Isolation and Characterization of Sendai Virus Temperature-Sensitive Mutants

Copyrighti 1974 AmericanSocietyforMicrobiology Printed inU.S.A.

Isolation

and

Characterization

of

Sendai

Virus

Temperature-Sensitive

Mutants

ALLEN PORTNER, PRESTON A. MARX, AND D. W. KINGSBURY

Laboratoriesof Virology andImmunology, St. Jude Children's Research Hospital, Memphis, Tennessee38101 Received forpublication 27September1973

Ten temperature-sensitive mutants ofSendai virus, a paramyxovirus, were

isolated and partially characterized. Themutantsreplicatedinchicken embryo

lung cells at 30C, but not at 38C; wild-type virus grew equally well at both

temperatures. Complementation tests divided the mutants into seven groups. Sixgroupssynthesizedneither infectious virusnorRNA when incubatedat38C

fromthebeginningofinfection.Temperature shift-up experimentsdemonstrated

thatthree ofthesecomplementationgroupswereblocked inearlystepsrequired

for RNA synthesis, but these gene functions were not needed throughout the

replicative cycle. In contrast, the other three RNA-negative complementation

groupswere defectivethroughout the replicative cycle infunctions required for

virus-specific RNAsynthesis. Onlyone mutant, whichcomplemented allofthe

above, synthesized RNA but not infectious virus when placed at 38C; the

hemagglutinin ofthis mutantfunctionedonlyatthe permissivetemperature.

Temperature-sensitive (ts) mutants of

ani-mal viruses have been useful instudying virus

replication (6, 7). With respect to

paramyx-oviruses, a number ofNewcastle disease virus

(NDV) nonconditional-lethal mutants as well

as ts mutants havebeenisolated,butthesehave

been used mainly to study genetic interactions

(4, 8, 9). We have isolated andcharacterized a

number of Sendai virus ts mutants to learn

more about biochemical events in

paramyx-ovirusreplication. Preliminary genetic and

bio-chemical analysesof 10 ofthese mutants are the

subject ofthis report.

MATERIALS AND METHODS

Virus and cells. A clone ofwild-typeSendai virus wasplaque purified from the Enders strain of Sendai virus and was free from incomplete virions (17).

Stocksof viruswereprepared byinfecting10-day-old

embryonated hens' eggs with 0.01 hemagglutinating unit and incubating for 72 h at 37C. Methods for preparing chicken embryo lung (CEL) cell cultures, forgrowing Sendai virusinthesecells, andforplaque assay were described (5), but in the present work, Eagleminimalessential medium was used as growth mediumand in theplaque overlay.

Virions were labeled with radioactive amino acids asdescribed (18), except that labeled precursors were added 16h after infection andvirions werecollected 32 h later. Culture fluids containing released virus were centrifuged for 10 min at 3,600 x g toremove cells and debris. Viruswaspelleted (78,000 x g, 5C, 30min)andresuspendedin 0.01Msodiumphosphate (pH 7.2) for fractionation experiments.

RNAextraction, rate-zonalcentrifugation, and

radioactivity determinations. These methods have allbeen fully described (13).

Selection ofts mutants. The methods for muta-genization of wild-type virus and isolation of ts mutants weresimilar to thosedescribed forSindbis virus (2) and vesicular stomatitis virus (14). After mutagenization with

N-methyl-N'-nitro-N-nitrosoguanidine (2), the remaining infectious titer was 10-3 of the initial titer. Mutagenization by

5-fluorouracil (FU)(14)wasdonebytreatingCEL cell

monolayerswith200jgofthecompoundperml for 1

h, by infectingwith10plaque-formingunits(PFU)of Sendai virus per cell, and by allowing the virus to growfor 48 hin the presenceofFU. Aftertreatment with mutagens, the virus stocks were diluted to produceafewplaquesat 30C.Afterincubation for7 to 10 days, well-isolated plaques were picked,

sus-pended in 1 ml ofphosphate-buffered saline (PBS) containing 5% fetal calf serum, and replated at

permissive(P) temperature (30C)andnonpermissive

(NP)temperature(38 C).Tenisolateswere tempera-ture-sensitive, whereas all others tested were not. Stocks of each ts isolate were

grownl

in CEL cell monolayers at 39C for further testing. Virus was harvested when thehemagglutinin titerwas27/mlor greater.

Complementation tests. Complementation tests weredoneinCEL cell monolayer cultures containing about106cellsatthe timeofinfection.Sendai virusts mutants were diluted in PBS to give an input multiplicityof5PFU/cell in asingle infectionor2.5

PFU/cell ofeach mutant in mixed infection. Virus wasadsorbed for 30min at25C, the monolayers were washed with PBS, and5 ml ofgrowth medium was added.Afterincubationat 38Cfor 4h, cultureswere washedagain, prewarmedgrowth medium was added, andmonolayerswerereincubatedat38Cfor 48 to 72 298

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SENDAI VIRUSTEMPERATURE-SENSITIVE MUTANTS

h. The mediumwasthen assayedforplaque formation

on CEL cell monolayers at 30C. Complementation levelswerecalculated accordingtoBurge and Pfeffer-korn (3).

Isolationof virion glycoproteins. Sendai virions

werefractionated bytreating them (1mg ofprotein

perml) with2%TritionX-100 and1MKClfor20min

at 25C (15, 16). The mixture was centrifuged, at

100,000xgfor 30minat5C, and the glycoproteins in the 100,000 x gsupernatantwereseparatedfromthe

smallest virion polypeptide by dialysis and centrifu-gation (15, 16).

Acrylamide gel electrophoresis.Thismethodwas

previously described(18).

Neuraminidase assay. Fetuin wasused as a

sub-strate. Freesialic acidwas measuredbythe

thiobar-bituric acid procedure(19, 20). Duplicate samplesof virionsor the glycoprotein fraction from virions (15,

16)werediluted with0.2M sodium phosphate buffer (pH5.9)to0.05ml, and0.05ml offetuin (6.25mg/ml) inthesamebufferwasadded. Theincubation temper-ature was30or38C.

RESULTS

Isolation ofts mutants. With nitrosoguani-dine and FU mutagenesis, about 1.5 and 1.0%, respectively, of the plaque isolates contained useful ts mutants. When mutant stocks were

tested at 38 and 30C, ratios of plaques

pro-ducedranged from less than 1.6 x 10-' (ts 271) to 2.5 x 108 (ts 935) (Table 1). Only a few

plaques from the 38 C plateswerenot

tempera-ture-sensitive when picked and tested again at

38 and30C, indicating that themutantshadlow back mutation frequencies and were slightly

"leaky." The frequency of wild-type revertants

in mutant stocks ranged from 3.8 x 10-'to4.5

x 106.

Growth of ts mutants at P and NP temperatures. To examine the growth of ts mutants, cultures were infected with an input

multiplicity of about 5 PFU/cell and incubated

at P and NPtemperatures. Growth curves are

shown forwild-type virus (Fig. 1A) andmutant

TABLE 1. Efficiency of plating of wild-typevirus and

tsmutants

38C/30 C ratio

Virus (PFU/ml)

Wild-type 1.0

ts74 ... 2.4 x 10-'

ts 105 ... 1.2 x 10-'

ts 245 1.2 x 10'

ts271 ... 1.6 x 10'

ts348 ... <2.7 x 10-5

ts557 ... 6.0 x10'

ts595 ... <1.0 x10-7

ts642 ... 1.0 x10-5

ts840 ... <8.0 x10-v

ts935 ... 2.5 x 10-B

-i 2

-0 24 48 72 96 120 0 24 48 72 96 120

HOURS AFTER INFECTION

FIG. 1. Growth of wild-type Sendai virus and mu-tant ts271at30and38C. CEL cell monolayerswere

infected with an input multiplicity of 5 PFUIcell. After adsorption (30 min at 24C), monolayers were

washed andincubatedat30or38 C.Atintervals after

adsorption, culture fluids were removed from the

monolayers and assayed forinfectious virusat30C by theplaquemethod. A, Wild-type virus; B, mutant ts

271.0,38 C: 0,30C.

ts 271 (Fig. 1B). Wild-type virus grew equally

well at 30and 38 C. Mostts mutants werelike ts 271, producing virus at approximately the

same rate at 30C as wild-type virus, but little or no virus at 38 C. Typical amounts of virus released 48 h after infection by each mutantare

shown in Table 2. All mutants synthesized substantialamountsof virus at30 C, andsome

(ts 74, 105, 245, and 271) grew better than

wild-type virus at 30 C. In contrast to growth under permissive conditions, mutant yields at

TABLE 2. Growthof Sendaivirusmutants at30and 38 Ca

Yield(PFU/ml) 38C/30 Cratio

Virus 30 C 38 C (PFU/ml)

Wild-type 2.0x 107 6.0 x 107 3

ts74 3.4x 10' 1.8 x 102 5.3x 10-7

ts105 2.0x 108 <101 <5.0x 10-8 ts245 6.3x 107 <101 <1.6x10-7 ts271 3.6x 10 1.2x 103 3.3x 10-B

ts348 6.3x 105 <101 1.6 x 105

ts557 5.0x 10' <101 <2.0x 10-4 ts595 1.6x 107 <101 <6.3x10-7

ts642 2.5 x 107 5.0x10' 2.0x 10-'

ts840 4.0x 105 <10l <2.5x 10-5

ts935 2.0x 106 <101 <5.0x 10-aCEL cellmonolayers wereinfected withan input

multiplicityof5to10PFU/cell. After 30minat24 C, cultures werewashed and thenwere incubated at30

or38C for 48h.Culture fluidswerecleared of debris

by low-speed centrifugationandthenwereassayedfor

infectivity bytheplaquemethodat30 C.

A

30C

7

8~~~~~~_\-~3 C -_

6_

5-4

2 _ .A

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therestrictive temperaturewerelessthan0.2%.

Most of the virus produced atNPtemperature wasofthemutant genotype,unable toproduce virus when tested again at 38 C, indicating slight "leakiness." Additionally, these results confirmed the previous observation that the

mutants havea low reversionfrequency towild type.

Complementation. The results of

com-plementation experimentsareshown in Table 3.

On the basis of thesedata, wehave assigned the mutantstosevencomplementationgroups(Ato

G). Five complementation groups (A and D to

G) arerepresented by single mutants(245, 105, 348, 74, 271). Group B is represented by two mutants (557, 642), and group C by three mutants (595, 840, 935). Within a group, each mutant complemented all other mutants, but

not mutants in the same group. Thus, the

groups arenonoverlapping.

Virus-specific RNA synthesis by ts mutants.Theability of Sendai virusts mutants to synthesize the various species of virus-specific RNA (1, 13) under restrictive conditions

wasexamined. After adsorption of virusat24C for 30 min, infected monolayerswereincubated at 38 C for 48 h. At this time, when viral RNA synthesis is maximal for wild-type virus (13), cultures were treated with actinomycin D and

labeled with [3H]uridine in the presence ofthe

drug. The RNAswereextracted and sedimented

insucrosegradients. Results shown in Fig. 2are

representative of each type of physiological

behaviorobserved, although amember ofevery

complementation group was examined.

Similar amounts ofviral RNA were

synthe-sized by ts mutant 271 (group G) atP and NP

temperatures (excluding the adsorption period) (Fig.2A). However, thiswasthreefoldless than

wild-type virus under similar conditions. The distribution of radioactivity was the same as

that found when wild-type viruswasincubated

under the same conditions (13), i.e.,

virus-specific 18S messenger RNA and 50S genomes were made. Thus, we have designated

com-plementationgroupG RNA+. Mutants

belong-ing to complementation groups Ato F synthe-sized little or no RNA when incubated at the restrictive temperature from the beginning of' infection, and we have designated them

RNA-(Fig. 2B, C, and D).

To distinguish viral gene functions required

forRNA synthesis throughout replication from thoserequired only early in the cycle, tempera-ture shift (30 to 38 C) experiments were done.

Infected cultures wereincubated atthe P tem-perature for 48 h and then wereshifted to the NP temperature for 24 h. In all cases, virus

production ceased within 24 h. After the 24 h shift-up, the cultureswerelabeled with [3H

]uri-dine for 1 h in the presence ofactinomycin D,

and the RNAs were extracted and analyzed as

described above. As expected, ts mutant 271

(group G, RNA+) synthesized normal amounts

ofviral RNA after shift-up (Fig. 2A). Both the

mutant and wild-type virus synthesized more

RNA after being shifted from the P to NP

temperature.

Mutants belonging to groups A, B, and C

(RNA-) synthesized substantial amounts of 18

and 50S RNA after being shifted to the NP

temperature for 24 h (Fig. 2B). When the intervalatthe NPtemperaturewasextendedto

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48 h before labeling (data not shown), the

TABLE 3. Complementation levelsa

Mutant Mutant

935 840 642 595 557 348 271 245 105 74

74 31 5 48 8 120 8 2,050 1,390 83

105 7 5 2 38 6 3 40 22

245 65 1,220 15 460 140 660 30,000

271 14 6,750 10 3,300 6,600 1,175

348 50 20,000 2 13 48

557 7 90 0.8 16

595 0.07 0.3 8

642 20 14

840 0.3

935

aComplementation testsweredone as described in Materials and Methods. Complementation levels were calculatedaccordingtoBurgeand Pfefferkorn (3). All valuesgiven are the average of three experiments. These levels weresignificantly greater than 1 by the Student's t test at P < 0.05. Complementation levels divided the mutants intosevencomplementation groups: group A, mutant 245; group B,557and642; group C, 595, 840, and 935; groupD, 105; groupE, 348; group F, 74; and groupG, 271.

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z

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2

I-_x

z

0

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FRACTION NUMBER

FIG. 2. Sedimentation ofvirus-specific RNA from cells infected by Sendai virus mutants. CEL cell cultures wereinfected withabout5PFUIcell.After30min at 24 C one group of cultures(0) was incubated for 48 h at 38C. Another group(0)wasincubatedat 30C for 48 h and then was shifted to 38 C for 24 h. Each group then wastreatedfor 1 h with 50 ugof actinomycin D per ml and labeled for 1 h with 50

,Ci

of[3H]uridineper ml in the presence of the drug, all at 38C. RNA was extracted with sodium dodecyl sulfate-phenol (13) and

centrifugedat 20,000 rpmat 20Cfor16h in 34-mllinear 15 to30%osucrose gradients (13). A,Infection with mutant ts 271 (complementation group G); B, mutant ts 557 (complementation group B); C, mutant ts 74

(complementationgroupF);andD,mutant ts 105(complementation group D).

amount of virus-specific RNA made was the

same as after the 24 h shift.

Thus,

these

mutants are defective inearly functions

essen-tial for viral RNAsynthesis, but these functions

are not required laterin thereplicativecycle.

Mutants ts 348 and 74 (groups E and F,

RNA-)

synthesized

low levels ofvirus-specific

RNAaftershift-upfor 24h(Fig.2C). ViralRNA

synthesis decreasedevenfurther (incontrast to

groups A to C) as the interval at the NP temperature was extended to 48 h (data not

shown).

The group D mutant (ts 105, RNA-) was

clearly separated physiologicallyfrom groups E

and

F.

Aftershift-upfor24h,ts105

synthesized

little or no virus-specific RNA (Fig. 2D). (The

peak ofRNAfound atabout 5Swas also found

in uninfected cells.) When the shift intervals

weredecreasedto 2 or 4 h(datanot shown), ts

105still showeda marked declineinviral RNA

synthesis, indicating that this mutant ceases

RNAsynthesis rapidly under restrictive

condi-tions.

(When

examined in the same way,

mu-tants

belonging

to groupsE and F

synthesized

normalamounts ofviralRNA.) From these data

it is apparent that mutants belonging to

com-plementation groupsD, E, and F aredefective

in functions required for virus-specific RNA

synthesis throughout the replicative cycle, but

the defect in group D may be more

intimately

related to the synthetic machinery for viral RNA.

Mutant ts 271 has a

hemagglutinin

that

does notfunctionatNPtemperature. Mutant ts 271

synthesized

RNA, but not infectious virus, at the NP temperature. When the

ability

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PORTNER, MARX, KINGSBURY

ofthis mutant to agglutinate erythrocytes was

examined, agglutinationoccurredatP, butnot

at NP temperature. Wild-type virus and the

other mutants agglutinated chicken

erythro-cytes atbothtemperatures

(Table

4). When the

mixture of virus and erythrocytes was shifted

from NP to P temperature, the erythrocytes

became fully agglutinated. When the

ag-glutinated mixture then wasshifted back from P to NP temperature, the hemagglutinin

pat-ternwasagainlost. These resultsindicated that

the hemagglutinin of mutant ts 271 did not

function atNP temperature, but that function

was restored at P temperature. However, the

inability to hemagglutinate chicken

erythro-cytes does not exclude the possibility that the

hemagglutinin was still capable ofbinding to

receptors.

Sendai virions contain at least six

polypep-tides (12). By

analogy

with other

paramyxovi-rions, the largest glycopolypeptide with an ap-parent molecular weight of 70,000 probably

represents the hemagglutinin (15, 16). The

inability of mutant ts 271 toagglutinate chicken

erythrocytesatNPtemperaturemight be dueto

analteration in someothervirionproteinwhich

makesthehemagglutinin nonfunctional.

There-fore, we tested isolated glycopolypeptides (15,

16) of ts 271forthetsphenotype. The prepara-tions contained all of the hemagglutinating

activity, and mainly virion glycopolypeptides

wereseen inpolyacrylamide gels (Fig.3,peaks2

and 5). When this material was tested for ts

hemagglutinin activity, the resultsweresimilar

tothose obtained withintact virions.Wild-type

TABLE 4. Hemagglutination by wild-type Sendai virus andmutantsa

Hemagglutination(perml) Virus

30C 38C 38C/30C

Wild-type 330 150 0.5

ts557 41 81 2.0

ts348 120 160 1.3

ts935 120 160 1.3

ts642 200 200 1.0

ts105 68 54 0.8

ts595 54 41 0.8

ts840 160 120 0.8

ts 74 160 68 0.4

ts245 240 68 0.3

ts271 200 <2 <0.01

aChicken erythrocytes (0.5% inPBS, pH 7.2) and

viruseswereseparatelywarmed in a water bath at 30 or 38C, mixed, and maintained at the respective temperatures. The test was performed in tubes, and titers representthereciprocal of the highest dilution

showinghemagglutination at 30 min after mixing.

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DiSTANCEMOVED (mm)

FIG. 3. Polyacrylamidegel electrophoresis of Sen-dai mutant ts 271

[3Hlglycopolypeptides

prepared as described in Materials andMethods.

['4C]polypep-tides from wholewild-typeSendaivirions were added beforeelectrophoresis. Thenumberingsystem is that

ofMountcastle et. al. (12). Migration isfrom leftto

right.a 3H;

O,

4C.

virion glycoproteins isolated the same way

ag-glutinated chicken erythrocytesat30and38

C,

whereas ts 271 glycoproteins were unable to

agglutinate

erythrocytes

at 38 C

(data

were

identical to those shown in Table 4). We

at-temptedto

identify

which of thetwo

glycopoly-peptides from

wild-type

or mutant ts 271 was

the

hemagglutinin,

but we were unable to separate the

glycopolypeptides

by

centrifuga-tionin sucrose

gradients (15, 16). However,

it is

clear that thets

hemagglutinin

resides inoneof

thesolubilized viral

glycopolypeptides,

andnot

inanother vironcomponentwhich

might

affect

hemagglutinating activity

inintactvirions.

Ifthesame

glycopolypeptide

has

neuramini-dase and

hemagglutinin

activities

(15,

16),

then

mutant ts 271

might

have a tsneuraminidase. To test this

possibility, equivalent

amounts of

glycopolypeptideswere isolated from

wild-type

virus and ts 271, and neuraminidase activity

was measured at the P and NP temperatures

(Fig. 4). Therateofsialic acid releasebyts271

enzyme was about the same at 30 and 38C,

indicating that the neuraminidase was not

tem-perature-sensitive. Increasing the temperature

to 46C did not change the results. However,

comparedwithwild-type, the mutant

neurami-nidase functionedmuch lessefficiently (Fig. 4),

even at theP temperature.Additionally, when

ts 271andwild-typevirions weretestedforheat

stability at 54C, it wasfound that the mutant

hemagglutinin and neuraminidase activities

were more heat labile than wild-type virus.

After 90 s at54C, wild-typevirionshad35% of their original hemagglutinin and 30% of their

original neuraminidase activities, but mutant

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SENDAI VIRUSTEMPERATURE-SENSITIVE MUTANTS

2.

2.0k

0.5-60 120

M NUTES

FIG. 4. Theeffect of30 and 38 Conneuraminidase activitiesoftheglycoprotein fraction from wild-type Sendai virus andts271.Replicatetubescontaining50

hemagglutinatingunitswereplacedat30or38 C. The

wild-typeviruspreparationcontained 5 ugof protein

perml,andts271contained 10 Mgof proteinperml.

Atintervals, onetube ofeach wasremoved, rapidly

chilled, andassayed forreleasedsialic acid(19, 20).

virions retainedonly3% of theirhemagglutinin andless than 1%of their neuraminidase

activi-ties. These data are consistent with the idea

that a single glycopolypeptide has

neuramini-dase and hemagglutinin activities, but other

explanationsare notruledout.

DISCUSSION

The Sendai virus genome has a molecular

weightof 6 x 106 (10). Thevirionpolypeptides have molecular weights from 40,000 to 80,000 (12). Thus, the genome may contain enough informationforeightto ten polypeptides.

Com-plementation experiments (Table 3) indicated

thatourSendai virusts mutantsweredefective

in seven different cistrons; thus, this small

seriesofmutantsmayalreadyrepresentmost of the viral gene functions. However, the number

of complementation groups may be high, be-causelowcomplementationvalues could reflect

intracistronic complementation or

complemen-tationbetweenmutants with multiple

tempera-ture-sensitivelesions.

The complementation groupings were

con-firmed in part by the physiological properties of

the mutants. We were able to classify the mutants into four categories according to their

ability to synthesize virus-specific RNA under

restrictive conditions. Complementation group

Gwas RNA+. Complementation groups A to F

synthesized neither viral RNA nor infectious

virus when incubated at the NP temperature

from the beginningof infection. However, after

shift-up late in infection, groups A, B, and C

synthesized substantial amounts of

virus-specific RNA, demonstrating that three gene

functionswererequired transientlyin the

repli-cative cycle for RNA synthesis to be initiated.

Groups D, E, and F appearedtobe defective in

functions required continuously for viral RNA

synthesis. The reason for the slow decline in

RNA synthesis with groups E and F after

shift-up is notapparent. One

possibility

isthat

at NP temperature the temperature-sensitive

defect prevents the formation of a protein

required for RNA synthesis, but once the

pro-tein is formed it remains functional aftershift from 30 to 38 C. Such mutants have been

described forSemliki forestvirus (11). Mutant

ts105 (groupD) showedarapid decline inviral

RNA synthesis after shift-up, suggesting that

itsgene productturns overrapidlyor isunable

to maintain a functional configuration after

shift toNP conditions.

Itmay seemsurprising thataviruswithsuch

a small amount of genetic information should

possess six functions related to RNA synthesis.

But the three

early

functions represented by

complementationgroupsA,B, and Care

proba-bly involvedinpreparing the viraltranscriptive

machinery for productive interaction with the

cell (entry,

"uncoating,"

transport, etc.),

be-cause

they

are not

required

later ininfection. It

should be

emphasized

that thesefunctions are

only "early" with respect to RNA synthesis:

with respect to virus production they are also

"late," according to the results of shift-up

experiments. This suggests that theyrepresent

virion structural components.

One structural elementofvirionswhose

func-tion maynotberequired forviral RNA

synthe-sis is the hemagglutinin, asexemplified bythe

RNA+ phenotype of mutant ts 271. But it is

possible thatother mutants of thispolypeptide

will be discovered which will affect RNA

syn-thesis.

Recently,

we showed that virion

enve-lope glycoproteins (12) and the smallest virion

polypeptide, also thought to be an

envelope

38C

30C WT

38C 271 ,,

n

30C

0 A \

Al

/

0X

/ lll

303

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component (10), inhibit virion transcriptase in vitro (P. A. Marx, A. Portner, and D. W.

Kingsbury, manuscript submitted for

publica-tion). Thus, these polypeptides are all

candi-dates for aroleinregulating viral RNA

synthe-S1S.

RNAsynthesis functions continually required

during infection, for which complementation

groups D, E, and F are candidates, would

include transcription and genome replication.

Transcriptive complexes isolated from virions

andinfected cells containtwopolypeptides, the

nucleocapsid structure unit (molecular weight

60,000) and thelargest virion polypeptide

(mo-lecular weight 75,000) (18; P. A. Marx, A.

Portner, and D. W. Kingsbury, manuscript

submitted for

publication).

Bothofthese may

be needed for transcriptase activity.

Polypep-tides involved in genome

replication

have not

been identified.

It isclear thatwe cannot exclude any

virus-specified

polypeptide

from an involvement in

paramyxovirus RNA

synthesis

at this stage in

our

understanding. Hopefully,

additional work

onthebiochemistry ofSendaivirus ts mutants

will contribute useful answers to these

ques-tions.

ACKNOWLEDGMENTS

Ruth Ann Scroggs and Andrew W. Moseley provided experttechnical assistance.

This research was supported by Public Health Service research grant AI-05343fromtheNational InstituteofAllergy and InfectiousDiseases,by traininggrantT01-CA-05176 and Childhood Cancer Center research grant CA-08480 from the National Cancer Institute, andby ALSAC.D. W.Kingsbury received Career Development Award HD-14,491 from the National InstituteofChild Health and HumanDevelopment.

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3. Burge,B.W., andE. R. Pfefferkorn.1966. Complementa-tionbetweentemperature-sensitive mutants of Sindbis virus.Virology30:214-223.

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