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Temperature-sensitive mutants of reovirus type 3: evidence for aberrant mu 1 and mu 2 polypeptide species.

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JOURNAL OFVIROLOGY, JUlY 1976,p. 174-179 Copyright©D1976 AmericanSociety forMicrobiology

Vol. 19, No.1 Printed inUSA.

Temperature-Sensitive

Mutants of Reovirus

Type

3:

Evidence for Aberrant ,l and ,u2 Polypeptide Species

RISE K. CROSS' AND BERNARD N. FIELDS2*

Departments of Cell Biology and Medicine, Albert EinsteinCollege ofMedicine, Bronx, New York 10461

Received forpublication3February1976

Ananalysis of reovirus-specific polypeptides incells infected with

tempera-ture-sensitive mutantsunder permissive and nonpermissive conditions revealed

the presence of (i) all the knownviral polypeptides and (ii) aberrant migration of

the,ul and ,u2 polypeptides in four groups of mutants.

Temperature-sensitive (ts) mutants of the

Dearingstrain of reovirus type 3 havebeen

iso-lated andclassified into seven groups (A to G)

onthe basis of genetic recombination analyses

(2, 4). Fields et al. (5) examined the abilityof

five groups of ts mutants to synthesize viral

polypeptidesatthenonpermissive temperature

by usingimmunological precipitationto detect

thosepolypeptides presentinsmallquantities.

Mutants of groups A toE wereshownby

poly-acrylamide gel electrophoresistobecapableof

inducing the synthesis of the three major size

classes ofreovirus-specific polypeptides (X,

t,

and (r). Greaterresolution of thesepolypeptides

was needed inorder to undertake a closer

ex-aminationof thespecificgeneproductsof thets

mutantsandthereby, perhaps,tobetter define

thenatureofthetslesion. The methodof poly-acrylamide gel electrophoresis using sodium

dodecyl sulfate inadiscontinuousTris-glycine

buffer (11) has been shown to resolve more

clearly thereovirus-specific polypeptides in

in-fected cells (1, 3;R. K. CrossandB. N. Fields,

Abstr. Annu. Meet. Am. Soc. Microbiol. 1974,

V21, p. 204).

MATERIALS AND METHODS

The methods used for this workare described in detail in theaccompanyingpaper(3).

RESULTS

Electrophoresis of ts mutant polypeptides

on sodium dodecyl sulfate-polyacrylamide

gels. To examine the virus-specificpolypeptides

synthesized by representative mutants of

groups A to G at the nonpermissive

tempera-ture (39C), cytoplasmic extracts of infected

cells were treated with immune sera, and the

IPresent address: The Rockefeller University, New

York, N.Y.10021.

2Presentaddress: The Department ofMicrobiologyand

Molecular Genetics, Harvard Medical School, Boston, Mass.02115.

precipitated 35S-labeled polypeptides were

an-alyzed by electrophoresis on Tris-glycine gels

(Fig. 1A and 2). When cells were infected at

39C inthe presence of actinomycin Dand the

polypeptides were precipitated from

cytoplas-micextracts withacetone, an appreciable

back-ground of hostcellular protein synthesis made

it considerably more difficult to identify the

viral polypeptides (Fig. 1B).

Mutants of all of the groups (A to G) were

foundtobe capable of inducing the same

poly-peptidespecies asthewild-type(ts+) strain

un-der nonpermissive conditions (Fig. 1A and 2).

The nomenclature used to denote virus poly-peptides is identical to that described previ-ously (3, 14). Both of the nonstructural species,

,u4

and (r2A, as well as all of the capsid

polypep-tides found in whole virions were synthesized

by the mutants. The absence of ,2 polypeptides

in cells infected with ts357, 320, and 453 is due

to the lack of processing of ,ul to ,u2 in this

particular experiment. This was often seen

whenmultiplicities in the low range were used

(note the relatively low amounts of the other

viral polypeptides synthesized by these three

mutants in this experiment). In Fig. 2, the

processing of ,1 to ,u2 and normal amounts of

the other viralpolypeptidesisillustrated. The

polypeptidespecies synthesized by the mutant

strains at the permissive temperature (31 C)

were also virtually indistinguishable from

those of the wild type (not shown). Careful

examination of the virus-specific polypeptides

synthesized at both 39 and 31C, however,

re-vealed that mutants of four of thegroups (A, D,

F, and G) possessed abnormal ,ul and ,u2

poly-peptides; i.e., both the uland itscleavage

prod-uct ,u2 migrated more rapidly on Tris-glycine

gels than the respective speciesof wildtype.

Analysis ofj, peptide regionof ts mutants

-evidence foraberrant polypeptide species. To

verify these ,ul and ,u2 migration differences,

viralpolypeptideswereanalyzedby

electropho-174

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ts MUTANTS OF REOVIRUSTYPE 3 175

A<

_

mc

*s

-_0- - -

-_ _ _

+ hN N e00o s (

+e

CNn

Oo fl ICe 0 N eC eC

0 Ln I n 00 C43 U L

.2- (V)m c7LO) (m ) 'q

XI

/X2_

-- r.^

v..

r;I

3 I- -*

3 F%A"4.

/-'1

I-L2 Hi4

B 2

U

7D

DO

to 'z ttro} Lo Nt t

(-N m- 'It (V) r)t COto tItz

-"

r14.

b&4

am _. am _W" on

a

_"

I_

4,ro,.,,I

:,..0a No w

X~.% &.

two

^* _ b'TA-.^

_w

4*

a1

a-2

a-2A0

5

q6

r U

ar3mses

- Z _ e 6

o _ s

_ _

_o _ ww 4

FIG. 1. Analysisofthe viral-specific polypeptides synthesized by ts+ and mutants ofgroups A to G at the nonpermissivetemperature. A total of1 x 107 cells was infected with ts+ and mutants ofgroups A to G at 39 C inthe presence of0.5pgofactinomycin D per ml. At 8 h postinfection, cells were concentrated to 1 x 107cellsl

ml in growth medium containing 0.05x (5% of the normalamount) methionine and pulsed for 1.5 h with 50

MCi of [35S]methionineper ml. Cytoplasmic extracts wereprepared and divided intoequal aliquots, and

polypeptideswereprecipitated by immune sera or by acetone as described previously (3). Electrophoresis was carried out on 10% Tris-glycine gels for 15 h at 50 V. These and subsequent gels were prepared and electrophoresed in a slab gel apparatus (3). (A) Gel autoradiogram of immune-precipitated polypeptides; (B)

gelautoradiogram of acetone-precipitated polypeptides. The direction of migration is from top to bottom.

resis under conditions that maximized

resolu-tionof the ,u polypeptide regions. The results of

these experimentsclearly showed that the

elec-trophoretic behaviorofthe,1 and,2 polypep-tides of mutants of groups B (ts352), C(ts447),

and E(ts320)wasidenticaltothat of the uland

pu2

species of the wild type (Fig. 3). The two

other group B mutants, ts405 and ts271, have

also been found to possess the wild-typea1 and

,u2

polypeptide phenotype. On the other hand, the electrophoretic behavior of the ,ul and ,2

polypeptides of the group D mutant ts357 and

the group A mutant ts201 was reproducibly

aberrant (Fig. 3). The groupA mutant

,1

and

p2species migrated slightly more rapidly than

the respective species of the wild type. The

group D mutant species migrated even more

rapidly than both the group A mutant and the wild type.

The electrophoretic behavior of the pu

poly-peptidesofgroupsAand D wasthencompared

with that of groups F andG, whichwere also

foundtosynthesizeaberrant,upolypeptides. As

shown inFig. 4, the migrations of the ,1 and

,2 polypeptides ofthe group D mutant ts357

and the group Dmutant ts453 wereboth

simi-larly aberrant. However, whereas the group D

mutant ts357 (and the G mutant) synthesized

gl

and p2 species that migrated faster than

those of thets+ strain, the other group D

mu-tant,ts585,inducednormally migrating ,ul and

,2polypeptides (Fig. 5).

Acomparison of themigrationof thep1 and

,u2polypeptides species of thegroup A mutant

ts201 and the group F mutant ts556 has

indi-cated thatthey both contained similarly

aber-rant species, which migrated slightly ahead of

the wild-type strain (Fig. 6). In addition, the

VOL. 19, 1976

*St...

'.'

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176 CROSS AND FIELDS

LO

4r

__

0

(N

1-1

LU

0

(N

CY)

cV)

A3

PL2

NW

-

4

0-1

02

O2A0

0-3

Om-O_

FIG. 2. Analysis of the viral-specific polypeptides synthesized by ts+ and mutant strains at the nonpermis-sive temperature. Infected cells were concentrated to 5 x 106 cells in growth medium containing 0.05 x methionine and 50 pXi of[35S]methionine at 5 h postinfection at 39 C. After a 1 h pulse, cytoplasmic extracts werepreparedand treated with immune sera. Gel electrophoresis was carried out on 10%polyacrylamidegels

for16h at 45 V. Gel autoradiograms ofpolypeptides of ts+ and mutants ofgroups A, D, G, and E are shown. The direction of migration is from top to bottom.

four other mutants of group A (ts340, ts16,

ts329, and ts438) thathave been examined

ex-hibited ,ul and ,2 polypeptides with the same

electrophoretic mobilityas theprototype group

A mutant ts201.

DISCUSSION

The ,ul and ,u2 polypeptides of reovirus ts

mutant strainsfall into three classes based on

theirelectrophoretic behavior:(i)thosethat

mi-grate asthe wildtypedo(groups B,C, E, and

D-ts585), (ii) those that migrate more rapidly

than the wildtype (groups A andF), and (iii)

those thatmigrateevenmorerapidlythanthe

preceding classes (groups D-ts357 and G).

These three classes of,ul and ,u2 polypeptides

(or,uphenotype) have been designated ,u+, u-,

and ,--, respectively.

With theexceptionof these slight buthighly

reproducible migration differences, analysis of

the polypeptides synthesized by the mutants

has demonstrated that thesame species

synthe-sized in cells infected with the wild type are

synthesizedincells infected withmutantsfrom

eachgroup atthe nonpermissivetemperature.

Mutantsof eachgroup arecapable of directing

thesynthesis of viral structural polypeptidesas

wellasthemajornonstructuralspecies, ,u4and

o-2A.This resultisentirelyconsistentwiththe

finding that all 10 species of mRNAare

tran-J. VIROL.

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[image:3.505.109.413.72.427.2]
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ts+

201 (A)

352(B)

447(C)

357(D)

II

I

320(E)

FIG. 3. Analysis ofthe y polypeptide region of

prototypemutantsofgroupsAtoE.Atotalof1 x1Of cells wasinfectedwith ts+ andts mutantstrainsat

31 C in thepresenceof0.5gofactinomycinDperml. At 18 hpostinfection, infectedcellswereconcentrated

to1 x 107 cellslml andpulsed in growth medium

containing 0.05x methionine and 50 ,UCi of [35S]-methionine per ml for 1 h. Cytoplasmic extracts

wereprepared, polypeptideswereprecipitated by

ace-tone, and electrophoresis was carried out on 10% Tris-glycine gelsat90 Vfor18 h. Under these condi-tions,the (r-sizedpolypeptides migrate offthegeland

the,u-sized polypeptides migratetothe lower thirdof

ts+

357(D)

453(G) /

320(E

I

~~~~I

I\\~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

II

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

~~~~II

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I~~~ ~~ ~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

III

FIG. 4. Comparison ofthe ,,u polypeptides ofthe group D mutant (ts357) and the group G mutant (ts453).Theexperimental detailsaredescribedinthe legend toFig. 3. Microdensitometer tracingsofthe gelautoradiograms oftheX-and

A-sized

polypeptides

ofts+and the groupD mutant(ts357), the group G mutant(ts453), and theE mutant(ts320)areshown.

the slabgel. Microdensitometer tracingsof gel

auto-radiograms of theX-andAz-sizedpolypeptides ofts+ and prototype mutantsofgroups A toEareshown. The directionofelectrophoresisisfromlefttoright.

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178 CROSS AND FIELDS

X1/X2

ts 352

(B)

ts357

(D)

ts585 (D)

p,4

[image:5.505.63.256.55.387.2]

x3 IL2

FIG. 5. Comparison ofthe ,upolypeptides ofthe twogroupDmutants, ts357 and ts585. The experi-mentaldetailsaredescribed in thelegendtoFig.3. Microdensitometer tracings of the X- and ,-sized polypeptidesof the group B ts352 and the two group D mutants, ts357 andts585,areshown.

scribed inmutant-infected cells under

nonper-missive conditions (2, 7) and with the

demon-strationof viral antigens and structuresin

non-permissively infected cells by

immunofluores-cence andelectron microscopy (6). The failure

to observe mature viral structures incells

in-fected with some of the mutants (B, C, D, and

G) at the nonpermissive temperature (6),

al-though the appropriate polypeptides are syn-thesized, suggested a posttranslational defect

eitherincleavage, modification, or assembly of

viralpolypeptides. Although it was not possible

tostudythe rate of cleavage of the ul

polypep-tide to the ,u2 species (for discussion of this

problem, see accompanying paper [3]), these

results conclusively demonstrate that the abil-ity to yield the ,u2 product is not impaired at the

elevated temperature. Itislikely that the

syn-thesized polypeptides are incapable of

assum-ing a functional configuration at the

nonper-missive temperature, thereby hindering the

normal assembly process and resulting in the

accumulation of particles whose completion is

blocked.

Synthesis of the aberrant ,1 and u2

polypep-tides at the permissive temperature does not

restore the wild-type electrophoretic behavior.

The expression of the u- and u-- phenotypes

is, thus, not temperature dependent. These

aberrant species are present not only in

cyto-plasmic extracts of permissively infected cells

but also are incorporated into matureinfectious

virions (Cross and Fields, unpublished data).

Wunner and Pringle (13) have similarly

de-scribed a ts mutant of vesicular stomatitis virus with two polypeptides of altered electrophoretic

migration but whose viability atthe permissive

temperature isunimpaired; the ts phenotype is

directly related to the altered mobility of only

one of theseproteins.

Controlleddigestion of groupDvirions with

chymotrypsin has also suggested that mutants of this group have an altered structural ,u2

polypeptide (9). One of the group D mutants

(ts357) also yielded retarded M2 and Li hybrid

double-stranded RNA species composed of a

P4

ts

ts201(

ts556(1

,A

+

~~~~~~~~~~2I

I+

I

al

I3

I

~~~~~~II

I

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

F)I

FIG. 6. Comparison ofthe ,u polypeptides of the group A mutant ts201 and group F mutant ts556. Theexperimental detailsaredescribedinthelegend toFig.3.Microdensitometer tracingsof thegel auto-radiograms of theX- andA-sizedpolypeptides of ts+ and the group A mutant ts201 and the group F mutantts556 areshown.

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mutant(+) single-strandedRNA strand anda

ts+ (-) single-stranded RNA strand (8). Since

the group D mutants appeared topossess

ab-normal,2polypeptides andoneof the mutants

yielded a retarded M2 hybrid double-stranded

RNA molecule, Schuerch and Joklik (12)

con-cluded that the group D-specific mutation to

temperature sensitivity was in theM2segment

(which waspresumed to code for this gene

prod-uct). However, in light ofthe above findings

that other mutant groups (A, F, and G) in

addition to group Dmutant ts357 alsopossess

aberrant ,2polypeptides(although theydo not

yield retarded M2 hybrid RNA species), it is no

longerclear that this is a group D-specific

de-fect. Thus, although the linkage ofaretarded

hybridto analtered gene product is consistent,

it does not necessarilyconstitute proof that

hy-brid retardation (as concluded in references 8

and 12)signifiesagroup-specifictsmutation.

The findingthat the ,uland ,u2polypeptides

are simultaneously affected lends additional

proof to the precursor-product relationship of

those polypeptides deduced from pulse-chase

experimentsby Zweerink and Joklik (14).

The precise biochemical alteration causing

the aberrant migration of the ,ul and u2

poly-peptides is not known. Whether the faster

mi-grationactually indicates that these species are

smaller than the respectivespeciesofthe

wild-type strainhas notbeendetermined. Since the

,u1

and

,2

polypeptidesarephosphorylated and

glycosylated (10; G. Krystal, J. Perreault, and

A. F. Graham, Virology, in press), variability

in modification, perhaps related to differences

inproteinconformation, couldaffect their

elec-trophoretic mobility. Biochemical studies

aimedatdistinguishingbetween these

possibil-ities arecurrently in progress.

ACKNOWLEDGMENTS

This researchwassupportedby Public Health Service

research grantAI-10326 from theNational Institute of

Al-lergy and InfectiousDiseases.

LITERATURE CITED

1. Both, G. W., S. Lavi, and A. J. Shatkin. 1975. Synthesis of all the geneproductsof the reovirus genome in vivo

andin vitro.Cell4:173-180.

2. Cross, R. K., and B. N. Fields. 1972. Temperature-sensitive mutantsof reovirus type 3: studies on the synthesis of viral RNA. Virology 50:799-809. 3. Cross,R. K., and B. N.Fields.1976.Reovirus-specific

polypeptides: analysis using discontinuous gel elec-trophoresis. J. Virol. 19:162-173.

4. Fields, B. N., and W. K. Joklik. 1969. Isolation and

preliminarygenetic andbiochemical characterization oftemperature-sensitive mutants of reovirus. Virol-ogy 37:335-342.

5. Fields, B. N., R. Laskov, and M. D. Scharff. 1972. Temperature-sensitive mutants of reovirus type 3:

studies onthesynthesis of viral peptides. Virology

50:209-215.

6. Fields, B. N., C. S. Raine, and S. G. Baum. 1971. Temperature-sensitive mutants ofreovirus type 3:

defectsinviralmaturation asstudied by

immunoflu-orescenceandelectronmicroscopy.Virology 43:569-578.

7. Ito, Y., and W. K. Joklik. 1972. Temperature-sensitive mutantsofreovirus. I.Patterns ofgene expressionby

mutantsofgroupsC,D,and E. Virology 50:189-201. 8. Ito, Y., and W. K.Joklik. 1972. Temperature-sensitive mutants ofreovirus. II. Anomalous electrophoretic

migrationofcertainhybrid RNAmolecules composed ofmutantplusstrand andwild-typeminusstrands.

Virology50:202-208.

9. Ito, Y., and W. K. Joklik. 1972. Temperature-sensitive mutantsofreovirus. III. Evidencethat mutantsof

group D("RNA negative")arestructuralpolypeptide

mutants.Virology50:282-286.

10. Krystal, G., P. Winn, S. Millward, and S. Sakuma. 1975.Evidenceforphosphoproteinsinreovirus.

Virol-ogy 64:505-512.

11. Maizel, J. V., Jr. 1971. Polyacrylamide gel electropho-resisof viralproteins, p.176-246.In K.Maramorosch andH.Koprowski(ed.), Methodsinvirology, vol.5.

Academic Press Inc., New York.

12. Schuerch, A. R., and W. K. Joklik. 1973. Temperature-sensitive mutants of reovirus. IV. Evidence that anomalouselectrophoreticmigrationbehaviorof cer-taindouble-strandedRNAhybrid speciesis mutant

group-specific. Virology 56:218-229.

13. Wunner, W. H., and C. R. Pringle. 1974. A tempera-ture-sensitive mutant ofvesicular stomatitis virus with two abnormal virus proteins. J. Gen. Virol.

23:97-106.

14. Zweerink, H. J., and W. K. Joklik. 1970. Studies on the

intracellular synthesis ofreovirus-specific polypep-tides.Virology41:501-518.

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Figure

FIG.1.polypeptidesgelelectrophoresedcarriednonpermissiveinmlMCi the Analysis of the viral-specific polypeptides synthesized by ts+ and mutants ofgroups A to G at the temperature
FIG. 2.forsiveThemethioninewere Analysis of the viral-specific polypeptides synthesized by ts+ and mutant strains at the nonpermis- temperature
FIG. 4.groupgeloflegend(ts453).mutant ts+ Comparison of the,,u polypeptides of the D mutant (ts357) and the group G mutant The experimental details are described in the to Fig
FIG. 5.polypeptidesDMicrodensitometertwomental mutants, Comparison of the ,u polypeptides of the group D mutants, ts357 and ts585

References

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