Structural polypeptides of simian rotavirus SA11 and the effect of trypsin.

0022-538X/81/010156-05$02.00/0

Structural

Polypeptides

of

Simian Rotavirus

SAil and

the

Effect of

Trypsin

ROMILIO T.ESPEJO,* SUSANALOPEZ, ANDCARLOSARIAS

Instituto deInvestigacionesBiome'dicas, UniversidadNacional Aut6noma deMexico,Mexico20,Distrito

Federal, Mexico

Analysis of purifiedsimianrotavirushas shown that it contains fewer structural

polypeptide classes than previously reported. Two polypeptides (molecular weights, 62,000 and 28,000) commonly found in purifiedrotaviruseswere,infact, produced by cleavageofalargerstructuralpolypeptide (molecular weight,about 88,000) by trypsin, whichisusually employedtoincrease theyieldofrotaviruses in tissue culture.

Trypsin-uncleaved,

double-shelled rotaviruses are probably composed of onlyfivepolypeptideclasses;three in the innerlayer,and two inthe

outerlayer.

Several workers have analyzed the polypep-tides ofrotaviruses obtained either from stool

samples of different animal species orfrom

in-fected cellculturesby polyacrylamide gel

elec-trophoresis(9, 11, 13, 14, 16-19, 21, 23, 24).These

studies haveyielded different datawithrespect

tothe numberofpolypeptide species (5to10)in

rotavirus particles and their apparentmolecular

weights.

Eight (9, 11), nine (19, 21),orten(13)different

polypeptide specieshave been described in

pur-ified tissueculture-grownsimian rotavirusSAil. However,allof thesereportsagreeonthe exist-ence ofatleast eight polypeptides, designated

VP1,

VP2,

VP3,

VP4, VP5,

VP6,

VP7,and VP8

by

Rodger

etal. (19).

Inthispaper,weshow thatinSAil,VP5 and VP8 are trypsin cleavage products of

polypep-tide VP3. We discuss the possible implications

of thiscleavage in the enhancement of infectivity ofrotaviruses and the number of SAil virion structuralpolypeptides.

MATERIALS AND METHODS

Virus. Simian rotavirus(SAil),obtainedoriginally

from H. H.Malherbe, UniversityofTexas,wasgrown

in MA104 cells aspreviously described (9). Trypsin

was used in all preparations unless otherwise

indi-cated.Topreparelabeledvirusforstructuralanalysis,

minimalessentialmediumcontainingalow

concentra-tion ofL-methionmie (0.3 mg/liter) and 33

ACi

of

L-[3S]methionineperml(>500 Ci/mmol;NewEngland

Nuclear Corp., Boston, Mass.) was replaced at 6 h

postinfection. Viruslabeled with14C-aminoacidswas

obtainedbyadding,at6hafter infection,1mCi ofa

U-3H-labeledL-aminoacidmixture(NewEngland

Nu-clear, Corp. Boston, Mass.) To obtain

trypsin-un-cleavedvirus,thecells were washedfour timeswith

phosphate-buffered salineafteradsorptionof the

tryp-sin-treated virus and then three times more before

addition ofthe radioactive medium.

156

Purification of virus. Cleaved viruswaspurified

aspreviouslydescribed(9). Uncleaved viruswas

pur-ified at 0 to 50Cas previously described (9) except

that instead ofsedimenting the virus in the precipitate

obtained withpolyethylene glycol in the discontinuous

gradients of CsCl and sucrose, itwasresuspended in

100 to200

pl

of TSM buffer(0.01 MTris, 0.15 M NaCl,

0.001M MgCl2.6H20, pH 8.2) and then sedimented

in alinear15 to45%sucrose(in TSM buffer) gradient.

Aftercentrifugation at 19,000 rpm for 145 min in the

SW40rotor ofa Beckman ultracentrifuge, the

gra-dients werecollected, and the radioactivity peak due

tothe viruswaspooled. The virus was subsequently

centrifugedtoequilibriumat40,000 rpmfor 17 h after

layering on top of a CsCl solution made in TM buffer

(0.01 MTris, 0.001MMgCl2, pH 8.2) with a density of

1.36 g/cm3. After centrifugation, fractions were

col-lected and counted, and those fractions containing the

radioactive virus were pooled and dialyzed against

TSM buffer.

Polyacrylamide gel electrophoresis of

poly-peptides. Sampleswereanalyzedinsodium dodecyl

sulfate-polyacrylamidegels (11%polyacrylamide, 0.3% bisacrylamide) by using the Laemmli (12)

discontin-uous Tris-glycine buffer system. Protein was

disso-ciatedby treatmentwithLaemmli sample buffer (12)

in aboiling water bath for2 min. Polypeptides were

stackedat 10 mA/gel, and then the electrophoresis

was continued at 20mA/gel until the bromophenol

blue ran off thegel. Gelsweresubsequently fixed and

stained with 30%methanol-10% acetic acid solution

containing0.06% (wt/vol) Coomassie blue and then

processed forfluorography by the method of Bonner

andLaskey (4).

Treatmentof virions with proteases. Purified

labeledvirions in TSMbuffer were treated either with

10

jig

ofchymotrypsin (type II; Sigma Chemical Co.,

St. Louis, Mo.) per ml for 20 min at 25°C or with

variousamountsoftrypsin treated with

diphenylcar-bamyl chloride to inactivate contaminating

chymo-trypsin(type XI;Sigma Chemical Co., St. Louis, Mo.),

asindicatedin thefigure legends for20minat 25°C.

Peptidemapping ofSA1l polypeptides. Peptide

map analysis of SAll polypeptides was performed

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using either Staphylococcus aureus V8 protease (Miles Laboratories, Inc., Elkhart, Ind.) or

chymotryp-sin asdescribed by Cleveland et al. (7). Briefly,

sam-ples of SAll structural polypeptides containingatotal

of about106 cpm were fractionated in sodium dodecyl

sulfate-polyacrylamide gels, and the appropriate bands were identified by autoradiography of the wet gel and subsequently excised. The resultant gel slices

wereplaced in the sample wells of a second sodium

dodecyl sulfate gel (5-cm-long 4% acrylamide stacking

gel, 15%acrylamide separating gel) and overlaid with

50ugofalbumin in25

id

ofCleveland sample buffer

(7); the slices then were subsequently overlaid with

either10,gof

chymotrypsin

or 100ug of V8 protease

insample buffer without bromophenol blue. Digestion

proceeded directly in the stacking gel during

subse-quentelectrophoresis, and the current was turned off

for30minwhen the bromophenol blue dye neared the

bottom of thestacking gel, as described by Cleveland

etal. (7).

Treatmentof virus withEDTA. Virus in TSM

buffer was incubated with 0.01 M EDTA and 0.1%

Triton X-100 for 30min at room temperature. The

treated virus wasthencentrifuged to equilibrium in

CsCl as mentioned above. After centrifugation, the

tworadioactivepeaks corresponding to the outershell

polypeptides (p = 1.3g/cm3) and the single-shelled

particles (p= 1.39g/cm3) were collectedseparately,

diluted 1:5with water, andprecipitated with

ammo-niumacetate-ethanol (20). The precipitates obtained

after

centrfugation

were resuspended in Laemmli

sample buffer and subjectedtoelectrophoresis.

RESULTS

Structural

polypeptides in differently

purified SAll

rotavirus. When

the

polypep-tides

present

in

purified SAll rotavirus obtained

after infection in the

presence

of

trypsin

were

analyzed by polyacrylamide gel electrophoresis,

using the Laemmli buffer

system, a pattem

sim-ilar

to

those

previously reported

for

SAll (6,

9, 13,

21)

was

observed. These

patterns,

although

not

identical,

agree on the

existence of

at

least

eight different

structural

polypeptides.

How-ever, when

SAll

was

obtained either from

cells

exhaustively washed after infection with

trypsin-treated virus

or

from

cells infected in

the absence

of

trypsin

and

then

purified

at a

low

tempera-ture,two

polypeptides,

called

VP5

and

VP8

(13),

01

and 2

(21),

or5and8

(9) by

different

authors,

were

barely

distinguishable,

whereas

the

amount

of

a

larger

polypeptide,

probably

that

called

VP3,

was

notably

increased

(data

not

shown).

(The nomenclature

originally

used

by

Rodger

et

al.

[19]

and

adopted by

Masonetal.

[13]

will

be

used.) Since

thesum

of

the

estimated

molecular

weights

of VP5 and VP8 is closetothatof VP3

(62,000, 28,000,

and

88,000,

respectively [13,

21]),

the

possibility

that VP5 and VP8 were

produced by

cleavage

of VP3

by

trypsin

was

explored.

Effect

of trypsin in SAll. When

purified

virus,

obtained

under

conditions that render

a

relatively larger

amount

of

VP3,

was

treated

with purified

trypsin,

a

precursor-product

rela-tionship

was

suggested between

VP3 and both VP5 andVP8, whereas the other

polypeptides

seemed

toremain

unchanged (Fig. 1).

This

spe-cific

cleavage of

VP3

with

trypsin

was

only

ob-tained with intact virus. When

EDTA-disrupted

particles

were

incubated with

trypsin,

VP3

and

several other structural proteins

were

exten-sively degraded (data

not

shown).

Outer and inner

capsid polypeptides.

Since it has been

reported that VP5 and

VP8 are

found

only in double-shelled

particles,

whereas

VP3 and VP4 are

also found

in

single-shelled

particles (13,

19,

21),

the

polypeptide

composition of the

outer

and inner capsid

of

uncleaved

purified rotavirus

was

examined.

Sin-gle-shelled particles banding

at 1.39

g/cm3

and

outer

capsid proteins banding

at 1.30

g/cm3

were

produced by

EDTA

treatment (8), and after

separation from each other by centrifugation

to

equilibrium

in a

CsCl gradient, they

were

ana-lyzed by polyacrylamide

gel electrophoresis.

Fig-ure 2

shows that

polypeptides VP3 and VP7

associated with

double-shelled particles

were

re-leased

by

EDTA treatment,

whereas

VP1, VP2,

and

VP6 were present in

high-density,

single-shelled particles.

Effect of

chymotrypsin.

Since

most

trypsin

2 3 4 5 6

FIG. 1. Effect of trypsin on

purified

SAlI

virus.

[3SJmethionine-labeled

SAlI,obtained and

purified

avoidingthe action of trypsin, was treated with 0

(track1), 1(track 2),2(track 3),5

(track 4),

10

(track

5), and100(track 6)pg

of trypsin

(diphenylcarbamyl

chloridetreated)per ml

for

20minat

25°C,

and the

virus wassubsequently

analyzed

by

polyacrylamide

gelelectrophoresis.

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[image:2.496.268.438.396.573.2]

158 ESPEJO,

LOPEZ,

AND ARIAS

2 3

VPI + _ ;:

VP3

VP5

VP VP7

VP8

FIG. 2. Polypeptides in double-shelled virus (track

1), outer layer (track 2), and single-shelled virus

(track 3).3H-amino acids-labeled SAlI, obtained and

purified avoiding trypsin action on progeny virus,

wastreated with EDTA, and the released

polypep-tides were separated from single-shelled particles (p

=1.39) bycentrifugation to equilibrium in CsCL.

Un-treated virus, polypeptides released by EDTA, and

single-shelled virus were then analyzed by polyacryl-amide gel electrophoresis.

preparations used

toincrease the

yield

of

rota-viruses contain

contaminating

chymotrypsin

ac-tivity

(see

reference

17and most

manufacturer

information)

whichcould also cleave some

struc-tural

polypeptides of

SAl1,

the

effect of this

enzyme on

SAll

was

examined. After

treatment

with

chymotrypsin, besides

two

polypeptides

migrating

like VP5

and VP8

which

were

proba-bly produced by contaminating trypsin activity,

another

polypeptide,

not observed

after

treat-ment with purified trypsin, was produced. The presence

of

this

polypeptide

which has a

similar

electrophoretic migration

to that of VP4 sug-gests

that

the

faint

band of

protein observed

in some

purified SA1l

virus and

considered

to be VP4may have been due to the action of

chy-motrypsin.

Comparison

of

SAll polypeptides by

partial digestion.

Other evidence that VP5 and VP8 are derived from VP3 was obtained by comparison of the peptides generated from these proteins by both chymotrypsin and S. aureus V8 protease (Fig. 3). When this analysis was used to compare other structural proteins of

SAl1,

a similar

pattern

was also found for the glycosylated polypeptides VP7 and VP7a (Fig.

4),

inagreement with

preliminary

observations

mentioned

by Mason et al. (13).

DISCUSSION

Polypeptide

composition of

SAil

virion seems to be

considerably

simpler

than what has been

[image:3.496.80.225.70.245.2]

reported. Previous reports indicated the exist-ence of 8 (9, 11), 9 (19, 21), or 10 (13) polypep-tides in purified particles. All three reports agree onthe existence of viral polypeptides VP1, VP2, VP3, VP4, VP5, VP6, VP7, and VP8. We show

FIG. 3. Comparison of [36S]methionine-labeled

polypeptides VP3, VP5, and VP8 by polyacrylamide

gelelectrophoresis of thepeptides generated by

diges-tionwith proteases. Polypeptide VP3 wasobtained

from trypsin-uncleaved, purified SAlI after

poly-acrylamide gelelectrophoresis; VP5 and VP8were

isolated from trypsin-treated SAlI. VP5 (track 1),

VP3(track 2), and VP8 (track 3) weretreated with

chymotrypsin (A) and S.aureusV8 protease (B).

A ts

2 1 2

FIG. 4. Comparison of [35S]methionine-labeled

polypeptides VP7 and VP7a by polyacrylamide gel

electrophoresisofthepeptides generated by digestion

with proteases. Polypeptides VP7 and VP7a were

obtainedfrompurified SAlI after polyacrylamide gel

electrophoresis. VP7(track 1) and VP7a (track 2)

weretreated withchymotrypsin (A)andS. aureus V8

protease (B).

J. VIROL.

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[image:3.496.258.454.141.276.2] [image:3.496.294.412.371.580.2]

inthis manuscript that

SAil

virions are

proba-bly made of five polypeptides-VP1,

VP2, VP3,

VP6, and VP7;

VP3 and VP7 being unique of double-shelled particles, and VP5 and VP8 being produced by cleavage of either VP3 or VP4 by

trypsin.

The observation that

VP5

andVP8 are

cleavage

products explains their absence among

the polypeptides

obtained after in vitro

transla-tion

of SAl mRNA

(13) or SAl1-denatured

RNA

(21).

A

protein observed

in infected

cells

but

not in pure viruses has beendesignated

OlA

because it

wasconsidered a likely precursor of

01

orVP5

(23).

Since RNA segment

5

of

SAl

codes

for

OlA

(21), itcould be assumed that this

RNA

segment

codes for

a structural protein.

The

evidence

from

this studysuggests that

SA11

RNA

segment 5

probably

codes for a

nonstruc-tural protein.

That

OlA

is not aprecursor of VP5

is in

agreement

with

theobservation of Matsuno

and

Mukoyama (14) that a polypeptide present

in

Nebraska calf diarrhea

virus-infected

MA104

cells, called NCVP1, with similar migration

to

01A,

remained

unchanged during

a

2-h chase

period

performed after

pulse-labeling.

The

polypeptide cleaved

bytrypsin has been

called

VP3 even

though

its correspondence with

polypeptide

VP3

of

Mason et al. (13) or

13

of

Smith

et

al.

(21) could not be firmly established. We have

called

this polypeptide VP3 because

when

a

fourth band

was

occasionally

observed among

the

larger viral polypeptides,

this

mi-grated ahead

of the

polypeptide cleaved by

tryp-sin.

However,

we

have

some

doubts

on

consid-ering

VP4 a

structural

component

of

the

virion

because

a

similar

polypeptide

can

be

produced

after

chymotrypsin

treatment

of

SA11,

and

most

trypsin

preparations

used

to

increase the

yield

of rotavirus

contain this

contaminating activity

(see reference

17

and

most

manufacturer

infor-mation).

Polypeptide

VP7a

observed

by

Mason et

al.

(13) and also

by

us

in the

present

study

is

probably

VP7

glycosylated

toadifferent extent as

suggested by

comparison

of the

products

ob-tained

after

partial digestion

of both

polypep-tides.

Trypsin

treatment is knownto enhance the

infectivity of SAl

two- to

ninefold

(10).

We

have

observed

an

enhancement of the

infectivity

of

purified

virus of

eightfold

after

trypsin

treat-ment

(5

,ug/ml

per 20 min at

2500)

without

noticeable

effectonthevirus other than

cleavage

of VP3 in VP5 and VP8

(data

not

shown),

and it is

likely

that this

cleavage

is thecause of the increased

infectivity

of the treated virus. Mat-sunoand

Mukoyama

(14)

reported

that

trypsin

treatment of

Nebraska

calf diarrhea virus

af-fected all

eight

polypeptides

and caused the appearance of severalnewbands. Their different

results

might be due to the difference in the temperature

(37°0)

and

type

of

trypsin

(acety-lated) employed.

Thespecificity of the cleavage observed with

trypsin

seems

mainly

due to

the

availability

of VP3 sites to the enzymebecause VP3 and other

SAll

structural proteins

arecut atmany

sites

if

the

outer

capsid of

the virus is

released

with

EDTA before

enzyme treatment (data not

shown).

Enhancement

of

infectivity

by

trypsin

has

been

widely observed with rotaviruses found

in

different species (1-3,

5, 15, 22,

23). Since

most

rotaviruses

analyzed

contain a

polypeptide

with

electrophoretic migration similar

to

polypeptide

VP5

of SAl

(9, 14, 16-18, 19, 23, 24), it is

possible that cleavage

of one of the

larger

rota-virus

structural polypeptides,

withproduction of a

polypeptide

with

similar electrophoretic

mi-gration

to

VP5, is

a

general mechanism

of en-hancement

of

infectivity

in these viruses. The

cleavage described here

is

analogous

to

that

observed for the

F and HA proteins

of

para-myxoviruses

and

myxoviruses, respectively, that

activates the

infectivity

of

these viruses,

proba-bly

at

the level of virus

penetration

(for

a

review,

see

reference

6).

The N

terminus

generated by

the

cleavage

on

Fl

of

paramyxoviruses

and on

HA2

of

influenza

viruses showsa

significant

ho-mology,

and

oligopeptides

with

specific

amino

acid

sequences that mimic the

active sites

on these

proteins

have been shown to be

effective

inhibitors of virus

replication

in tissue

culture.

These inhibitors have been

suggested

as a

novel

tool

to

control virus infections

(6). The

true

analogy of the activation by

trypsin between

rotaviruses and

paramyxoviruses

or

myxoviruses

requires further studies which

might offer

an-other

approach

to

the control of

rotavirus

infec-tions.

ACKNOWLEDGMENTS

This workwaspartially supported bygrant 1539fromthe Programa Nacional Indicativo de Salud of theConsejo Na-cional de CienciayTechnologia.

It isa pleasure to acknowledge the excellent technical assistance ofP.Alvarez and P. Romero.

LITERATURE CITED

1. Almeida,J.D.,T.Hall,J. E.Banatvala,B. M.

Tot-terdell,andL.L. Crystie.1978.Theeffect oftrypsin

onthegrowthofrotavirus. J.Gen.Virol. 40:213-218.

2. Babiuk,L.A.,K.Mohammed,L. Spence,M.Fauvel,

and R.Petro. 1977. Rotavirusisolation and cultivation in the presence oftrypsin.J.Clin.Microbiol. 6:610-617. 3. Barnett,B.B., R. S.Spendlove,andM. K.Clark.1979. Effectofenzymesonrotavirusinfectivity.J.Clin. Mi-crobiol. 10:111-113.

4. Bonner,W.M.,andR. A.Laskey.1974. Afilmdetection method fortritium-labeledproteinsand nucleic acids in

polyacrylamidegels.Eur. J. Biochem. 46:83-88.

5. Bryden,A.S.,H.A.Davies,M. E.Thouless,andT.

H.Flewett.1977.Diagnosisofrotavirusinfectionby

on November 10, 2019 by guest

http://jvi.asm.org/

160 ESPEJO, LOPEZ,

cell culture. J. Med. Microbiol. 10:121-125.

6. Choppin, P. W., and A. Schied.1980.Theroleof viral glycoproteinsinadsorption, penetration and pathogen-icity of viruses. Rev.Infect. Dis. 2:40-61.

7. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol.Chem. 252:1102-1106.

8. Cohen,J., J. Laporte, A.Charpilienne, and R.

Scher-rer. 1979.Activation of rotavirus RNA polymerase by calcium chelation. Arch. Virol.60:177-186.

9. Espejo, R. T., E.Martinez, S. Lopez, and0.Munoz. 1980. Differentpolypeptide composition of two human rotavirus types.Infect.Immun.28:351-354.

10.Graham, D. Y., and M. K. Estes. 1980. Proteolytic enhancementof rotavirus infectivity: biologic mecha-nisms. Virology101:432-439.

11. Kalica, A. R., and T. S. Theodore.1979.Polypeptides of simian rotavirus(SA-ll)determinedbyacontinuous

polyacrylamide gelelectrophoresis method. J. Gen.

Vi-rol. 43:463-466.

12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London)227:680-685.

13. Mason, B. B., D.Y.Graham, andM. K.Estes. 1980. In vitro transcription and translation of simian rotavirus SAll geneproducts. J. Virol. 33:1111-1121.

14. Matsuno,S., and A. Mukoyama. 1979. Polypeptides of bovine rotavirus. J.Gen. Virol. 43:309-316.

15.Moosai,R.B.,P.S.Gardner,J. D.Almeida, andM.

A. Greenaway. 1979. A simple immunofluorescent

technique forthedetection of human rotavirus. J. Med. Virol. 3:189-194.

16. Newman, J. F. E., F. Brown,J.C. Bridger, and G. N. Woode.1975.Characterization of a rotavirus. Nature (London) 258:631-633.

17. Obijeski, J. F., E. L. Palmer, and M.L. Martin. 1977. Biochemical characterization of infantile gastroenteritis virus(IGV). J. Gen.Virol.34:485-497.

18. Rodger, S. M., R. D. Schnagl, andL.H. Holmes. 1975. Biochemical andbiophysical characteristics ofdiarrhea viruses of humanand calf origin. J. Virol.16:1229-1235. 19. Rodger,S. M., R. D.Schnagl, and I. H. Holmes. 1977. Further biochemicalcharacterization, including the de-tection of surface glycoproteins of human, calf, and simian rotaviruses. J. Virol. 24:91-98.

20. Rueckert,R. R.1965.Studiesonthestructure of viruses

of the Columbia SK group.II.Theprotein subunits of ME-virus and other members of the Columbia SK group.Virology 26:345-358.

21. Smith,N.L.,I.Lazdins,and I. H.Holmes. 1980. Coding

assignments of double-stranded RNA segments of SAll rotavirus established by in vitro translation. J. Virol. 33:976-982.

22. Theil, K. W., E. H. Bohl, and A. G. Agnes. 1977. Cell culturepropagation of porcine rotavirus (reovirus-like agent).Am.J. Vet. Res. 38:1765-1768.

23. Thouless, M. E. 1979.Rotavirus polypeptides. J. Gen. Virol. 44:187-197.

24. Todd,D., and M. S. McNulty.1977.Biochemical studies

on areovirus-likeagent(rotavirus) from lambs. J. Virol. 21:1215-1218.

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