Chemical Methylation of RNA and DNA Viral Genomes as a Probe of In Situ Structure

0022-538X/81/110482-09$02.00/0

Chemical

Methylation of RNA and DNA Viral Genomes

as a

Probe

of

In

Situ Structure

MINORUYAMAKAWA, AARON J. SHATKIN,ANDYASUHIRO FURUICHI*

Roche Institute of MolecularBiology, Nutley, New Jersey07110

Received3April 1981/Accepted12June1981

We used[methyl-3H]dimethylsulfatetoprobethegenomestructuresof several

RNAand DNA viruses. Wecompared sites of modification in nucleic acids that

were methylated chemically before and after extraction from purified virions.

With both single-stranded and double-stranded substrates alkylation occurred mainly attheN7 position of guanine. However, adenine NI atomswere differ-entially accessibleinsingle-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirusmRNAand deproteinized

genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae

methylated insitu yieldedRNAs with ratios of0.04 to 0.08, indicating that the intraviriongenomes weredouble stranded. Weobtained ratios of 0.26 and0.35for

the RNAs ofdimethyl sulfate-treated brome mosaic and aviansarcomavirions,

respectively,whichwasconsistent with partial protection ofadenine Ni sites by

structural proteins or genome conformation or both. The ratios of

1-methyl-adenosine to7-methylguanosineforvacciniavirus DNAs methylated in situ (0.10)

and after phenol extraction (0.14) wereless than the ratios for 4X174 and M13

DNAs (0.39 to 0.64) but considerably greater than the ratio observed with

adenovirus DNA (0.002 to0.02). The presence ofa single-stranded region(s) in

the vaccinia virusgenomewasconfirmedbySi nuclease digestionof[methyl-3H]

DNA;the released radiolabeledfractionhadaratioof0.41,compared with0.025

for the residualduplexDNA. Inadditiontothestructure-dependent accessibility

ofadenineNi, methylationofadenine N3wasseveralfold lowerin the intravirion

genomes of vaccinia virus, 4X174, and adenovirus than in the corresponding

extracted DNAs. Chemicalmethylation ofvirions and subviralparticles should

be useful for in situanalysesofspecific regionsof RNA and DNAgenomes,such

asthe sites ofproteinbinding duringvirusmaturation.

Animal virus genomes varywidely in compo-sition and structure. Different groups contain

single-stranded DNAs

(parvoviruses),

single-stranded RNAs

(picornaviruses),

double-stranded DNAs (adenoviruses), and

double-strandedRNAs

(reoviruses) (14).

Inmostcases

the basic structures of these genomes were

es-tablished aftertheywereextracted frompurified

virionsbytreatmentwithphenol,sodium

dode-cyl sulfate, or other protein denaturants. The

importance ofcharacterizing viral genomes in situby directmethods, suchaschemical

modi-fication,has been shownbystudies of the

defec-tive parvovirus adeno-associated virus. The

DNA extracted from purified preparations of this virus is double stranded (25), but intact virions containeitherplusorminusDNAsingle

strands whichreadilyannealtoform

base-paired

duplexes upon deproteinization (18, 24).

Re-cently,weobserved thatpurifiedreovirus cores,

like adeno-associated virus (17), fluoresce red 482

whentheyarestainedwith acridine orange and

examinedunder UV

light.

Underthesame

con-ditionsphenol-extracted reovirus genome RNA

gave agreenishyellow

color,

which istypicalof

double-stranded nucleic acids (2, 6). Since red

fluorescence is a characteristic that is usually

attributed to single-stranded polynucleotides

(16), thisfindingraised the possibility that the double-stranded genome RNA extracted from reovirus may be derived from polynucleotide

chains that exist in situascomplementary single

strands.

Dimethyl sulfate (DMS),a potent alkylating reagent, reactswithnucleic acids under neutral pH conditions(13, 27).In contrast tomanyother

alkylating agents that react only with

single-stranded nucleic acids, DMS methylates both

single-anddouble-stranded

polynucleotides;

the

methylationsitesareconfined tothe base

moi-etyof the targetnucleotides,andtheyvarywith substrate strandedness. For example, DMS

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treatment of double-stranded DNA results in methylation mainly on the N7atomof guanine within the major groove and the N3 atom of adenine within the minor groove of the helix. The adenine Ni position is protected by base pairing. Insingle-stranded DNA the Ni position rather than the N3 position of adenine is readily methylated by DMS, in additiontothe guanine N7 atom (13, 19, 27). By an analogous compari-son of DMS-susceptible sites in phenol-ex-tracted reovirus genome RNA and single-stranded viral mRNA, we found that methyla-tion of the Niposition of adenine is also largely blocked in double-stranded RNA. Byusing the availability of the adenine Ni position for meth-ylation by DMS as an indicator of base pairing, we confirmedthat the reovirus genome is also

double stranded in situ. Studieswith a variety

of otherRNA and DNA viruses indicated that

methylation patterns reflect overall genome

structure,suggesting that this simple method is useful forprobing viruses and subviral particles.

MATERIALS AND METHODS

Viruses. Human reovirus type3Dearing strain was

purifiedfrominfectedmouseLcellsasdescribed pre-viously (26). Reovirus cores were prepared by

digest-ingpurified virions (2 mg/ml) with chymotrypsin (1

mg/ml)at45°Cfor30minin50mM

Tris-hydrochlo-ride buffer (pH 8.0) containing 50 mM KCI. The

resulting viruscoreswerecollected by centrifugation

at10,000xgfor15minat4°C, washed by suspension

in10mMHEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) buffer (pH 7.1) containing 50 mM

KCl,centrifuged again,andfinally resuspended in 10

mMHEPES buffer (pH 7.1).

Cytoplasmicpolyhedrosis virus (CPV)wasisolated

from polyhedralinclusion bodies extracted from the midguts of infected silkworms (Bombyx mori). After

purification by successive sedimentations in sucrose

and CsCldensity gradients(3), the finalsuspension of

purified CPV was dialyzed against 10 mM HEPES

buffer(pH7.1).

Aviansarcomavirus(ASV)B77 and brome mosaic

virus (BMV) were kindly supplied by J. M. Bishop

(UniversityofCalifornia-SanFrancisco) and P. Kaes-berg (University of Wisconsin-Madison), respectively.

These viruses were also dialyzed against 10 mM

HEPES buffer (pH 7.1) and stored at

40C.

Adenovirus type 2 and vaccinia virus strain WR

werepurified from infected HeLacellsbypreviously

describedprocedures (7, 11). BacteriophageM13 and

adeno-associatedviruswerekindly provided by Grace

Ju(Roche Institute of Molecular Biology) and Barrie

Carter (National Institutes of Health), respectively.

OX174

virus and DNA were purchased from Miles Biochemicals.

Viralnucleic acids. Genome RNAs were obtained

by extracting purified virions with distilled phenol

followed by ether extraction and ethanol precipitation.

To obtain DNA genomes, viruses were dialyzed

against10 mMcacodylate buffer (pH 7.0) and treated

for 30 minat 37°C with a solution containing 0.5%

sodium dodecyl sulfate andproteinase K (1mg/ml)

predigested for 2 hat37°C.Anequalvolume ofphenol

wasthen added, and after three extractionsatroom

temperature, the DNA was ethanol precipitated,

washed twice with 80%ethanol,anddissolved inwater.

Methylation of nucleic acids with

[methyl-3H]DMS.Genome RNAs (30to100,ug)wereincubated

at room temperature in reaction mixtures (0.2 ml)

containing0.1 M phosphate buffer (pH 7.0), 0.2 M

NaCl, and10mM[methyl-3H]DMS (specific activity,

200 tCi/umol;NewEngland Nuclear Corp.) (9). After

3h orlonger, the mixtureswerediluted twofold with

waterandphenolextracted. Theincorporationof

3H-labeled methyl groups into RNA was measured by

precipitating asample ofanextracted mixture in 5%

trichloroacetic acid andcountingonnitrocellulose

fil-ters. Theusual levels of incorporation were -4,000

and -6,000 cpm from 3H-labeled methylgroups per

,ugfor the extracteddouble-stranded reovirus genome

RNAs andsingle-strandedviralmRNA's,respectively.

Incorporationwasabout 20% less for RNAs

methyl-ated in reovirus cores and intact CPV. To eliminate

unreacted[3H]DMSfrom radiolabeledRNAs, reacted

sampleswereethanolprecipitated and passed through

Sephadex G-100 in 20 mM Tris-hydrochloride (pH

7.5). Theradioactive material in the excluded volume

wasethanolprecipitated,and thepurifiedRNAswere

dissolved in5mMsodiumacetatebuffer(pH6.0).

Viral DNAswerealkylatedinsituorafterphenol

extraction from virions in incubation mixtures (0.2ml)

containing 10 mM cacodylate or sodium phosphate

buffer(pH7.0),200

lCi

of[3H]DMS(0.5 mM;specific

activity,2.1 Ci/mmol; NewEnglandNuclearCorp.),

and40 to 50,tg ofextracted DNA oran equivalent

amount asvirions.Incubationwas at0°Cfor17h;in

thecaseof in situmethylation,DNAwasextractedas

described above.

Digestion of RNA and separation of

3H-methyl-labeled ribonucleosides by electropho-resis. 3H-methyl-labeledRNAs (-50,ug)in0.1ml of

5mMacetate buffer(pH6.0) were digestedto

mon-onucleotides by incubating them with 100iLg of P1

nuclease(YamasaShoyu Corp.)at37°Cfor1h.Before

enzyme treatment,duplexRNAswereheatdenatured

(100°C for 1 min and rapid chilling). P1 nuclease

digestswereneutralized byaddingTris-hydrochloride

buffer (pH 8.0) and then incubated with 8.6 U of

bacterialalkaline phosphatase (Worthington

Diagnos-tics) permlfor 90min at 37°C. Theproducts were analyzed byhigh-voltagepaperelectrophoresisin 10%

pyridine-acetate buffer (pH 3.5) containing 1 mM

EDTA(4). Todistinguishbetween7-methylguanosine

(m7G) and 1-methyladenosine

(m'A),

which were

poorly resolvedinthissystem,m7Gwasconvertedto

m7G* [i.e., the ring-opened derivative

2-amino-4-hydroxy-5-N-methyl

carboxamide-6-(N-fi-ribofurano-syl)pyrimidine] by incubation in7NNH40Hfor2h

at room temperature. Under these conditions m7G,

which hadanetchargeof+1,wasconverted

quanti-tatively to thering-opened compound, which had a

netchargeof zero;m'Awaspartially(17%)converted

tom6A. To determine thecontentofmethylated

cy-tosine,whichwas notresolvedfromm'A by

electro-phoresis, radiolabeled RNA samples that were

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gested with P1 nuclease and phosphatase were ana-lyzed by paper chromatography in n-butanol-NH40H

(100:1). Polyadenylic acid, polycytidylic acid, and

polyguanilic acid were also 3H methylated and

ana-lyzed inparallel. TheRfvalues form'A,m6A,

meth-ylated cytidine, and m7G were 0.2, 0.3, 0.44, and 0.04, respectively.

Resolutionof'H-methyl-labeledDNAbases by

columnchromatography.After chemical

modifica-tionby[3H]DMS,DNAwasprecipitated with ethanol,

washed two times with80% ethanol, dried, and

hydro-lyzed in 7 NHCl04 for 1 hat 1000C.Theresulting

digest was analyzed by chromatography on a Dowex

50 x 4column (0.4 by 20 cm); 0.5-ml fractions were

eluted with0.3Mammoniumformate at a flow rate of

4.5ml/h (20).The marker compounds used included

m7G,m'A,and m3A (Vega Fox Chemicals), aswell as

3-methylcytosine and5-methylcytosine (PL

Biochem-icals).

Sinucleasedigestion. Phenol-extractedvaccinia

virus DNA was methylated by exposure to[3H]DMS

and waspurifiedby gel filtration on a Sephadex G-100

column (0.8by25cm) in50mMsodium acetate buffer

(pH6.0)containing 7Murea.The DNAwas

precipi-tated with ethanol, washed two times with 80%

ethanol, dried, and dissolved inwater. For each

en-zymedigestion a reaction mixture (0.5 ml) containing

30mMsodiumacetatebuffer(pH4.5), 0.3 MNaCl, 5

mMZnCl2,[methyl-3H]DNA,and 200 U ofSinuclease

(MilesBiochemicals) wasincubated for2h at370C.

The Si-resistant and -sensitive DNA components

were separatedby gel filtrationon aSephadex G-50

column(0.8by25cm) in10mMammonium

bicarbon-ate(pH 7.6). Fractions(0.5ml) containingthepeaks

ofradioactivity were pooled and lyophilized. Acid

hy-drolysis andanalysisofmethylated bases by Dowex

columnchromatographywereperformedasdescribed

above.

RESULTS

Methylation

of RNAgenomes.To

charac-terize the sites of DMS

alkylation

in

double-stranded and single-stranded RNAs, genome

RNA thatwas

phenol

extracted from

purified

reovirus and viral mRNAsynthesized invitroby

reovirus cores were incubated with 10 mM

[methyl-3H]DMS

as described above. Under

these conditions

incorporation

of 3H-labeled

methylgroups into virus RNAs was nearly linear

for3to4hand then continuedat aslowerrate.

Thereacted RNAswereisolated from the

incu-bation mixtures

by

Sephadex

G-100

gel

filtra-tion, and the

3H-methylated

nucleosides ofthese

RNAswere

analyzed by

high-voltage

paper

elec-trophoresis after successive

digestions

with P1

nuclease and

alkaline

phosphatase (4). The

di-gests of both the double-stranded genomeRNA

(Fig. 1A) and the single-stranded viral mRNA

(Fig. 1B)

yielded

most of the

radioactivity

as

material that

migrated

in

positions

correspond-ing

to the

positions

of marker ribonucleosides

mG and

m1A.

Bothm7Gand

m1A

contained a

x

_

-S

5 ~~~~~~~B

ee

2

~ h

E._

5

o

O, ,

5

C C

10FRACTION20

3

FIG. 1. Analysis of methylated residues in RNAs.

(A) Reovirus double-stranded genome RNA was

phenol extracted from virions, purified by Sephadex

G-100column chromatography, and chemically

meth-ylated by exposure to [methyl-3H]DMS, as described

in thetext. (B)Reovirus mRNA synthesized in vitro

by using virus-associated RNA polymerasewas

meth-ylated asdescribed above. The RNA preparations (50

ag in(A)and (B) weredigested withP1nuckeaseand

alkaline phosphatase, and theresulting3H-labeled

nucleosides were analyzed by high-voltage pagper

electrophoresis. (C)and(D)The materialin fractions

2 through 6 of(A) and(B), respectively, was eluted,

treated with 7 NNH4OH,andre-electrophoresed.

positivechargeunder these conditions (pH3.5),

and these two compounds migrated together

toward the cathode. Toidentifythe

3H-methyl-ated residue(s), fractions 2 through 6 (Fig. 1A

and B) were eluted from the paper inwaterand analyzedbyelectrophoresis aftertreatmentwith 7 M

NH4OH

for 2hatroomtemperature. These conditions degraded m7G completely tothe ring-opened, uncharged derivative m7G* and also partially converted

m'A

to m6A (4, 8). As Fig.

10

shows, the predominant 3H-methyl-labeled

nucleoside in DMS-treated reovirus

double-stranded RNA was m7G (97%), whichwas con-verted by alkali treatment to the ring-opened derivative

m7G*.

A small portion of the

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[image:3.497.291.442.63.365.2]

METHYLATION OF VIRAL GENOMES IN SITU 485

activity (3%) migrated in the original position (Fig. 1C, fractions 3 and 4). This minor 3H-la-beled compound presumably was m1A since it comigrated with m6A after alonger treatment

with 7 MNH4OH(datanotshown).

Incontrast tothedouble-stranded RNA, the

3H-methyl-labeled materialderived from

reovi-rus single-stranded mRNA was resolved into three prominent, distinct peaks by electropho-resis aftertreatmentwith P1nuclease,

phospha-tase,and alkali(Fig. 1D). Theradioactivitynear

the origin (fractions 20 through 24) was m7G*

(61% of the total), and the small peak of radio-activity (7%) in fractions 15 and 16 corresponded

to m6A. The radioactive material (31%) that comigratedwithauthenticm'Atoward the cath-ode consisted of m1A andmethylated cytidine. The m'A was converted to m6A by prolonged alkline treatment (87% conversion after 18 h [datanotshown]). Under thesameconditions,

morethan75% ofthemethylated cytidine

deriv-ative, whichwasthe mainproduct inchemically

methylated polycytidylic acid, remained

un-changed. The methylated cytidine derivative did

notcomigrate with marker3-methylcytidineor

5-methylcytidine during paper electrophoresis atpH 3.5orduringpaperchromatographyin

n-butanol-NH4OH (100:1). Althoughthe methyl-ated cytidine derivative was not identified, its contributiontothemethylated nucleoside com-position of the RNAswasassessedby chroma-tographyasdescribed above(Table 1). Like the otherminorradioactivecomponents, the

3H-la-beledmaterial in Fig. 1D, fractions9 and10, in thepositionof5-methylcytidinewasnot identi-fied.

These data demonstratedaclear difference in the chemical methylation patterns of double-stranded and single-stranded RNAs. Although

the plus strands of reovirus double-stranded

RNAareapparently identical insequencetothe corresponding single-stranded viral mRNA's (12), thepresenceofthesestrands inbase-paired duplexes limits methylation almost exclusively

to the guanosine N7 position, compared with additional accessible sites on adenosine and cytidine residues in the mRNA. The ratio of m1Atom7G fordouble-strandedRNAwas0.03

(i.e., 14-fold less than the ratio obtained for mRNA). Sincetheextentofguanosine methyl-ationwasnearly identical in bothtypesofRNA

(5% ofthe totalguanosineresidues), the 1.7-fold increase in methylation of mRNA compared

withgenomeRNAresulted fromthe differential incorporation ofmethyl groups intoadenosine residues and,toasmaller extent, cytosine

resi-duesinthesingle-stranded molecules. The

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lim-itedextentofmethylation presumablywasdue

TABLE 1. Methylation of virus RNAs in situ'

Total % Ofradioactivity in amtof thefollowing

prod-radio- ucts: Ratio

Sample activity ofm'A

ana- mA + 7 to

m7G

lyzed mC mC m7G

(cpm) m

Reovirus double- 3,345 3.0 <0.1 97.0 0.03 strandedRNA

Reovirus mRNA 5,827 38.6 12.0 61.4 0.43

Reovirus cores 3,808 7.0 93.0 0.08

CPVvirions 11,298 3.4 96.6 0.04

BMVvirions 21,467 25.1 5.4 74.9 0.26 ASVvirions 1,593 29.3 4.8 70.7 0.35 aPhenol-extracted reovirusdouble-strandedgenome RNA, reovirus mRNA, reovirus cores, and purified virions were labeledbyincubatingmaterialequivalent to 30 to 100 ug of RNA with[methyl-3H]DMSasdescribed inthe text. After purification,the extents oflabeling were asfollows:reovirus double-stranded,3,100cpm/g,reovirus mRNAsynthesized invitro, 6,950 cpm/llg; reovirus cores, 4,300cpm/pg;CPV, 4,825 cpm/pg, BMV, 4,474cpm/g,and ASV, 880cpm/yg. Sampleswereanalyzedformethylated nucleosidesby paper electrophoresis after P1 nuclease andphosphatasedigestion andquantitativeconversion ofm7Gtom7G in 7 NNH40H. mAincludedm'A and itsalkalinedegradationproductm"A, which were separated by paperelectrophoresis.The methyl-ated cytidine derivative (mC) whichcomigrated with m'A during electrophoresiswasquantitatedby paper chromatog-raphy of samples digested with P1 nuclease and phosphatase asdescibed in the text.

to a

decreasing concentration

of

DMS,

which

wascaused

by evaporation

and

degradation

dur-ing the modification reaction.

Methylation

by DMSwas nextusedwith

ge-nome RNAs in virus

particles.

Reovirus cores

wereprepared from

purified

virions

by

chymo-trypsin

digestion (1).

Another member of the

Reoviridae,

insect

CPV,

wasusedas

intact,

pur-ified virions. BMV and ASV werealso treated.

After the

particles

wereincubatedwith

[methyl-3H]DMS

for24hat roomtemperaturetoobtain

maximum

modification,

viral

RNAs

were

phenol

extracted and freed ofresidual

alkylating

agent

by

Sephadex

G-100

gel

filtration and ethanol

precipitation.

The

purified

RNAswere

analyzed

fortheir

methylated

nucleosidecompositions by

paper

chromatography

and

electrophoresis

after

digestion

with P1 nuclease and alkaline

phos-phatase.Ineachcasethe

electrophoretic

pattern

wassimilartothe

pattern

shown in

Fig. 1A;

i.e.

essentially

all of the3H-labeled ribonucleosides

migrated in fractions2

through

6, the

positions

ofm7G and

m'A

markers. The material from

each

digest

waselutedfrom the paper, treated

with alkali to convertm7G to the

ring-opened

structure, and

reanalyzed by

electrophoresis.

The resultswithreoviruscores

(Fig. 2A)

could

be

compared

directly

withthe resultsobtained

withdeproteinized reovirus genome RNA

(Fig.

1C) since

equivalent

amounts of nucleic acid

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(W)

2

x

0 2

'~8

cn 6

4

2

1.0 D

0.5-0 to 20

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(0 FRACTION

FIG. 2. Methylation ofgenomeRNAs in situ.

Pur-ified particles were methylated by using

[methyl-3HJDMS,asdescribedin the text.RNAswerephenol

extracted andpurified by gel filtration, andsamples

weredigestedtothe nucleosidelevel withPlnuclease

andphosphatase. m7Gwasconverted tom7G*, and

m'Awaspartiallyconvertedtom6Abyexposureto7

NNH40H beforepaperelectrophoresis. (A)Reovirus

cores. (B)CPV.(C)BMV. (D)ASV.

were reacted with DMS. The methylated

nu-cleosidepatternswerealmostidentical forRNAs

modified in situ and after extraction. In each

case more than 90% of thealkylation occurred

at the N7 position of guanine. Similar results

wereobtained withintact CPV(Fig.2B). Thus, genome RNAs packaged within reovirus cores

or intact CPV yielded patterns of chemical

methylation similartothose obtained with

de-proteinized double-stranded RNAs.

Further-more,theextentsofguanosine methylation were

similar (-5% oftheguanosine residues) for

reo-virusand CPV genomeRNAsalkylatedin situ

andafter extraction. Incontrast to the two

rep-resentative members of the Reoviridae,

meth-ylation of BMV and ASV resulted in the

for-mation of significantly higher levelsof

methyl-ated adenosine relative to methylated

guano-sine, whichwas consistent with thepresence of

single-stranded genomes in these two viruses

(Fig. 2C and D). It should be notedthatthere

was no apparent disruption of virus particles

under the conditions whichwe used forchemical

methylation and that the mRNA

guanylyltrans-ferase and nucleotide phosphohydrolase

activi-ties associated withpurified reoviruscoreswere

notaffected by the DMStreatment (30).

The methylated nucleoside compositions of

the RNAsamples are summarized in Table 1.

Thediagnostic ratios of

m'A

tom7Gwere0.03,

0.08,and0.04 forphenol-extracted reovirus

ge-nome RNA, intact reoviruscores, and CPV

vi-rions,respectively. These valueswerewellbelow

thevalue of0.43 obtained with reovirus

single-stranded mRNA. The genome RNAs of BMV

and ASV yielded ratios of0.26 and 0.35 (i.e.,

values which were intermediate between the

value for double-stranded reovirus RNA [0.03]

and the value for

single-stranded

viral mRNA

[0.43]). The viral genomes of BMV and ASV

apparently

contained some adenosine residues

that were inaccessible to the

methylating

re-agent, possibly dueto shielding by

intramolec-ular base

pairing

or intermolecular structures

(23) in the case of ASV

diploid

RNA. Also

association of thegenomeRNAs with structural

proteins in virions

probably

accounted forsome

protectedsites (10, 21).

Analyses

of

chemically methylated

viral

DNAs. To assessthe generality of using DMS

methylation

patterns todetermine viralgenome

strandedness, weexposed a

variety

of DNA

vi-ruses tothe radiolabeled

alkylating

agent. Nu-cleic acids wereextracted and acidhydrolyzed,

and the

resulting

baseswere

analyzed by

Dowex

column

chromatography.

We

compared

viral ge-nomes methylated in situ and viral genomes

afterextraction from virions. AsFig.3Ashows,

adenovirusDNAwas

methylated mainly

on

gua-nineresiduesatthe N7position

and,

to alesser

extentontheadenine N3

position.

Theseresults

and the

striking

absence of

1-methyladenine

(m'A)

incontrast tothe

prominent

m1A

peak

in

DMS-reacted

4X174

DNA (Fig.3B andD) are

consistent with a

completely

double-stranded

structure for DNA extracted from adenovirus.

Interestingly,

DMS treatmentof intact adeno-J.

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virions yieldedDNA that containedasignificant

m'A fraction (Fig.30). Thissuggestedthat the genome in situ contained destabilized regions whichwereabsent from thedeproteinized DNA.

The methylation patterns of phenol-extracted and intravirion

OX174

DNAswerealso

noticea-bly different. Therewas anincreased

accessibil-ity of the adenine N3 sites after deproteinization (Fig. 3B), suggesting that there was some pro-tective effect of the virion proteins, either

di-rectly by shieldingorindirectly byalterationof thegenome confonnation. Similarresultswere obtained with M13, another single-stranded DNA bacteriophage (Table 2). As this table shows, in each of the genomes examined the accessibility of the N3 atom of adenine was enhancedinpurifiedDNA.

In theparvovirus,adeno-associatedvirus,the relativeamounts of m1A andm3A confirmedthe predominantly single-strandednatureof the

ge-E

C~~~~~~~~~~~~~~~~~~~~~~~~~~C

50,2-

B

D 50

300

120

4

-~~~~~~~~~~~~~~~~~~~~~~~~~-2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

20 40 60 100 20 40 60 100

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FRACTION

FIG. 3. Separation of31H-methyl-labeled bases byDowex column chromatography. ViralgenomeDNAs

weremethylated,acidhydrolyzed, andanalyzedasdescribed in thetext.AdenovirusDNAwasmethylated

after extraction (A) and in situ(B). X174 virus DNAwasmethylated afterextraction(C)and in situ(D).A26o, Absorbanceat260nm.

TABLE 2. Methylation ofvirusDNAsa

Totalamtof radioac- % Ofradioactivityin the follow- Rtio

Sample tivity analyzed per 40 ing products:

Agof DNA(cpm)

m1A

m3A m7G

m1A/m7G

m3A/m7G

Adenovirus DNA 66,396 0.2 7.6 92.0 0.002 0.082

Adenovirus 62,142 2.2 0.8 97.0 0.022 0.008

4X174virusDNA 143,969 26.4 6.1 67.5 0.391 0.090

4X174 113,327 35.7 1.7 62.6 0.570 0.027

M13 virusDNA 219,686 29.7 6.0 64.3 0.461 0.092

M13 virus 154,271 38.1 4.5 58.9 0.646 0.076

Adeno-associatedvirus DNA 45,976 7.9 7.4 84.7 0.093 0.087

Adeno-associatedvirus 40,168 36.8 1.1 62.1 0.593 0.017

Vaccinia virus DNA 23,558 11.7 6.7 81.6 0.143 0.082

Vaccinia virus 22,106 8.9 1.5 89.6 0.100 0.017

aChemicalmethylationofviriongenomesin situorafter phenol extractionwas

performed

as described in the

text.3H-methyl-labeled baseswereanalyzed by Dowex chromatography. 40,1981

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[image:6.497.59.451.506.635.2]

AND FURUICHI

nome in virions and the duplex structure of extracted DNA. A high proportion of m1A was

obtained inDNAfrom DMS-treatedintact

vir-ions; isolated adeno-associated virus DNA

yieldedanincreased amount ofm3Aand a low

percentage of

m1A

(Table 2).

Vaccinia virus DNA exposed to DMS either

in the virionor after phenol extraction yielded

consistently high levels ofm1A compared with

the amounts obtained with adenovirus

double-stranded DNA (Table 2). The higher ratio of

m'A

tom7Gwasapparentlynotdue to

contam-inating RNA or single-stranded DNA since the

same value (0.14) was obtained after RNase

treatmentand repurificationof

high-molecular-weight vaccinia virus DNA. This was

accom-plished bytreatmentwith pancreaticRNase (10

,ug/ml,

370C,

15min) followed by sedimentation

(SW27rotor, 52,000 x g, 3 h,

2000)

through a 5

to20%sucrosegradient(0.05 MTris buffer, pH

8, 1M NaCl, 0.001 MEDTA, 0.15% Sarkosyl)

onto a 50% glycerol cushion. (Under the same

conditions

4X174

DNA remained nearthe top

of the gradient.) These results suggested that

vaccinia virusgenomesin situ containunpaired

adenine residues that retain DMS-accessible

Ni

sites after extraction, possibly dueto their

pres-ence in

single-stranded

or readily denaturable

stretches

(for

example,

in duplex regions

dis-torted by superhelical twisting or in the loop

structures attheDNA termini [5]). To test for the presence ofsingle-stranded regions,

depro-teinized vaccinia virus DNA was radiolabeled

with [methyl-3H]DMS, treated with

single-strand-specific nucleaseSi, and filtered through

Sephadex G-100. Most of the radiolabeled DNA

wasresistanttoSidigestion andwasrecovered

in the excluded volume (Fig. 4, inset, peak I).

However, approximately 20% of the

radioactiv-itywasreleasedaslow-molecular-weight

mate-rial thatwasretardedduring gel filtration (peak

II).Peaks Iand IIwerepooledseparately, con-verted totheirconstituent bases by acid

hydrol-ysis, andanalyzed by Dowex column

chromatog-raphy. Peak I contained radiolabeled m3A and

alow level of

m'A,

in additiontothe mainpeak

of7-methylguanine (Fig. 4A). PeakIIyieldeda

higher proportionof

m'A

andnodetectable m3A

(Fig. 4B).The ratios of

m'A

to7-methylguanine

forpeak I (0.025) and peak II (0.41) were

con-sistent with the values expected for

double-stranded and single-stranded DNAs,

respec-tively.

DISCUSSION

Alkylation of nucleic acids by exposure to

DMS at a neutral pH is an extremely useful

A m7G

-L m3A

0.6 0.4

0.2

I

.0.025

m1A

0---

4m7G.0.41

-%I

0 20 40

mIA

B J-~

20 40 60 80 100

FRACTION

FIG. 4. Chromatography ofanSI nucleasedigest of 3H-methylatedvaccinia virusDNA. Vaccinia virus

DNA (45pg) wasmethylated and separated fromunreacted[3H]DMS by Sephadex G-100filtration. The

DNAwasdigestedwith SInuclease andfilteredthrough SephadexG-50asdescribed in the text(inset).The

Si nuclease-resistantDNAfraction (peak I)and the included materialfromthedigestedDNA(peak II)were

pooled separatelyand lyophilized.Methylatedbases obtainedbyacidhydrolysiswereseparated byDowex

columnchromatography. (A)PeakI. (B) peakII.

I0

8

6

41

0.8

0.4

)Q

x

a.

C-0.8

0.4

J. VIROL.

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METHYLATION OF VIRAL GENOMES IN SITU 489

procedure for molecular structure studies and

has provided the basis for rapid chemical se-quencing of DNA (15) and,morerecently, RNA (22).We used this method forananalysis of the

structures of viral genomes. Both RNA and

DNAweremodified chemically in situ by

expos-ing virus particles to [methyl-3H]DMS. Al-though onlya small fraction of thetotalbases were modified under the experimental

condi-tions whichweused, the availability ofareagent

withahigh specific radioactivity facilitated the

studies.

Methylation occurred mainlyonpurine

resi-dues. In eachcasem7Gwasthemajor 3H-labeled

product, together with m1A, minoramountsof methylated cytosine, and (in DNA) m3A. As previously observed for DNA (13), single-stranded and double-single-stranded RNAs could be distinguished onthe basis oftheaccessibility of

the adenine Ni positions. In double-stranded molecules, hydrogen bonding of adenine-uracil base pairs shielded the adenine Ni sites. Con-sequently, the yield of m'A from phenol-ex-tracted reovirusgenomeRNAwasconsiderably

lower than the yield obtained with single-stranded RNA. Our results confirmed that the RNA genomes of human reovirus type 3 and insect CPV are double stranded in situ. The genomeRNAsof intact BMV and ASVyielded relatively high amounts ofradioactivity in the

m1A fraction, as expected for single-stranded RNAs, but the valueswereless than thevalue

observed for reovirus mRNA. Since reovirus mRNA contains extensive amountsof intramo-lecular structure (28), which tendsto decrease the availability of adenine Ni sites, it seems

reasonabletosuggestthat the loweraccessibility of adenine Ni atoms in the single-stranded RNAs ofBMVand ASVwasduetoprotection by structural proteins. Clearly, it should be pos-sible by comparative sequence analyses of ge-nomes modified in situ and after deproteiniza-tion(10, 21, 23)toestablish whetherdiminished adenine Ni accessibility is primarily due to

shielding byassociated proteinsordue to

con-fornational effects.

The potential of this approach for probing specific regionsofaltered structure inviral

ge-nomes is suggested by our preliminary results

with vaccinia virus DNA. Obviously, resolution

canbeimproved markedly by using cloned DNA

fragments (29), as has been done with simian

virus40origins of DNA replication andT anti-gen (R. Tjian, Curr. Top. Microbiol. Immunol., in press). Extension of the DMS methylation methodtostudies of RNA viruses and subviral nucleoprotein complexes should allow identifi-cation ofbinding sites of structuralproteins and virion-associatedenzymes.

ACKNOWLEDGMENT

We thank Alba LaFiandra for purified reovirus.

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