Characterization of the Coat Protein mRNA of Southern Bean Mosaic Virus and Its Relationship to the Genomic RNA

JOURNALOFVIROLOGY, July 1981, p.87-92

0022-538X/81/070087-06$02.00/0 Vol.39,No.1

Characterization

of the

Coat Protein

mRNA

of

Southern Bean

Mosaic Virus and Its

Relationship

to

the

Genomic

RNA

AMIT GHOSH,* TINEKERUTGERS, MANG KE-QIANG, ANDPAUL KAESBERG

Biophysics Laboratory, GraduateSchool,andDepartmentof Biochemistry, College of Agricultural and Life Sciences, University ofWisconsin-Madison, Madison, Wisconsin 53706

Received20February 1981/Accepted13April1981

RNA isolated from southernbean mosaicvirions contains, insmallamount,a

subgenomic RNA (molecular weight, 0.38x106) thatservesinvitroas anmRNA

for southern bean mosaic viruscoat protein. The RNA has a5'-linked protein

indistinguishable from the protein linked to the 5' end of

full-length

genomic

RNA. Its basesequence, determined to91bases from the 3' end, is identicalto

the 3'-terminalsequenceofthe genomic RNA. The resultssuggestthat thecoat

protein messenger sequence exists as a "silent" cistronnear the 3' end ofthe

genomic RNA.

The RNA contained in southern bean mosaic

virus

(SBMV) virions is heterogeneous (16, 21);

RNAs

of

many

lengths

exist

in

addition

to

full-length RNA

(Mr,

1.4x

106).

Four

major

proteins

could be translated from these RNAs in vitro

(17):

(i)

two

related

proteins of molecular

weight

105,000

(P1) and

75,000

(P2) induced by

full-length RNA;

(ii)

coat

protein

(P3; molecular

weight, 29,000) induced

by

a sucrose

gradient

fraction

containing predominantly RNAs

in

the

molecular-weight

range

of

0.3x

106

to0.4x

106;

and

(iii)

a

14,000-molecular-weight

protein (P4)

induced

by RNAs of all size

classes, including

full-length

RNA and the

coat

protein

messenger

fraction. Diener found that

SBMV

full-length

RNA

is

infectious

(4).

This

implies that the

information for all four

proteins

must

be located

in the

full-length

RNA and

that the

coat

protein

gene must

be

present as a

"silent" cistron. In

this

reportwe

show that the

coat

protein

cistron

is

located in the

full-length

RNA

near

the 3' end

and that it is

expressed

by

a

subgenomic

mRNA

which has

thesame

protein

linked

to

its 5' end

as

does

full-length

RNA.

MATERIALS

AND METHODS

Isolationof SBMV and its RNA. SMBV

(bean

strain) wasisolated from Phaseolus

vulgaris

L. cv. Bountiful

essentially

bythe method ofHull

(7).

ExtractionofRNAwasdoneaccordingtoZimmern (22). The RNAwas purifiedfurtherbydensity gra-dient centrifugation. The region of the RNA that contained thefull-lengthRNAof molecular

weight

1.4 x 106 and the region that contained RNA able to induce coat protein synthesis in vitro were

pooled

separatelyand concentratedbyethanol

precipitation

(16).

Invivo

labeling

of virus.The

preparation

of

3P-labeled virus andRNA hasbeen described before(6).

Enzymedigestion conditions. Enzyme digestion

conditionshavebeen described before (6). Proteinase K (Boehringer Mannheim Corp.) digestion was as describedbyFlanegan et al. (5).

Fingerprinting ofSBMV RNAs. Fingerprinting

wasdone by standard procedures (1). The first-dimen-sion electrophoresis was done on cellulose acetate at pH3.5. The second dimension was homochromatog-raphyon aDEAE-cellulose thin-layer plate (CEL 300 DEAE/HR 2/15). The homochromatography mixture used was a3%solution, in 7 M urea, of yeast RNA thathad beenhydrolyzed for 25 min.

3'- and 5'-endlabeling ofSBMV RNA. 3'-end labeling was done with cytidine 3',5'-[5'-3P]bisphos-phate ([5'-3P]pCp) (2,000 Ci/mmol; New England Nu-clearCorp.) byuseof T4 RNA ligase, as described by Dasguptaetal. (2).

5'-Endlabelingwith

[y-3P]ATP

andT4 polynucle-otide kinasewasdoneby the method ofSilberklang et al.(18).

Reductivemethylation.RNAfrom thecoat pro-teinfractionwastreated with['4C]formaldehyde(45.8

mCi/mmol; New England Nuclear Corp.), using the

procedures of Rice and Means (15) and Means and

Feeney(9).

Polyacrylamide gel electrophoresis.Terminally

labeled RNAs from thecoatproteinfraction or ribo-some-selected RNAs (10 to20

gg)

orboth were

ana-lyzedon8%polyacrylamidegels containing 7 M urea.

Theelectrophoresiswasfor48hat12.5V/cm,

accord-ingtoPeacock andDingman (13).

To elute the RNAs, bands were cut from the gel, homogenized withasterile siliconized glass rod, and takeninto siliconizedvialscontainingtheappropriate volume of0.25 MNaCl. Themixturewasmade 0.1% with sodium dodecyl sulfate. The vials were shaken gently at room temperature for several hours. The mixture was then filtered through membrane filters (0.45 nm, type HA; Millipore Corp.) to remove gel particles. An appropriateamountofcarrier calf liver tRNA was added to the filtrate, and the RNA was precipitatedwithethanol.

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Analysis ofdifferentlylabeled RNAson a5%

poly-acrylamide gel after glyoxal denaturation was done

essentially as describedby

Desselberger

andPalese

(3). Acridine orangestainingofgelswasdonebythe procedure of McMaster andCarmichael(8).

Polyacrylamide-sodiumdodecylsulfategel

electro-phoresis (19) of in vitro-labeled proteins has been described before(6).

Translation. In vitrotranslation,measurementof

aminoacid incorporation, and electrophoretic analysis

of the synthesized proteins were performed as de-scribedby Salerno-Rifeetal.(17).Incaseof

transla-tionofgel-elutedRNAswhichhad beencoprecipitated

withcalf livertRNA,the reactionmixtureswere cor-rected for thatamountof tRNA.

Ribosomecaptureof mRNA. Labeled RNAwas

incubated in the rabbitreticulocyte cell-free transla-tion system understandard reaction conditions. After 10 minofincubationat30°C,the reactionmixturewas diluted withanequalvolume of ice-cold 10 mM Tris-acetate (pH 7.6)-100 mM potassium acetate-8 mM magnesium acetate. Thediluted reaction mixturewas

layeredonto 2ml ofsucrosesolution(30%sucrose,10

mMTris-acetate,[pH7.6],100 mMpotassiumacetate,

4mMmagnesiumacetate) andcentrifugedfor4hat 36,000 rpmand40C inaSpinco40 rotor. The ribo-somal pellet was dissolved in 0.2% sodium dodecyl

sulfate-0.1 M Tris. RNA was then extracted three timeswith water-saturatedphenol.The final aqueous phase was made0.2 Mwith sodiumacetateand the

RNAwasprecipitatedwith2volumes ofethanol.The

majority of the ribosomal RNAwasremovedby su-crosegradientcentrifugation (16). Fractions contain-ing labeled RNAwere pooled and concentrated by

ethanolprecipitation.

Control experiments indicated that RNAs in the coatprotein fractionareunable topenetrate the su-crosecushion in the absence ofribosomes,i.e.,unless ribosome bound.

Determinationof the 3'-end sequences. RNase

digestions and gel electrophoresis were done as de-scribed before(2).

RESULTS

Fingerprint analysis of the

coat

protein

messenger

fraction.

The

sucrose

gradient

fraction

of

SBMV RNA that

induced the

syn-thesis of both coat

protein

and P4

(16)

in the in

vitro

translation

systems

contains

a

considerable

number of different RNA

species (16).

To

deter-mine thenature and origin of these

RNAs,

uni-formly

32P-labeled

coat

protein messenger

frac-tion was

digested with RNase T1,

andits

finger-print was

compared

with that

obtained

from

purified

1.4 x

106-molecular-weight

full-length

RNA.

The

resulting autoradiograms

(Fig. la and

b)

show that all of the

oligonucleotides

from the

digest

of the coat protein

messenger fraction

were present in

the

digest of

full-length

RNA.

This

indicated that

the

sequences

ofthe

major

RNA

species

in

the

coatprotein

messenger

frac-tion were subsets

of the

full-length

RNA

se-quence.

Identification

ofcoat

protein mRNA

and P4

mRNA.

To

determine which of the RNAs

in

the coat

protein messenger fraction served

as

the

mRNA's

for the coat

protein

and

for

protein

P4,

the coat

protein messenger fraction

was

la-beled at the 3' end

with

[5'-32P]pCp, using

T4

RNA

ligase.

The

RNAs

were

separated

on an 8%

polyacrylamide gel. Three major and

several

minor

bands

were visible. Of the

three

major

bands,

the

largest

one

(designated 1)

wasmore

intense

than the others

(designated

2 and

3)

(Fig. 2,

lane

a).

The material

from the

major

bands was

eluted,

andthe

extracted RNAs

were

added

separately

to

the

reticulocyte translation

system.

Only

the

RNA

eluted from band 1

in-duced coat

protein synthesis

(Fig. 3). The RNA

from this

band also induced the

synthesis of

a

small amount of protein P4. In contrast, RNA

eluted from bands 2 and 3 induced synthesis

only

of

protein

P4.

To

eliminate

the

possibility that translation

was

induced by

a

co-migrating nonlabeled RNA,

a

ribosome

capture

experiment

was

performed

(see above)

with the

3'-labeled

coat

protein

mes-senger

fraction. The

ribosome-selected RNAs

were

analyzed

again by

polyacrylamide gel

elec-trophoresis.

Bands of

labeled

RNA

appeared

in

a

b

FIG. 1.

Fingerprint

analysis of32P-labeledSBMV

RNA fr-om the coatProteinfraction ofthe sucrose

densty radentrun and itscomparisonwith thatof

32P-lbeled genomic

full-length

RNA. See text for

further explanations. (a) Coatprotein

fraction;

(b)

full-length

RNA. Thefirst

dimnension

was

electropho-resisoncelluloseacetate,pH 3.5; the second

dimen-sionwas

homochromatography

onaDEAE-cellulose

thin-layerplate.

B,Positionof

bromophenol

blue. J. VIROL.

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VOL. 39, 1981

2-

3-FIG. 2. Analysis ofSBMV coatprotein fr-action

RNA on a denaturing 8%polyacrylamide gel. (a)

Coatproteinfr-actionRNAlabeledatthe 3' ends with

32P; (b) coatproteinfr-action RNA labeled in vitro with ['4CJformaldehyde; (c) coat protein fr-action

RNA labeled in vitro with [14C]formaldehyde

fol-lowedby proteinaseKdigestion.

_lm

-CP

Jo

_NQ

-

P4

a

b

C d

FIG. 3. Polyacrylamideslab gel electrophoresisof translationproducts, in the rabbit reticulocyte

sys-tem,oftheRNA bandseluted fromthe gel in Fig.2.

Lanes: (a-c) productsmade by RNAs1 through3of

Fig.2;(d) products obtainedwhencoatprotein frac-tionRNAwastranslatedunderidentical conditions.

CP,Coatprotein;P4, proteinP4.

positions, 1, 2, and 3, and their translation

con-firmedourprevious results. On the basis of these

results, we identified the band 1 RNA as the

mRNA forcoatprotein.

The molecular weights of the mRNA's in

bands 1, 2, and 3 were estimated from a gel

electrophoretic analysisafterglyoxal

denatura-tion of the RNAs (3, 12) in the presence of

molecular-weight

standards. The molecular

weights of RNAs in bands 1, 2, and 3 were

determinedtobe

approximately

0.38x

106,

0.19

x

106,

and 0.17 x

106,

respectively (data not

shown). The 3'-labeled RNAs from bands 1, 2,

and3weresequenced.Thesequencesofthe first

91 nucleotides from the 3' ends of the RNAs

from bands1,2,and3wereidentical(Fig. 4)and

were the same as that found in the 3'-end se-quenceof thefull-lengthRNA(6).Theseresults,

togetherwith the

fingerprint

data(Fig. 1),

indi-SBMV COAT PROTEIN mRNA 89

cated that the

genetic

information

for both

coat

protein

and

protein

P4

is

located

in

the 3' part

ofthe

full-length

RNA.

5' terminus of

coat

protein mRNA. To

investigate

thenature of the 5' terminus of coat

protein mRNA,

the coat

protein messenger

frac-tion was labeled

either

with

[5'-32P]pCp, using

T4 RNA

ligase,

or, after a

phosphatase

treat-ment,

with

[y-32P]ATP,

using

polynucleotide

ki-nase.

The RNAs

werethen

analyzed

on an 8%

polyacrylamide gel. It

was

found that

only band

1

RNA, which

was an

efficient substrate for

T4

ligase, could

not

be

labeled by

polynucleotide

kinase at all. This

indicated that

the coat

protein

mRNA

probably had

a

blocked 5' terminus.

Attempts

to

label the

coat

protein

mRNA,

using guanylyl

transferase and

[a-32P]GTP

(10)

after

periodate

oxidation and

fl-elimination

to

removeany

putative 5'

pm7G,

were

also

unsuc-cessful.

A

further

attempt to

identify the 5'-blocking

group was

made

by

complete

hydrolysis of

uni-formly 3P-labeled

coat

protein

fraction RNA

with a

mixture of RNases

T2,

T1, and

A

followed

by

analysis of the

products

by

thin-layer

chro-matography, according

to

the

method of

Nishi-mura

(11). In

addition

to

four major

mononu-cleotides, only

two

other spots,

one

due

to

free

phosphate and the other

at

the

origin,

were

found. There

were no

other

spots

characteristic

of

an

m7GpppX-like

structure

(data

not

shown).

The

translational

studies, like

the

structural

studies, also suggested that

the

blocking

group at

the

5'

terminus of

the coat

protein mRNA

was not acap structure.

Translation of the

coat

protein

messenger

fraction in

the

reticulocyte

lysates

in

the

presence

of

the

cap

analog

7-meth-ylguanosine-5'-monophosphate

(m7Gp)

resulted

in

about 30%

stimulation of

the amino

acid

in-corporation

activity, whereas

the

translation

of

brome mosaic virus RNA

4

(a

capped mRNA)

showed

a

90% inhibition

(Fig.

5). Such

stimula-tion in

incorporation

in the

presence

of

cap

analogs

in

the

reticulocyte lysates

has been

ob-served for other viral

mRNA's

which have

a

protein linked

to

their

5'

ends

(14).

Since

full-length

SBMV

RNA has a

protein

linked

to

its

5'

end

(6)

and since

in

all

plant

viruses examined

to

date the

genomic

and

sub-genomic RNAs of

a

particular

virus

always

have

the sametype ofstructure attheir termini

(20),

welooked for thepresence ofa

protein

onthe5'

end of thecoat

protein

mRNA. Reductive

meth-ylation, using

["C]formaldehyde,

of the coat

protein messenger fraction and

subsequent

gel

analysis

resulted in the

appearance

of

"4C

label

at the coat protein mRNA

position

in the gel

(Fig. 2, lane b). Treatment of the

'4C-labeled

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90 GHOSH ET AL.

I

:ii

I

:i

I

:ii

a

b

c

FIG. 4. Sequencing

gel

pattern

showing

3'-endsequence

of (a)

RNAfrom band1, Fig. 2;

(b)

RNA

from

band2,Fig.2; (c)RNAfrom band3, Fig.2. The RNAslabeled at the3'endwerepartiallydigested with

differentenzymesandthenrunon a 12% thingel. Lanesfrom left toright, in eachpanel, are: untreated

control;RNAtreated withRNaseU2,RNase T1 formamide,RNasePhy M, pancreaticRNase. Twoseparate

loadingsofthesamplesweremade. Panel I is the shorterrunshowingbasesfromthe3'end; panelII is the

longer run.

coat

protein

messenger

fraction with

proteinase

K, however, caused the

'4C

label

to

disappear

from the

coat

protein mRNA

(Fig.

2,

lane c).

Staining

of the

gel

with acridine

orange

showed

that

the RNA itself

was

still

intact. These results

suggested

the

presence

of

a

protein

linked

to

the

5' end

of the

coat

protein mRNA.

The

protein

was

analyzed by gel

electropho-resis after

a

complete RNase digestion

of the

"4C-labeled

coat

protein

mRNA fraction. An

electrophoretic

comparison

of

the released

pro-tein with the

'4C-labeled

genome-linked protein

(6)

showed that the two proteins comigrated

(Fig.

6,

lanes

a and c). The two proteins were

compared further

by digesting them

partially

with

protease Staphylococcus aureus V8 and

analyzing

the products

formed

on a

polyacryl-amide-sodium

dodecyl sulfate gel (19). They gave

rise

to an

identical cleavage

pattern

con-sisting of

three bands, indicating that the two

proteins

are

identical

(data not shown). The

presence ofa

protein

presumably

linked

to

the

5'

terminus of the

coat

protein

mRNA

indicated

that the

coat

protein mRNA is

a

subgenomic

RNA,

synthesized

in

vivo and

encapsidated

in

minor

amounts

in the virions.

DISCUSSION

Our results show that the

coat

protein

gene

is

present as a

silent cistron in the

genomic

RNA

and that it is

expressed

viaa

subgenomic

mes-senger

which has the

same

protein

linked

to

its

5' end

asthe

genomic

RNA. Protein

P4

could be

translated

from thecoat

protein

mRNA and

also

from

smaller RNAs

having

the

same

3'-end

se-quence.

From

denaturing

gels,

the

molecular

weight

of thecoat

protein

mRNAwas

estimated

to

be

0.38 x

106.

This may be

insufficient

to

accommodate the information for both

thecoat

protein

(molecular weight, 29,000) and the

P4

protein

(molecular weight, 14,000)

independ-ently.

This

implies

that the

genes

for the

coat

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SBMV COAT PROTEIN mRNA 91

0

--0

c

0

0

01.-c

0

a

-o

o

c

0.5 1.0 1.5 2.0

mM pm7G

FIG. 5. Effect of m7Gp5'ontranslationalefficiency

of thecoatproteinmessengerfraction RNAin rabbit reticulocyte lysate. Reactions wereprimed with (0)

SBMVcoat proteinfractionRNAor(0) brome

mo-saic virus RNA 4at 50pg/ml. The potassium ion concentrationusedwas40mM.

a

b

c

FIG. 6. Comparison ofthe invitro-labeled protein from thecoatproteinfraction with that associated

with the fuU-length RNA. (a) SBMV-coat protein

RNA labeled with ['4CJformaldehyde; (b) SBMV-coatprotein RNA labeled with ["Clformaldehyde

foUowed byproteinaseKdigestion; (c) SBMV full-length RNA labeledwith[4Clformaldehyde.Inevery

case the sample was treated with RNase A before electrophoresis.

andP4proteinsoverlap,and since theseproteins

didnotsharecommontryptic peptides

(17), they

would have to be coded in different

reading

frames.

SBMV differsfrom most other plantviruses in

having

aprotein linked to the 5' end ofits

genome.Unlike theviruses withgenome-linked

protein

(20),

however,

it does notsynthesizeall

of itsproteins

through

apost-translational

cleav-age mechanism.

Rather,

it

adopts

the

strategy

ofexpressing its internal cistrons

through

the

mediationofsubgenomicRNAs.

Turnip

rosette

virus,

which hasagenomicRNAwithaprotein

attached

to

it,

also

synthesizes its

coat

protein

from

a

subgenomic

messenger

(B.

A. M.

Morris-Krisinich,

70thAnn.

Rept.,

John Innes

Institute,

Norfolk,

England,

1979).

It

thus appears that

there

is not

necessarily

a

correlation

between

the terminal structure of the

RNA

and the

strat-egy

adopted by

the

virus

inthe

expression

of its

genetic

information.

ACKNOWLEDGMENTS

We thankTerrySalerno-Rifeforhelpfuldiscussions. This work waasupportedbyPublic HealthServicegrants AI-15342 andAI-01466andCareer AwardAI-21942 from the National Institutes of Health andbygrant7800002 from the Science and EducationAdministration of theU.S.

Depart-mentofAgriculture.

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