Coding assignments of double-stranded RNA segments of SA 11 rotavirus established by in vitro translation.

0022-538X/80/03-0976/07$02.00/0

Coding

Assignments of Double-Stranded

RNA

Segments of

SA

11

Rotavirus

Established

by In

Vitro Translation

MICHAEL L.SMITH,* IEVA LAZDINS,ANDIAN H. HOLMES

Departmentof Microbiology, University ofMelbourne, Parkville, Victoria3052,Australia

The segmented double-stranded (ds) RNAgenomeof the simianrotavirus SA

11,afterdenaturation,canbetranslated inacell-free protein synthesizingsystem. Of the 11genome segments,9 canberesolved onpolyacrylamide gels andthus

could be individuallyisolatedand translated, providinga meansof identifyingthe

polypeptide encoded by eachsegment.On the basis of electrophoretic mobilityof

products in sodium dodecyl sulfate-polyacrylamide gels, the probable gene-coding assignments of dsRNAsegments 1 to 6weredetermined. RNA segments 1to4

code for polypeptides II, I2, I3, and

I4,

respectively; segment 5 codes for a

polypeptide very similar in mobility toa minorpolypeptide present in SA 11-infectedcells,

01A;

andsegment6codes for the major inner-capsid polypeptideIs.

Rotaviruses are a common cause ofenteritis in the young of many, if not all,

mammalian

species (8, 10), including

humans.

Biochemical

and

biophysical

studies have

shown that the

rotavirus

genome consists of 11 segmentsof

dou-ble-stranded (ds) RNA (11,

21, 25, 32,33). The

simian rotavirus

SA 11

(14, 22, 26) is

readily

cultivable in cell culture and is thus

a useful

model rotavirus for detailed laboratory

study.

Polyacrylamide

gel electrophoresis of genomic

RNA

produces

10

bands,

1band

being composed

of

two

RNA

segments

(segments

7

and

8)

of

equal mobility (20).

Structural

polypeptides of

purified SA

11

virus have been well

character-ized and

aresimilar to

those

of

other rotaviruses

(22,

32). Few

studies

of

the virus-coded

polypep-tides in

infected

cells

have

so

far

emerged (16,

29);

however,

it appears that in addition to

nine

structural

polypeptides,

there are atleast three

nonstructural

polypeptides.

Recently,

a method for

assigning

cognate

RNA

and

polypeptide species

for reovirus was

published (17). Reovirus

also containsa

dsRNA

segmented

genome

(24). By isolating

individual

dsRNA segments,

denaturing them, and then

adding

them to acell-free translationsystem, we

could

analyze

the

proteins produced,

and

com-pare them withtruereovirus

polypeptides.

This

enabled

coding assignments

tobe made.

With this method the

polypeptides

encoded

by

each of the first six dsRNA segments of

SA

11 rotavirus have been determined. We regret

that,

duetothe

difficulty

of

separating

segments

7, 8, and 9, and the apparent failure of our

translation system to

synthesize

products

re-latedto rotavirus

glycoproteins,

we are

unable

to present

assignments

for the

remaining

seg-mentsatthisstage.

MATERIALS AND METHODS

Cells andvirus.Thefetal rhesusmonkey kidney

cellline MA104was agift from S. Matsuno. Cultures

were grown inEagle minimum essential medium

con-taining nonessential aminoacids,10% fetal calf serum,

andantibiotics(penicillinandstreptomycin).

SA 11 virus waskindly supplied by H. Malherbe

andwasplaque-purifiedtwice in MA104cells.A

first-passagestock (1.1 x

107

PFU/ml)wasused throughout

thisstudy. Virus stockswereinfected celllysates

pre-pared by freeze-thawing andwerestored at-70°C.

Virusgrowthandpurification.Confluent

mono-layers of MA104 cellswerewashed with

phosphate-bufferedsaline and infected withadilution of

plaque-purifiedvirus stock in virusdiluent (Hanksbalanced

saltsolution,0.01 M

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES], 0.02% gelatin) at a

multiplicity of infection between0.1 and1 PFU/cell.

After an adsorption period of 1 h at 37°C the

inoculumwasdecanted and maintenance mediumwas

added(minimalessentialmedium, 0.05% bovineserum

albumin). The cells and culture fluidwereharvested

at3dayspostinfection,atwhich timegrosscytopathic

effects were evident. Initial fluorocarbon extraction

wasperfonnedby the method ofRodgeret al. (21).

Theresultant fluidwascentrifugedat24,000 rpm for

2.5hat4°C inaSpincoSW25.2 rotor, the virusbeing

pelleted through8ml of 35%(wt/wt)sucrosein0.002

MTris-hydrochloride (pH 7.5).Theconcentrated

vi-rus was sonicatedfor 15 sand then bandedinCsCl

and concentrated by centrifugation as described by

Rodgeretal.(21).

Plaque assay for SA11.The method ofMatsuno

etal.(15) fortheplaqueassay of Nebraska calf

rota-virus wasadapted byS. Rodgerin thislaboratoryfor

SA 11rotavirus(unpublished data). Briefly,confluent

monolayers of MA104 cells inplastic culture dishes

werewashedoncewithphosphate-bufferedsaline,the

virusinoculum (suitablydilutedinvirusdiluent)was

added, andviruswasallowedtoabsorbfor1hat37°C.

Thentheinoculumwasremovedandoverlaymedium

(minimal

essential medium, 0.05% bovine serum

al-976

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bumin,5,ugof trypsin[1:250, Difco Laboratories] per

ml, 0.5%agarose)wasadded. At3dayspostinfection

the cellswerefixed informol-saline and stained with

crystal violet, and theplaqueswerecounted.

DEAE-dextran (15, 28) was notfound tobe necessary, but

trypsin was essential. Plaques were clearly evident

whenobservedatlowmagnificationbeforefixingand

staining.

Preparation of

[36Slmethionine-labeled

SA 11

virus. Cellswereinfectedasdescribed above. At6h

postinfection, the maintenance mediumwasreplaced

by mediumcontainingonly 10% of the usual

methio-nineconcentration and 5,uCi of [3S]methionine(1,300

Ci/mmol;AmershamCorp.)perml. At3days

postin-fection theinfected cultureswereharvested,and the

virus waspurifiedasdescribed.

Preparationof labeled intracellular

rotavirus-coded polypeptides. Confluent monolayers of

MA104 cells in35-mmplastic petridisheswerewashed

withphosphate-buffered saline and infected withSA

11 virus at amultiplicity of10PFU/cell. Viruswas

allowed to adsorb for 1 h at 37°C, and then the

inoculumwasremoved and maintenance mediumwas

added. At10 or 12hpostinfection, the mediumwas

changedtomaintenancemedium without methionine.

After30minthemediumwasagain changedto

main-tenancemediumcontainingmethionineat 10%of its

normal concentration and 10,uCi of[3S]methionine

perml. Incubationwascontinued for2h, after which

the cellswerewashed twice with ice-cold

phosphate-buffered saline and dissolved in 200

pl

of Laemmli

sample buffer (12). DNA was sheared by passage

througha25-gaugeneedle.

Extraction of dsRNA. Purified SA 11 virus in

0.002MTris-hydrochloride (pH 7.5)wasmade upto

1ml withSTE (0.15 M NaCl,0.05M

Tris-hydrochlo-ride,1mMEDTA,pH7.5)buffer,and sodiumdodecyl

sulfate(SDS)wasaddedto afinal concentrationof 1%

(wt/vol). After 30min at 37°C the solution was

ex-tracted three timesat roomtemperature withanequal

volume of water-saturatedphenol.The aqueousphase

was made 0.3 M with respect to sodium chloride,

residualphenolwasremoved, and RNAwas

precipi-tatedat-20°Cby the addition of3volumes ofethanol.

The precipitate was collected by centrifugation,

washed three times with 90%ethanol, dried in air, and

dissolved in 50

pl

of distilledwater. The amountof

RNAwasestimatedby usingtherelation:oneunit of

optical densityat 260 nm=50,g of dsRNA per mlfor

reovirus(23). Convenientamounts(50or 100

Ag)

were

storedasethanolprecipitatesat-700C.

Fractionation of individual dsRNA species.

Preparative polyacrylamide slab gels (10%, 1.5 mm

thick) wereprepared by the method of Laemmli (12)

butwithoutastacking gel. Before loading, the RNA

wasdissolved in

Laemnli

sample buffer and heated to

70°C for 2 minto resolve RNAaggregates (27). The

gelwaspreelectrophoresed for1hat 40 mAwithlower

gel buffer, then the lower gel buffer in electrode

res-ervoirs wasreplaced with Laenunli reservoir buffer,

the RNAsamplewasloaded, and electrophoresis

con-tinued for20 h (room temperature, 25 mA). The gel

was stained with0.005%ethidiumbromidein 20 mM

sodium acetate(pH 7.8) (27) for 15 min, and the bands

werevisualized under UV light. The bands were

ex-cised withascalpelblade and elutedbydiffusion as

follows. Thegel pieceswere crushedthroughan

18-gaugesyringe needle into2 mlof STE buffer. After

beingshakenat roomtemperatureovernight, thegel

pieceswereextractedagain withafurther1mlof STE

buffer. The bufferphaseswerepooled andprocessed

asdescribed below.

Preparation of RNA segments for translation.

The RNAsegmentseluted fromgelsweretreated as

described by McCrae and Joklik (17). Briefly, this

involved extraction with isoamyl alcoholto remove

ethidiumbromide,thenphenolextraction to remove

monomericacrylamide,andfinallyethanol

precipita-tion. Theprecipitatewaswashed twice in 80%ethanol,

washedonce in 100% ethanol, dried in air, and

dis-solved in 6

p1

of 90% (vol/vol) dimethyl sulfoxide

(Me2SO) in water. The RNA was stored at -20°C

until required. For translation, 2

pl

of solution was

removed and heatedto30°C for5min,and then the

translation reaction mixturewasadded.

In vitro translation. Denatured dsRNA was

translated inawheat germ systemprepared essentially

asdescribedby Roberts and Patterson (19). The

re-actionmixture contained:20mMHEPES (pH 7.6),39

mMKCI,1mMmagnesium acetate,2mM

dithiothre-itol, 1 mMATP, 20,uM GTP,8mM creatine

phos-phate, 30Mgof creatinephosphokinase per ml, 0.2 mM

Spermidine,toeach of19unlabeled amino acidsat 20

to 30,uM, 5 to 20 MuCiof[3S]methionine (1,300 Ci/

mmol Amersham),10

pl

of wheatgerm (S-30) extract,

and about2Mgof individual denatured dsRNA

seg-ments. The final volume was 50

p1.

Reactions were

incubatedfor60min at30°C; then the reaction was

terminated by chilling to 0°C. Samples of 5

01

were

withdrawn, spottedontoglass fiber disks (Whatman,

GF/A), and processed for trichloroacetic acid-precip-itable radioactivity by the method of Roberts and

Patterson(19).

Analysis of translation products and viral polypeptides. Translation products and viral pro-teins were analyzed in SDS-polyacrylamide slab gels by using the Laemmli (12) Tris-glycine discontinuous

buffer system. The acrylamide/bisacrylamide ratio

was 30:0.8. SDS was notpresentin gels but only in

reservoir buffer (0.6%) andloaded samples (1%) before

commencement of electrophoresis, as suggested by

Wyckoffet al. (34). Protein samples to be analyzed

were treated with Laemmli sample buffer (12), (1%

SDS, 2% 2-mercaptoethanol, final concentrations) and heated to 100°C for 5 min before being loaded onto

thegel. Electrophoresiswasperformedat room

tem-perature withacurrentof25mA pergel. Fluorographs

wereprepared as described by Bonner and Laskey (1)

andLaskey and Mills (13).

Molecularweightdeterminations. The

molecu-larweights of SA11polypeptides were determined by

comparisonwith standard protein molecular weight

markersrun onthesameslab gel. The protein

stand-ards usedwere:,B-galactosidase(Escherichia coli,

mo-lecularweight 130,000), phosphorylase a (rabbit

mus-cle, molecular weight 94,000; Worthington

Biochemi-calsCorp.), bovine serum albumin (molecular weight

68,000; Commonwealth Serum Laboratories,

Aus-tralia), catalase (bovine liver, molecular weight

60,000),immunoglobulinG (heavy chain) (rabbit,

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978

SMITH, LAZDINS, AND

lecular weight 53,000; D. Jackson), L-lactic

dehydro-genase (bovine heart, molecular weight 36,000),

car-bonic anhydrase (bovine erythrocytes, molecular

weight 29,500), a-chymotrypsinogen A (bovine

pan-creas, 25,700), myoglobin (whale skeletal muscle,

17,200). All exceptimmunoglobulin G, phosphorylase

a, andbovineserumalbuminwereobtained from the

Sigma Chemical Co.

RESULTS

Translation of

unfractionated

dsRNA.

When

suitably

denatured, the dsRNA

segments

of the

SA

11 genome

stimulated the

incorpora-tion of

[35S]methionine into trichloroacetic

acid-precipitable material

in a

wheat

germ cell-free

protein

synthesizing

system.

Figure

1 presents

several

parameters

of

this reaction. Addition of

undenatured

dsRNA

up to 100

,tg/ml

did

not

inhibit

endogenous

activity (amino acid

incor-poration

in

the absence of

any

added

mRNA)

(9); however,

no

stimulation

of

[35S]methionine

incorporation

occurred

either.

Dimethyl

sulfox-ide

(Me2SO)

at the

concentration used in

this

study

(3.6%), did

not

inhibit

endogenous

activity

(results not shown),inagreement with the

find-ings of

McCrae

and

Joklik (17).

Denaturation with 90% Me2SO did not require heating to

50°C,

as was necessary for reovirus

dsRNA

(17). In fact, normal roomtemperatures

were

adequate, although equilibration

to 30°C

was

chosen for

convenience, because this was

the

temperature at which the wheat

germ

sys-tem was

incubated. Once optimal

concentrations

of KCl and Mg

were determined (39 and 1 mM,

respectively) they

were used throughoutfurther

experiments.

An

RNA

concentration (80

,tg/ml)

sufficient tosaturate

the

translation

system wasselected

on

the basis of

preliminary

studies(results not

shown) for

the

optimization

and

time

course

experiments.

Although high levels of

incorporation were

observed

upon

addition of

rotavirus RNA, the

specific

stimulation of incorporation

(i.e., the

ratio of

the incorporation in

a systemwith added

RNA

to

that

in a system

with

noadded RNA) wasnot as

high

as

expected

(maximum of

about

eightfold) due

to

significant

endogenous

incor-poration

by the particular wheat

germ

prepara-tion used

in

this

study.

Polyacrylamide gel analysis of

rotavirus-coded

polypeptides. Since identification of

§m A

3}B

s0C

IDD2125 125

2so 100 100

w

2

260 75 75

0 I

I40 50 50

20 25 25

20 40 60 s0 100 1 2 3 4 0 20 40 60

KCI mM M+ mM Time

(inin)

FIG. 1. KCIand

Mg2+

concentrationoptimaand timecourse

of

[35SJmethionine incorporation

curves

for

the translation ofdenatured rotavirus dsRNA. Translation assays were as described in the text. The

incubation timefortheconcentration

optima

curves was60min,

after

which5-,ul

samples

wereremoved and

theincorporationof[3SJmethionineinto trichloroacetic

acid-precipitable

materialwasdetermined.The KCI

concentrationoptimumwasdeterminedata

Mg2+

concentration

of

1 mM

(A),

and the

Mg2+

concentration

optimumwasdeterminedat aKCI concentrationat39mM

(B).

The timecourse

incorporation

curve(C)was

performedatKCIand

Mg2+

concentrations

of

39 and1

mM,

respectively.

For all threecurves, the dsRNA

concentration was 80pg/ml, and the

final

Me2SO concentration was 3.6%.

Symbols: 0, [35S]methionine

incorporationin thepresence

of

added denatured

dsRNA;

0,

[35S]methionine incorporation

in the absence

of addedRNA, andin C thisalso represents the

incorporation

in thepresence

of

undenatured rotavirus

dsRNA(i.e.,addedtothetranslation mixtureas anaqueoussolution)at50or100

pg/ml.

Eachofthese three

controlsgaveindistinguishableresults.

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[35S]methionine-labeled translation products of

denatured dsRNA

was to

be made

on

the basis

of

electrophoretic mobility, it

was

first

necessary

to

establish the migration

patterns

of

rotavirus

polypeptides

inthe

SDS-polyacrylamide slab gel

system

employed.

This

gel

system gave good

resolution of all

known rotavirus

polypeptides.

Figure 2 shows

typical patterns of SA

11virion

(structural)

pro-teins, and also

virus-specific proteins that appear

in

[3S]methionine-labeled

infected

cells.

No-menclature of these polypeptides

isbased upon

the systemof

Thouless

(29), in which viral pro-teins are

designated

by

whether

they are inner

(I)

capsid

structural,

outer

(0)

capsid

structural,

or

nonstructural

(NO)

proteins.

They are

num-bered from highest

to

lowest molecular weight.

SA

11

virus

possesses

five inner

(I,

through

15)

A rA..,A-_- n 4Wu _= ns:I1

anatour outer

(Ul

tnrougn

wJ4)

tides. In

our

modified

systemr

equivalent

toI3

and

14, respecti

tem

of

Thouless (29) (see Discu

ber and

pattern of the proteins

arations

of

purified

virus are ess

as

described

by Rodger

et

al.

(2'

structural

proteins,

virus-infecl

two

polypeptides

notseen in pi

thus

considered nonstructural

IC

UC

I,-.

2

F~

1

14

G~A

15

4~t

4

-...

-_!

:

_05

_

-*F'

.,:

.

.'

S.

-._

-FIG. 2. SDS-polyacrylamide gel

methionine-labeled, purifiedSA1.

virus-infected(IC)and uninfected

and translationproducts ofa w

without added RNA (-), orprog

fractionated Me2SO-treatedSA11

thesameastrack Vexceptthat itit

toshow theminoroutercapsidpc

04. Analysiswas on a10% slabge

TABLE 1. Molecularweight estimates of SA 11

rotaviruspolypeptidesa

Polypeptide mol wt(xlo-3)

II 130

12 93

I3 88

L4 82

O0 62

OIA 55

I5 42

02 36

NS, 33

NS2 31

03 28

04 26

aMolecularweight

estimates

were determinedby

polyacrylamide gel electrophoresis, using protein

standards of known molecularweight.

capsita

poIypep-

[Fig.

2]). A

protein

observed

in SA

11-infected

ie

14 and 15 are

cells

but

not inpure

virus

was

designated

OIA.

ively,

in

the

sys-

As first

suggested by Thouless, this protein is

ssion).

Thenum-

possibly

theprecursor to

O1;

however, since

OlA

entiaryvthe

same is a

minor

product

(see

Fig.

2 and 4), the rela-2). In

addition

to

tionship

between these two proteins

will be

dif-ted

cells

display

ficulttoelucidate. The molecular

weights

ofall

urified virus

and

SA 11 proteins are given in Table 1.

(NS1

and NS2

A minor outer capsid protein, 03, was

ob-served

only occasionally

in

virus-infected

cells.

This

protein

was

also difficult

to detect in

V

V

[35S]methionine-labeled

cells

infected with other

rotaviruses

(29).

However, it was

always

present

in

preparations of purified SA

11

virus

(Fig. 2).

S

Virus infection of

cells

considerably

reduced

host cell

protein

synthesis, allowing

virus-spe-lE

_

cific

proteins, including those-produced

in small

quantity,

to

be observed

clearly (Fig.

2).

01oom

Identification

of the in vitro translation

products

of unfractionated dsRNA. The

translation

products

of

denatured

rotavirus

dsRNA were

analyzed

on

polyacrylamide slab

gels

as

described

above,

and

the

results

are

shown

in

Fig.

2.

Products

identical

in mobility

*_

__.i_ to

11,

I2,

13,

I4, I5, and

NS2

were

observed.

A

E_

_

protein corresponding

in

electrophoretic

mobil-ity

to

NS,

was

only

occasionally

seen(not shown

in

Fig. 2). Proteins of equal mobility

tothe

major

03

WM

(02)

and minor (03 and

04)

outer

capsid

proteins

043

werenot

produced.

A

major

translation

product

(54,000

molecular

weight)

migrated

slightly

ahead

of

OIA.

analysis of

[35S]-

The fairly high endogenous

incorporation

of

Ivirus

(V

and

V');

[3S]methionine

in the wheatgerm system

pro-(UC)

MAJO04

cells;

duced

densely

labeled bands near thegel front,

heatgerm system which made translation products of low

molec-a,rammed with

un-dsRNA (+). V'is

ular weight

difficult

to observe.

a longer

exposure

Translation of individual

species

of

de-glypeptides

03

and

natured dsRNA. Total

genomic

dsRNA was

1. fractionated by

polyacrylamide

gel

electropho-,.:M";tK..

I

0:

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SMITH, LAZDINS,

AND HOLMES

[image:5.504.271.467.63.238.2]

resison a

preparative

scaleas described in the text. Atypical preparative scale gel is shown in Fig. 3.Not all RNA segments could be resolved,

because

segmnents

7and 8

have identical

mobil-ities in

this gel

system (20) and run

together

as

a

heavily staining band

(Fig. 3). Bands were

excised, and the

RNAwas eluted. The individual

RNA

segments

(segments

1 to 6) were then

denatured

and

translated

as described in the

text.

Figure

4 shows the translation products of

genome segments 1 to 4.

The

translation

product

of

the

denatured dsRNA

segment 1 is identical

in

electrophoretic mobility

to the virion

struc-tural

polypeptide

I,.

Similarly, dsRNA segments

2, 3,

and

4

produce

labeled polypeptides

corre-sponding

to

I2,

I3,

and

L, respectively. Although

there

are

endogenous bands

in

this

region which

migrate

to

positions close

to

the translation

products of

segments 2

and

4,

they

clearly do

not

interfere with the

assignments.

The translation

of

denatured dsRNA

segment

5

produces

a

labeled

protein of

similar

electro-phoretic

mobility

to

OIA

(Fig.

5). However,

whereas

segment 5appears to

be

translated very

efficiently

in

vitro,

01A

is

quite difficult

to

ob-serve in

SA

11-infected

cells.

Genome segment

6

codes for the major inner

capsid

polypeptide 15. This

segment

appears

to

3 }

...

4-

5-

6-

,Z8-

9-

10-

1I--FIG. 3. Preparative gelelectrophoresis ofSA 11

rotavirusdsRNA.RNAwasextractedfrompurified virus andanalyzedon10%oSDS-polyacrylamide

(La-emmli)slabgels. Thegelwasstainedwith ethidium

bromide andphotographedovera UVlightbox.The dsRNAsegmentsarenumbered in orderof

decreas-ingmolecularweight.

1_

0

Si

S2

S3

S4

4~~~'

FIG. 4. Wheat germ cell-free protein synthesizing

systemprogrammedwith denatured dsRNAsegments

1 through 4. Individual dsRNA segments were

re-coveredfr-om excisedgel bandsfr-om apreparative

RNAgelandpurified by organicsolvent extraction

and ethanol precipitation. After treatment with

Me2SOtheywereaddedtoawheat germtranslation

system, and the [35S]methionine-labeled products

were analyzed on a 10% SDS-polyacr-ylamide slab

geL IC,SA11virus-infectedMA1O4cellslabeled with

[35S]methionine; 0, wheat-germ system with no added RNA. Translation system wasprogrammed with dsRNAsegment1 (Si),segment2(S2),segment

3(S3),andsegment4(S4).

UC IC S6

_.,

't,. _ 11

40=

13-t

02

N06S,

4

FIG. 5. Wheatgerm cell-freeprotein-synthesizing

systemprogrammed with denatured dsRNAsegments

5 and 6. UC, Uninfected [36S]methionine-labeled

MA104cells; IC, SA 11 virus-infected,

[35S]methio-nine-labeled MA104cells; 0,noRNA addedto the

wheat-germ translation system; S5, denatured

dsRNA segment 5added; S6, denaturedsegment6 addedtothe translationsystem.Productswere

ana-lyzedon a12%o slabgel.

S5 0

A MO

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be

translated

efficiently

both in vitro and in

vivo,

unlike

segment 5.

DISCUSSION

McCrae

and

Joklik

(17) found that for

the

efficient translation of the denatured dsRNA

genome

of

reovirus,

a

rapid shift

in

potassium

ion

concentration,

soon

after the addition of

RNA

to

their wheat

germ system,

was

required.

If

this "salt

jump"

was not

performed,

none

of

the

high-molecular-weight (lambda)

polypep-tides

were

produced. This

wasnotnecessary

for

the translation of the rotavirus

genome

because

all

known

high-molecular-weight

proteins (I1

through

L) were

formed.

02

is known

to

be

a

glycoprotein (22).

No

product

equal in mobility

to

this protein

was

formed in

vitro, which is consistent with other

studies

(2,

4,

6) in

which mRNA's of viral

gly-coproteins

were

used

toprogram

cell-free,

pro-tein-synthesizing

systems.

Often the

unglyco-sylated form of the viral protein

was

produced,

which migrated

significantly ahead of the

gly-coprotein

(i.e. of lower

apparent

molecular

weight)

on

SDS-polyacrylamide gels.

This

pos-sibility

is

currently under

investigation

in this

laboratory.

This method of

directly translating

virion

ge-netic

material

clearly

indicates primary gene

products, and clarifies the published data

con-cerning

viral

polypeptides. Rodger

et al. (22)

described four

high-molecular-weight

rotaviral

polypeptides (e.g.,

p133,

plO2,

p99, and p92 for

SA 11), but others have observed only three

(16,

18). Thouless (29)

described

an

occasional

split-ting

of bands in the I2

or

I3

region and suggested

this may

be

due

to

cleavage, similar

to that of

reovirus

(36). Our results demonstrate

that there

are

indeed four distinct

high-molecular-weight

polypeptides and that they

are

all

primary gene

products.

It

is for this

reason that we found it

necessary

to

change the designation

ofthe major

inner

shell

polypeptide from

I4(29)

to

15.

The

assigmnent

ofdsRNA segment 5 to

OlA

is

quite

firm,

although

the

relationship between

OIA

and

01

has

not

been

clearly established.

Thouless (29) also showed

a

slight

difference in

migration between the 01 in infected

cells

and

the

corresponding virus

structural protein. Since

OlA

is produced in such

small quantity in

virus-infected

cells,

it will be

difficult

to compare it withthe translation product of

segment

5 and with

01

fromvirus

particles,

butthe

comparison

(for example

by

limited proteolysis

analysis)

ap-pears

feasible.

Unlike

Matsuno and

Mukoyama

(16), neither Thouless

(29) norwe were able to

detect a

polypeptide

ininfected

cells

which had the same

mobility

as the

structural polypeptide

01

of

purified

virus. Weconsider that the protein

designated

NCVP 1

by

Matsuno and

Mukoyama

(16) may correspond

to

OIA

inour

system.

In another recent

study (5),

translation of

transcription products

ofcalf rotavirus

appeared

to

yield only polypeptide

I5

and

possibly

NS1

and

NS2. However,

no

comparison between

la-beled translation

products

and

virus-infected

cell

proteins

was

done,

so

any

lower-molecular-weight bands

are

of uncertain identity.

Since

infectivity and

antigenic specificity

of

rotaviruses

depend

on

polypeptides of the

outer

capsid

(3, 30, 35),

it is

tantalizing that

most of

the successful

assignments

so

far

are

for inner

capsid proteins. From other studies in

progress

in

this

laboratory (I.

Lazdins, unpublished data),

it appears

probable that 04 is

a

glycoprotein,

as well as

02, and in vitro translation of these

may

require variations in

technique. These

assign-ments,

and the identification of the protein

carrying

type-specific antigenic determinants,

are of

high priority

to

assist

interpretation

of

epidemiological investigations based

on

electro-pherotypes (7,

11,

20;

S. Rodger, manuscript

in

preparation).

ACKNOWLEDGMENIS

We are verygrateful to J.Phillipsforhelpfuldiscussion on in vitrotranslation and forsupplyingthe wheatgermextract. Weappreciate the advice of M. McCraeontheapplicationof his RNA denaturationtechniquetorotaviruses. We thank C.

Adeneyfor technical assistance.

This work was supported by the National Health and Medical Research Council of Australia. M. Smith is the holder of a CommonwealthPostgraduateResearch award.

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