X106 2.5X106 3x 106 4X104 106 106

RE 31 2x 106 3x 106 2x106 5 x105 3x 105 5x 106

39 5x 105 4x 105 5x 105 103 1.5x104 5 x104

FEA 31 2.5x107 2x 107 2.5x107 2x 107 107 3x 107

39 7.5x106 107 106 5 x103 106 106

BEK 31 2.5 X106 106 2x 106 2x106 1.5X106 2.5x 106

39 7x 105 5x 105 1.5x105 2X103 3.5x 104 5X 104

L929 31 5x 106 5 X 106 5x 106 2 X 106 3.5X 106 3x106

39 5x 105 3.5x105 2x 105 102 1.5x105 5x102

NIH/3T3 31 106 2x 106 2.5x 106 2x 106 5X 105 5x 106

39 106 5x 105 5x 105 102 2x 105 1.5 x104

a Thetitersthatshowsignificant

differences

areindicatedin

italics.

proteinsinexperiments (not shown)in which ts Ch851-infected cells were labeled andharvested

inthe presenceofthe proteaseinhibitors tolyl-sulfonyllysyl chloromethylketoneand tolylsul-fonylphenylalanyl chloromethylketone. There-fore itwasconcluded that NVP1 wasprobably

ahostproteinwhich accumulated at39°C asa consequenceof the defectivematuration of mu-tant ts Ch851. Occasionally a faint band was

observedin the NVP1positioninmock-infected cells(e.g.,Fig. 3,tracks 1to4). Stimulation ofa specific host protein by a rhabdovirus has not

beenreportedpreviously,butenhanced

synthe-sis of two host proteins has been observed in

simianvirus40-infectedCV1 cells (8).

Finally, the isolation of tdCE mutants of

Chandipuravirussuggests that host factorsare

involved in thereplication ofallrhabdoviruses,

notonlyVSV(10).

ACKNOWLEDGMENT

D.A.G.wastherecipient ofaFellowshipfrom the Com-monwealthCommissionduringthecourseof thiswork.

LITERATURE CITED

1. Gadkari,D.A.,and C. R.Pringle.1980.

Temperature-sensitive mutants ofChandipuravirus. I. Inter- and intragroupcomplementation.J.Virol. 33:100-106.

2. Knipe, D.,H. F.Lodish,andD.Baltimore.1977.

Lo-calization oftwocellularformsof the vesicular stoma-titisviralglycoprotein.J. Virol. 21:1121-1127. 3. Knipe, D.,H.F.Lodish,andD.Baltimore.1977.

Anal-ysis of the defects oftemperature-sensitivemutantsof vesicular stomatitis virus: intracellulardegradationof specific viral proteins.J. Virol. 21:1140-1148. 4. Lafay, F.1974.Envelope proteinsof vesicularstomatitis

virus: effect oftemperature-sensitive mutation in com-plementationgroupsIIIand V. J. Virol. 14:1220-1228.

5.Marsden, H. S., I. K. Crombie, and J. H.

Subak-Sharpe. 1976.Control ofprotein synthesis in

herpes-virus-infected cells: analysis of the polypeptidesinduced by wildtypeandsixteen temperature-sensitivemutants of HSV strain17.J.Gen. Virol. 31:347-372.

6.Moreau,M. C. 1974.Inhibition ofavesicular stomatitis

virusmutantbyrifampin. J. Virol. 14:517-521. 7. Moreau, M. C., B.Sanzey, and Ch. Chany.1975. In

vitrotranscriptionofavesicularstomatitis virus mu-tant, p. 103-108.In Invitro transcription and translation of viral genomes, INSERM Colloquium, vol.47.

IN-SERM,Paris.

8.O'Farrell,P.Z., and H. M. Goodman.1976.Resolution

of simian virus40proteins in whole cellextractsby two-dimensionalelectrophoresis: heterogeneity of the major capsid protein. Cell9:289-298.

9. Pringle,C. R. 1977. Genetics of rhabdoviruses, p. 239-289. In H. Fraenkel-Conrat and R. R.Wagner (ed.), Comprehensive virology, vol. 9. Plenum Publishing Corp.,New York.

10. Pringle, C.R. 1978. The tdCE andthehrCEphenotypes: host range mutants ofvesicular stomatitis virus in whichpolymerase functionis affected.Cell16:597-606.

11. Szilagyi,J.F.,andC.R.Pringle.1972.Effect of

tem-perature-sensitive mutations on thevirion-associated RNAtranscriptaseof vesicular stomatitis virus. J. Mol. Biol.71:281-291.

12.Szilaigyi,J.F.,andC. R.Pringle.1979.Effect of

tem-perature-sensitive mutation on activity of the RNA transcriptase of vesicularstomatitisvirusNewJersey. J.Virol.30:692-700.

114 GADKARI AND PRINGLE J. VIROL.