RNA synthesis in cells infected with herpes simple virus. XIII. Differences in the methylation patterns of viral RNA during the reproductive cycle.

CopyrightC 1976 American Society for Microbiology Printed inVol. 20, No. 3U.S.A.

RNA

Synthesis in Cells Infected with Herpes Simplex Virus

XIII. Differences in the Methylation Patterns of Viral RNA During the

Reproductive

Cycle

MICHAEL BARTKOSKI AND BERNARD ROIZMAN* Committee on Virology, University ofChicago, Chicago, Illinois 60637

Received for publication 20 July 1976

Herpessimplex virus 1 (HSV-1) RNA labeled with

[methyl-3H]methionine

at

varioustimes during the infectious cycle and purified by hybridization to viral

DNAwasanalyzed for the presence of methylated nucleotides. The data indicate

the following.

(i)

RNA labeled from 0 to 14 h postinfection and accumulating in

the

cytoplasm contained internal base-methylated nucleotides and terminal

oligonucleotides

consistentwith thestructure7mG(5')ppp-(5')XmpYmpNp. Simi-lar methylatednucleotides and oligonucleotides were also found in viral RNA accumulatinginthe cytoplasm of cells treated with cycloheximide from thetime ofinfection.Previousstudies(M.Kozak andB. Roizman, 1974)have shown

that,

whereas the RNA accumulating in the 14-h infected cells contains all ofthe

sequences functioningasmRNA throughout infection, theRNA accumulating

inthe cytoplasm of cycloheximide-treated cells isassociated with polyribosomes

synthesizing

the earliest (a) groupofpolypeptides specified by the virus. (ii) Cytoplasmic viral RNA from cells labeled11to 14hpostinfectionaswellasthe total adenylated RNAinthe cytoplasm and polyribosomes labeledinthesame fashion contained the terminal oligonucleotide butnotthe internal

base-methyl-atednucleotide.

Previous reports from this laboratory have

shownthatinhumancellsinfected with human herpesvirus1 (herpessimplexvirus 1 [HSV-1]) viral polypeptides form at least three groups whose

synthesis

is

coordinately

regulated

and

sequentially

ordered (13, 14, 21).

Thus,

a

poly-peptides

are made first and reach maximum

ratesofsynthesis between2and4h

postinfec-tion. They induce the

synthesis

of the

second

or

,8

groupmadeatmaximumratesbetween5and

7hpostinfection.

These

shut off the

synthesis of

a

polypeptides and induce

the

synthesis

of the thirdory group.Thisgroup,

consisting

largely

of viral structural

polypeptides (12),

in turn shuts off the

synthesis of

the

/8

group.

Several lines of evidence indicate that the a and

J8

mRNA's,

are

relatively

stable and that the shutoff of

synthesis

of these

polypeptides

is a

specific

functionofone or morepandy

poly-peptides,

respectively.

Thus,

a

polypeptide

syn-thesiscontinuesformanyhoursifthe

synthesis

of 3

polypeptides

isblocked either

by

rendering

a polypeptides nonfunctional (14) or

by

inter-fering

with thetranscription of

(8

mRNA after a

mRNA

had accumulated inthe infected cell

(13, 21). However, a

polypeptide

synthesis

de-cays at a

relatively

rapid

rate oncefunctional

(8

polypeptides

aremade (13, 21). One expectation

ofasystem inwhich the

polypeptide

synthesis

iscoordinatelyregulatedinacascade fashionis that the mRNA's of eachgroupshare structural features thatwould permit differential regula-tion at the level of translation (21). In the search forsuch differential features, webegan studies on the presence and distribution of methylated nucleotidesinviral RNA extracted from

the

cytoplasm

of infected cells.

Methylated

nucleotides have been

demon-strated in mRNA of

eukaryotic

cells (1, 4, 20) and virus (17, 24, 30), butnotall viruses infect-ing eukaryotic cells

specify methylated

infor-mational RNAmolecules (7). The

methylated

nucleotides occur

internally,

usually in the form of

6-methyl

A, and atthe5' terminusas an

oligonucleotide

of the structure

m7G(5')-ppp(5')XmpYmpNp or

m7G(5')ppp(5')XmpNp,

where Xm and ym representthe 2'-O-methyl-ated derivatives of all four ribonucleosides (1). Although it. has been recently demonstrated that thepresence of m7Gatthe5' terminus is needed for the effective translation ofglobin,

vesicularstomatitis virus, and reovirus mRNA's

(2,

19), theexact functionof thesemethylated

sequences isstillunknown.

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In designing the experiments described in

this paper we took advantage of two

observa-tions. First, only a polypeptides are made in

cellsimmediatelyafter withdrawal of

cyclohex-imidepresent inthe medium from the time of

infection (13) or inthe presence of amino acid

analogues addedatthe time of infection(14). In

bothinstances transcripts arisingfromonly 10

to 14%of the DNAaccumulate in the cytoplasm

(16,21;N.Frenkel, H. Locker,and B.

Roizman,

manuscript in preparation). We have defined

thesetranscriptsasamRNA. Second, afterthe

onset of assembly and maturation of

viruses,

the transcripts accumulating in the infected

cell cytoplasm are homologous to 43% of the

viral DNA, and molecularhybridization

stud-iesshowthatthis RNAincludesalltranscripts

translocated into the cytoplasm, includingthe a mRNA sequences, even though the a

poly-peptidesare nolonger made (16, 21, 25).

MATERIALS AND METHODS

Cells and virus. The procedures for the

propaga-tionof HEp-2 (humanepidermoid carcinomano. 2) cells and the assay, production, andpertinent prop-ertiesoftheF strainof HSV-1 weredescribed else-where (6, 8, 15, 22).Inthesestudies, confluent

mon-olayers of HEp-2 cellswereinfected with HSV-1 (F)

at amultiplicity of10PFU/cell.

Labeling ofRNA. Infected cells were labeled at

the times specified inthetext with20to 25 mCiof

[methyl-3H]methionine

(Amersham/Searle Co.) per

ml ofmaintenance medium consisting ofmixture 199containing one-fifththe normal methionine

con-centrations, butsupplemented with 1%dialyzed calf

serum, 20 ,Meachadenosine and guanosine, and20 mM sodium formate toinhibit the incorporation of

[methyl-3H]methioninegroups intothepurinerings

(18).

Cell fractionation. Cells were scraped and har-vested in phosphate-buffered saline (5), washed

once,and resuspendedinlysing buffer(50 mM Tris-hydrochloride [pH 7.4]-50 mM KCl-1 mM MgCl2-0.5%Nonidet P-40 IBDHChemicalsLtd.]). After10 to 20min onice,the nucleiwereseparated from the cytoplasmicextractbycentrifugation. Sodium dode-cyl sulfate (SDS)wasaddedto afinalconcentration

of0.5%tothe cytoplasmic fraction.

ExtractionofRNA. RNA wasextracted from the

cytoplasmic fraction with phenol followedby reex-traction ofthenonaqueous residuewith Tris buffer, pH 9.0 (3). Theaqueous phaseswere combined and reextractedwithphenol-chloroform(1:1)followedby

chloroform-4% isoamyl alcohol. Inthe finalstep of

purification the RNA in a solventcontaining 0.025

M ammonium acetate was precipitated with ethanol.

FractionationofpolyadenylatedRNA. The bind-ing and elution of adenylated RNA from poly(U) immobilized on glass-fiber columns were done as

previouslydescribed (26).

Enzymatic digestionofRNA. RNA was digested

withT2RNase(30Ag/ml;SigmaChemicalCo.) in 10

mM sodiumacetate, pH4.6. P1 nuclease

digestion

(200 .g/ml; Yamasa, Ltd., Japan) wasdone in 10 mM acetate buffer, pH 6.0. For alkaline

phospha-tasedigestion(10 Ag/ml;SigmaChemicalCo.)after

P1 digestion, thepHwasraisedto8.2with Tris.

DEAE-Sephadex chromatography. The nuclease

digestion products were analyzed by chromatogra-phyonDEAE-Sephadex(A25)columns

equilibrated

with 20 mM Tris-hydrochloride (pH 7.4), 0.05 M

NaCl,and 7 Murea(29).Thedigestwaselutedfrom the columns withlineargradients of0.05 to 0.4 M NaCl in theTris-urea bufferandclearedcompletely

with1 MNaCl.

The eluent fractionswerepooledanddesaltedby

thebariumprecipitation proceduredescribedbyRoy

andBishop(23). The elution ofnucleotidesand

oli-gonucleotides withcharges rangingfrom -2 to -7

wasstandardizedwiththe aid ofpancreaticRNase

digestofEscherichiacolitRNA.

DNA-RNAhybridization. TheDNA-RNA

hybrid-izationswerecarriedout byoneoftwoprocedures.

Inthefirst, DNAwasimmobilizedonSepharoseas

described by Gilboa et al. (11). The hybridization

wasdone in 5x SSC(1XSSC contains 0.15 MNaCl plus0.015 Msodiumcitrate)and 50% formamideat

50°C. Afterhybridization, the DNA-Sepharose

col-umn was washed extensively with hybridization buffer. The

hybridized

RNA wasthen eluted with

98% formamide-10mMTris(pH7.4) followedbya1

M NaCl wash. The second procedure involved hy-bridization ofRNA to viral DNA fixed to

nitro-cellulose filtersasdescribedby Kieffetal. (15).The

hybridizations were done at 50°C for 48 h in 0.8 ml ofhybridization buffer containing0.75 M NaCl,

0.1 MTris(pH7.4), 0.5 mMEDTA,0.1% SDS, and 50%formamide. Afterhybridizationthefilterswere

soaked in 1 x SSC containing 0.5% SDS, reincu-bated for 1h,and then rinsed in 2 xSSC. The RNA

wasthen released by incubatingthe filters in95% formamide-30mM Tris (pH8.2)-0.1%SDS for1hat

50°C. Released RNA was precipitated with 2

vol-umesofethanol and stored at -20°C.

RESULTS

The studiesdescribedinthispaperconsisted

ofthree series of experiments. In the first we

examined the methylated nucleotides in

cyto-plasmic virus-specific RNA labeled with

[methyl-:H]methionine from the time of

expo-sure of cells to virus until 14 h postinfection.

Previous studies from this laboratory have

shown thatthecytoplasm of 14-h infectedcells

contains RNA sequences homologousto 43%of

DNA. This RNA represents all informational

sequences present incytoplasmthroughout

in-fection even though only a polypeptides are

madeatthattime (16, 21). In this seriesHEp-2

cells were infected with HSV-1 (F) at a

multi-plicityof 10PFU/cell and labeled with

[methyl-IHimethionine from 0 to 14 h postinfection as

described above. Atthattimetheinfectedcells

were harvested, and nuclear and cytoplasmic

fractions were separated as described. Viral

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RNA SYNTHESIS IN CELLS INFECTED WITH HSV 585

RNA was purified by hybridization to viral

DNA immobilized on Sepharose (11), eluted,

and then analyzed for thepresence of

methyl-ated nucleotides. In these analyses the T2

RNase digest of purified RNA was

chromato-graphedonDEAE-Sephadex in thepresenceof

7Murea(29). This procedureseparates

nucleo-tides and oligonucleonucleo-tides on the basis of the

number of negatively charged phosphates.

Thus, each internal phosphate contributesa-1

charge, whereas each free terminal phosphate contributesa-2charge.Themethyl-3Hlabel in

theHSV-1(F) RNAdigest chromatographedas

two peaks, which eluted at positions

corre-spondingto-2and -5to -6charge (Fig. 1A).

Peak 1, containing approximately 37% of the

totalelutedactivity(Table1),consisted of

base-methylated nucleotides. This conclusion is basedonitscharge(-2)andontheobservation

that subsequenttreatmentwith alkaline phos-phatase convertedittoanucleoside that didnot

absorb to the DEAE-Sephadex column (Fig.

1B). The material elutinginpeak1corresponds

in its properties to internal base-methylated

nucleotides (1, 24, 30). The -5 to -6 charged material inpeak 2 (Fig. 1A) wasdesalted,

di-gested with P1 nuclease, which degrades RNA to5'-nucleotides(9),and splitintotwoportions.

Oneportion, designated as the P1 digest, was

chromatographed on DEAE-Sephadex in 7 M urea. The otherportion,designatedasP1

alka-line phosphatase digest, was redigested with

alkaline phosphatase and then

rechromato-graphed asabove. The P1 digest eluted in two

peaks (Fig. 10). Thefirstpeak, containing

ap-proximately one-third of the label, eluted at a

position corresponding to a mononucleotide

witha -2charge.The secondpeak,containing two-thirds of the label,elutedataposition (-2

to -3) asexpected for a structure of the type m7G(5')ppp(5')Xm (10). The P1 alkaline phos-phatase digestion (Fig. 1D) did not, however,

affect the elution of the peak2 ofFig. 1C. We

interpretthese results to indicate that P1

nu-clease digestion released the twoterminal

nu-cleotides (Ymp, Np) of which the methylated nucleotideeluted in peak1 (Fig.1Cand D) and

that the remaining dinucleotides of the type

m7G(5')ppp(5')Xm elutedinpeak2(Fig.1C and

D). The results of these analysesareconsistent

54

40

301

210

1le

E

m 0

30

10

A X l;5 6

.

D

ilo

i20

30 40

Fraction

FIG. 1. Elution profile of HSV-l-specificRNA

di-gest on DEAE-Sephadex columns. The RNA was

labeled with [methyl-3H]methionine from 0to 14 h postinfection. (A) T2RNasedigest ofHSV-1 RNA.

(B) Rechromatography of peak1 of (A) after

desalt-ingandalkaline phosphatase digestion. (C)

Rechro-matography of peak 2 of (A) after desalting and

digestion with P1 nuclease. (D) P1 digest of (C) further digestedwith alkalinephosphatase. The

po-sitionofcharges-2to-7wasstandardized, usinga

pancreaticRNasedigest ofE. coli tRNA.

TABLE 1. Distributionofmethyl-3H-labeled nucleotidesinselected RNAsfromHSV-1-infectedcells [methyl- [methyl-3H]methionine-la-

3H]methionine-la-Labeling beledmononucleo- beled oligonucleotides Infected cell RNAdigested withT2RNase afterinfec- tidesindigest(-2 indigest(-5to-6

tion(h) charge) charge)

cpm %of total cpm % of total

Cytoplasmic HSV-1 specificRNAa 0-14 60 37 99 63

Cytoplasmic HSV-1specificRNAb 0-8 25 55 20 45

Adenylated polyribosomal HSV-1specific 11-14 2 5 48 95

Adenylated totalcytoplasmic 11-14 80 1 6,880 99

Adenylated polyribosomal 11-14 50 1 3,550 99

aHSV-1-specific RNA referstoRNAselectedbyhybridizationtoHSV-1DNAasdescribedintext. bFromcellsinfected and maintainedinthepresenceofcycloheximide.

04

a

VOL. 20, 1976

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with the hypothesis that HSV-1 RNA

accumu-lating in the cytoplasm has a modified 5'

terminus predominantly of the type

m7G(5')-ppp(5')XmpYmpNp, designated as cap 2,

al-though we cannot exclude the possibility that

modified5' terminiof thetype

m7G(5')ppp(5')-XmpNp, designated as cap 1, arealso present.

This conclusion is based on a comparison of

labelinpeaks1and2of Fig. 1C. Assumingthat each nucleotide has a single methylation, the predicted ratio oflabel in peak 1 to peak 2 of

Fig. 1C is 1:2for cap2. P1nucleasedigestionof

acap1structureshould yieldno countsinpeak

1. Since we are observing the maximum ratio we could expect for pure cap 2, the extent of contamination with cap 1 structures is prob-ablynotvery high.

Inthesecond seriesof experiments we

exam-ined RNA accumulating in the cytoplasm of

cells treated withcycloheximidefromthe time

ofinfection. As indicated in the introduction,

the RNA extracted frompolyribosomesorfrom

total cytoplasm of cells treated with

cyclohexi-mide is homologous to 10to 12% of viral DNA

(16, 21). On withdrawal of the drug, the cells

make only a polypeptides (13, 21). In these

experiments HEp-2 cells were infected with

HSV-1 (F) at amultiplicityof10PFU/cells, in

the presence ofcycloheximide (50 ,ug/ml), and

thenmaintainedinthe presence of thedrugfor

8h.The cells werethenfractionated, andRNA

wasextracted from the cytoplasm asdescribed

above. Cytoplasmic viral RNA purified by

hy-bridization toviral DNA wasdigested with T2

RNase and chromatographed as above. The methyl-3H-labeled material from

HSV-1-spe-cific RNA eluted from the columnin two peaks

(Fig. 2A and Table 1) in a pattern similar to

that ofthedigestof HSV-1 RNAlabeledfor0 to

14hpostinfection (Fig. 1A). The twopeaks with

-2 and -5 to -6 charges, respectively, were

collected and desalted for further analysis.

Peak 1 (Fig. 2A) was shown to consist of a

mononucleotide because subsequent digestion

with alkaline phosphatase converted it to a

nucleoside that did not absorb to the column

(Fig. 2B). Peak2(Fig. 2A) was digestedwithP1

nuclease and alkaline phosphatase. The

prod-ucts werethenchromatographedasabove(Fig.

2C). Approximately one-third of the methyl-3H

label activity was converted tonucleosides that

didnotabsorbtothe column (peak 2, Fig. 2C).

However, the remainingmethyl-3H-labeled

ma-terial retaineda -2 toa -3charge (peak 2, Fig.

2C), corresponding in its elution properties to a structure ofthe type m7G(5')ppp(5')Xm (10). It

appears from this analysis that HSV-1 RNA

madeinthe presence of cycloheximide contains

10-E

U,

a A

I

1 I

L ----F---

E----20

i1

i 10 20

Fraction

30 40

FIG. 2. Elution profile of HSV-1-specificRNA di-gest on DEAE-Sephadex columns. [3IH-methylimethionine from0to 8hpostinfection in the presenceofcycloheximide (50

Mig/ml)

asdescribed in the text. (A) T2 RNase digest ofHSV-1-specific RNA. (B)Rechromatography of peak 1 of (A) after desalting and alkaline phosphatase digestion. (C) Peak2of(A) rechromatographedafterdesalting and digestion with P1 and alkaline phosphatase.

both terminal and internal methylated nucleo-tides.

The objective of the third series of

experi-ments was to examine the mRNA specifying

the last or y group of viral polypeptides.

How-ever, because (i) the RNA sequences associated

with polyribosomes of infected cells making a

polypeptides are conserved and cosediment

with polyribosomes even after a polypeptide

synthesis ceases (16, 21) and (ii) current evi-dence strongly suggests that the templates for the three groups of polypeptides are intermixed

rather than clustered (P. Jones, G. S.

Hay-ward, and B. Roizman, J. Virol., in press),

itwasnot feasible to obtain purified y mRNA.

We have therefore examined only the RNA

labeled and entering into the cytoplasm late

in infection without prejudice as to whether

this RNA is capable of specifying all classes

or only y polypeptides. In designing this ex-periment we took advantage of the

observa-tionthatall or most species of viral RNA

accu-mulatinginthecytoplasm of infected cells are

adenylated and bind to poly(U)bound to

glass-fiber filters (26). Inone experiment the

(poly)A-containing RNA was hybridized to viral DNA, B

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RNA SYNTHESIS IN CELLS INFECTED WITH HSV

587

eluted,

and then

dige

described above. The el double

peak

of

radioa4

tetra- and

pentanuclec

(Table

1 and

Fig.

3A)

less than 5% ofthe to

position

ofmononucle

3A).

The

radioactivity

witha

charge

of-5to and

digested

with Pl

raphy,

this

peak elutez

charge

(Fig.

3B).

Th with a

"capped"

m7G(5')ppp(5')X'pYml

although

in this inst could also be

present.

base-methylated

nucle

entering

cytoplasm

I

questions

as to whetl fered from all other X

made atthat time. A(

the total

cytoplasmic

bound and eluted from

glass-fiber

columns.

T;

nylated

RNA

species

a

plasm

share with vir

ciency

ininternal bas

DISC] The salientfeatures

thispaperare asfollov

themRNA'sof

eukary

some viruses

infecting

RNA

accumulating

in bosomesof infectedcel

terminus. The modifi

in a

mRNA,

in

viral:

infection,

and inviral

ported

intothe

cytople

-2 -3

20]

.5

10]

Ei

A

ID

F

FIG. 3. Elutionprofile

gest on DEAE-Sephadex

labeledfrom11 to14 hp4

digest of HSV-specific RA ofmaterial eluting with

desaltinganddigestionu

linephosphatase.

,sted with T2 RNase as cause current methodology does not permit the

lutionprofile showedone recovery of adequate amounts of viral RNA, we

ctivity atthe position of have not sequenced the modified

5'-ologonu-)tides (-5 to -6 charge) cleotide. The chromatographic behavior of the

A minor peak, usually oligonucleotide and its digestion products

indi-)tal, was detected at the cates that the form m7G(5')ppp(5')XmpYmpNp,

otides (-2 charge) (Fig. commonly seen in many other mRNA species,

eluting from the column predominates on viral RNAs accumulating in

-6 waspooled, desalted, the

cytoplasm.

We do not know whether all

nuclease. On chromatog- modified termini are identical; these studies

dwith a -2and -2to -3 awaitpurificationofindividual mRNA's.

is pattern is consistent (ii) In conformity with mRNA of other

spe-5' end structure cies, the T2 RNase digest ofHSV-1 (F) RNA

pNp

as described above, yieldedbase-methylated mononucleotides.Two

Lance a cap 1 structure observations are of special interest. The first

The absence of internal relates to the fact that viral RNA sequences ,otidesinHSV-1(F)RNA

normally

made andtranslocated intocytoplasm late in infection raised immediately after infection contain internal ier HSV-1 (F) RNA dif-

base-methylated

nucleotides, whereasthe RNA

adenylated

RNA species made and translocated into the cytoplasm at ccordingly, we examined the time of peak synthesis of y polypeptides and

polyribosomal

RNA doesnotcontain

appreciable

amountsof these ipoly(U) immobilizedon nucleotides. We cannot atthis time determine able1shows that all ade- whether the absence of internal base-methyl-Lccumulatinginthecyto- atednucleotidesin RNAmade11 to 14h after

ral RNA species a defi- infection is the consequence of the absence of

;e-methylated

sequences.

signals

forbase

methylation

in

RNA,

inhibition

of the enzyme

by

a

virus-specific product,

or

USSION rapid turnover coupled with a lack of new

syn-;of the data presented in thesis of the enzyme because of viral inhibition

fs.

(i)

In conformity with of host

protein synthesis

(27, 28). The fact that

roticcells (1, 4, 20) andof all adenylated RNA, which includes both host

them (17, 24, 30), HSV and virus-specific RNA, lacked the

base-meth-the cytoplasmon polyri- ylated nucleotides suggests the absence of

ac-lls contains amodified5' tive enzyme, but this remains to be

deter-ad terminus waspresent mined. It is perhaps useful to note that among

RNAlabeled

throughout

the various possibilities left open by this

obser-RNA labeled andtrans- vation are extreme ones. The first is that the

asm lateininfection. Be- disappearance ofa host enzyme due to rapid

decay

and overallinhibition ofhostprotein syn-thesismayinfactserve a

regulatory

function in

-4 -5 -6 thatitimparts onthe RNAastructural

altera-tionwhichcouldserve todifferentiate this RNA

from otherspecies. At the otherextremeis the

possibility

that internal

base-methylated

nu-cleotides have no

regulatory

function.

Experi-mentsare inprogressto differentiate between

these

possibilities.

The secondobservation ofinterestrelates to

20

30 the fact that cytoplasmic viral RNA labeled

raction from 0 to 14 hpostinfection contained internal ofHSV-1-specificRNAdi-

methylated

nucleotides,

whereas the RNA

la-oflHSVm1nspeific

RNA

das

beledfrom 11 to 14h didnot. Wedonot know

columns. The RNA was

ostinfection.

(A) T2 RNase the

precise time

after infection when internal

iA.

(B)Rechromatography base methylation ceases; the data indicate, a -5 to -6 charge after

however,

that the RNA

containing

the internal

vith Pl nuclease and alka-

methyl-3H-label

is conserved; the data also

reinforce the conclusion reached from

experi---I'1---

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mentspublished earlier thataRNAsequences

are present in the cytoplasm of infected cells

many hours after a polypeptides cease to be

made (16, 21).

ACKNOWLEDGMENTS

Wewishtothank Aaron Shatkin and his associatesat

the Roche Institute for invaluable advice in the early

stagesof thesestudies.

These studies were aided by grant VC 103L from the American Cancer Society and Public Health Service Grants CA 08494and CA 19264 from the National Cancer Institute. M. B. isaPublic Health Servicepostdoctoral trainee (Al

00238) of the National Institute of Allergy and Infectious Diseases.

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