Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences.

0022-538X/81/050593-13$02.00/0

Identification and Mapping of Two Polypeptides Encoded

Within

the Herpes Simplex Virus Type 1

Thymidine Kinase

Gene

Sequences

CHRISM. PRESTON* AND DUNCAN J. McGEOCH MedicalResearch Council Virology

Unit,

Glasgow, Gll5JR Scotland

Received 7 November1980/Accepted 11 February 1981

mRNA's

homologous to the herpes

simplex

virus type

1 DNA restriction

endonuclease

fragment

BamHI p,

which contains the thymidine kinase gene,

have been identified

and mapped by hybrid-arrested translation and mRNA

selection. Such

mRNA's, when translated in vitro, directed the synthesis of

polypeptides of apparent molecular weights 43,000

(VI43) and 39,000

(VI39).

mRNA for

enzymatically active thymidine kinase was enriched by more than

20-fold after selection. Mapping was carried out with restriction endonuclease

fragments of BamHIp, and locations of the 5' and 3' termini of

VI43

mRNA

were

deduced.

Analysis

of nucleotide sequences around the 5' terminus revealed several

consensus

sequences

commonly found at the start of eucaryotic mRNA's and

which are

presumably involved in initiation of transcription by RNA polymerase

II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was

arrested

only by a 1,170-base-pair region of BamHI p. Since this region is

insufficient

for adjacent genes, coding sequences for VI43 and VI39 must overlap;

the

possible

relationship

of these two polypeptides is discussed. A virus-induced

product equivalent to VI39 was detected in infected

cells.

An

important

method of

mapping and

puri-fying

specific mRNA's is

to

combine

nucleic acid

hybridization with in vitro

translation.

In such

experiments,

an

mRNA

preparation is

hybrid-ized with a

purified fraction of genome DNA or

RNA.

Subsequent

analysis involves

either

recov-ery

from the

hybrids

and translation of selected

RNAs

or

determination of the

polypeptides

whose

synthesis

is inhibited

(termed

"hybrid-arrested

translation").

This

approach

has

al-lowed mRNA

mapping

in

myxoviruses

(16,

20),

papovaviruses (42),

adenoviruses

(12,

22, 23,

30,

38), and

poxviruses

(8).

The

techniques

should

be

similarly applicable

to

herpesvirus mRNA's,

since

herpes

simplex

virus

(HSV)-specific

RNA

can

be translated after

hybridization

to

viral

DNA

(14) and mRNA's have been enriched

by

hybridization

to

cellulose-bound DNA

frag-ments

(1, 2).

A

region

of the HSV type

1

(HSV-1)

genome

which

currently

commands

special

interest is

the

BamHI

restriction

enzyme

fragment

BamHI

p. This

fragment,

located at

approximately

29to

32

map units (46), contains the information for

expression of viral

thymidine

(pyrimidine

deox-yribonucleoside)

kinase

(TK)

when used to

bio-chemically

transform

TK-

cells

to a

TK+

phe-notype

(4, 7, 28, 29, 32, 37, 45,

47).

Furthermore,

cells transformed with BamHI p increase HSV

TK

lvels after

superinfection with TK- virus

mutants

(17, 21, 47),

showing that the

fragment

also contains sequences involved in activation

by

viral

polypeptides

(24, 35, 36).

Hybrid

plas-mids

containing

BamHI p have

recently

been

constructed, thereby

providing

a source

of

large

amounts

of pure BamHI p

DNA,

and

detailed

restriction

maps have been

produced

(7, 10, 47).

The limits

of sequences necessary for biological

activity have been defined by the demonstration

that

cleavage

with

EcoRI, BglII

and

SstI abolish

transformation,

whereas

a

2-kilobase

pair

(kbp)

Pvull fragment

retains full

activity

(7, 47).

Anal-ysis of

intertypic

recombinants has located TK

coding

regions

to 0.300 to 0.309

genome

map

units

(13), and the direction of

transcription

of

TK

mRNA has also been determined

(41).

With this

background,

studies

to

investigate

the

arrangement of mRNA

coding

regions within

BamHI p

were

initiated.

Apart

from its

biologi-cal

relevance,

this

fragment appeared

tobe

suit-able

for such an

approach

because

enzymatically

active

TK can be

synthesized

in vitro

(9, 34)

and

the

putative

TK

polypeptide, VI43,

is

the

most

abundant

product

upon translation

of RNA

ex-tracted from cells at

early

times of infection

(33,

35).

We have been

able

to

identify

and

substan-tially

purify

mRNA's

homologous

to

BamHI

p,

to

map within fine limits the

coding

sequences

593

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of

TK

mRNA,

andtoobtain nucleotidesequence

data for the controlregionsaroundthemRNA 5' terminus. The existence oftwo

polypeptides,

encoded

by overlapping

sequences within the TK gene, has also been established.

MATERIALS AND METHODS RNA isolation.

garly

RNA was extracted from BHK C13 cellsat 5hafter infectionat31°C with 20 PFU of HSV-1 strain17per cell(33).Late RNAwas

obtained from BS-C-1 cellsat 6h afterinfection at

370C.

Plasmid DNA. A stock of Escherichia coli HB101 containingplasmid pTK1 (HSV-1

BamHIp

fragment

inserted into pAT153[44])wasinitiallyprovidedby

P. Sanders. PlasmidpTK6wasobtainedbycleavage

ofpTK1withBgllI, followed bypartial digestionwith BamHI. Theproduct, consistingofpAT153plusthe smallBgJI-BamHIfragment (see Fig. 10),was reli-gated and transfected into E. coliHB101.The struc-tureof theplasmidin theresulting ampicillin-resistant

colonieswasconfirmedbyrestriction enzymeanalysis,

andonecolonywaspurified and usedasthesourceof pTK6 DNA.

Restriction endonucleasemappingofpTK1.A number of approaches, all based on recleavage of known DNAfragments,wereused torefine existing

mapsofpTK1.(SeeFig. 4, 7, and10for therestriction enzymesites usedtogeneratethe DNAfragmentsfor this study.) Fragments are described here by their approximate coordinates, inkilobasepairs, from the BamHI site upstream from TK mRNA (41). The correctedpositions of AluI sitesdefiningfragmentsA, E, and F differ from previousresults (47).

Isolation of DNA from polyacrylamide gels. Gels ofvarious polyacrylamide concentrations (5 to

10%) were cast and run in 50 mMTris-borate (pH 8.3)-1.25 mM EDTA. After staining with ethidium bromide and visualization underlong-waveUVlight,

therelevant bandswerecut outandhomogenized in

10ml of TE buffer (20 mM Tris-hydrochloride [pH 7.5]and2mMEDTA). The macerated gelfragments

weregently mixed for10 to16 hat4°C,polyacrylamide

wasremoved bycentrifugation, and thesupernatant

was passed through a glass-fiber filter disk. Sodium phosphate buffer (pH 6.8) wasadded to 0.1 M and DNA adsorbed to hydroxylapatite. After thorough washingat60°C with0.2Msodiumphosphate buffer, DNAwaseluted with 0.5 M sodium phosphate buffer.

E. coli rRNA(20,ug)wasadded, and the eluate was dialyzed against TNE buffer (TE buffer plus 0.1 M

NaCI)andprecipitatedinethanol.

Portions of isolatedDNAfragments were electro-phoresed, and negatives of theUV-illuminated,

ethid-iumbromide-stained bands were prepared. The nega-tives werescanned, using aJoyce-Loebl

microdensi-tometer, to assess the purity and concentrations of

DNA fragments. None ofthefragments used in the experiments reported here contained detectable con-taminationby others.

DNA sequencing. DNA sequences around the 5' terminus of the TK genewere determined by using thechemical degradation system of Maxam and Gil-bert(26, 27), and also by cloning fragments into phage

M13derivedvectors(11)andcopying the recombinant

single-stranded phageDNA in the presence ofspecific

chain terminators(39, 40;F.Sanger,A. R.Coulson, B. G.Barrell,A. J. H.Smith,and B. A.Roe, J. Mol.Biol.,

inpress).

For the chemicaldegradationsystem,pTK1 DNA

wascleaved with EcoRIorwithBglll,labeledatthe 5'termini withpolynucleotidekinase and

[y-32P]ATP,

andthen cleaved withHinfl. Theresulting single end-labeledfragments wereseparatedbyelectrophoresis

in polyacrylamide gels, and the appropriate bands

were recovered. All sequencing gels contained 9 M

urea.

EcoRI-cleaved fragments of pTK1 DNA were

cloned into the EcoRI site of the phage vector M13mp2am and propagated in E. coli JM101 (11).

Sequencing was carried out as described by Sangeret

al. (J. Mol. Biol., in press) with the short universal

primerofAndersonetal.(3).

Hybridarrest.Plasmid DNAs were cleaved with EcoRI, extracted with phenol, andprecipitated with ethanol. Twomicrograms of restricted plasmidDNA,

orthe molarequivalent amount of a particular isolated

fragment, wasdenatured in 85

pl

of 95%formamide (deionized) containing 60 mM PIPES [piperazine-N,

N-bis(2-ethanesulfonic acid)] (pH 7.4) at 95°C for 5

min. HSV-1-infected cell cytoplasmic RNA (6

pl;

8

mg/ml)wasadded, followed by addition of 8

p1

of 5 M

NaCl,and thehybridization mixture wasrapidly trans-ferredtoa58°Cbath. Afterincubation for 1 h, 1 ml of HSB (500 mM KCl; 20 mM Tris-hydrochloride [pH 7.5]; 1 mM EDTA) was added. Polyadenylic acid-containing RNAwasselectedon asmall oligodeoxy-thymidylic acid-cellulose column and precipitated withethanol afteraddition of E. coli rRNA.Pelleted RNAwasdried, dissolved in deionized water, and used for in vitrotranslation either directly or after denatur-ationby heatingat95°C for 2min.

mRNAselection.Thetechnique of Ricciardi et al. (38) was modified for selection of pTK1-specific mRNA's. A nitrocellulose filter (13-mm diameter; Schleicher and Schuell, Dassel, West Germany), loaded with 100ytgofplasmidDNA,wascutinto small pieces and incubated for2hat550C withearly or late cytoplasmic RNA, dissolved in hybridization buffer (40%o formamide [deionized], 600mM NaCl, 50 mM PIPES [pH 7.4], and1mMEDTA). The hybridization mixturewasthen removed andprecipitated after ad-dition of 1 volume of water and 2.5volumes of ethanol

toyield thenonhybridized fraction. The filter pieces were incubated at550C for 10 min inhybridization buffer, then washed 10timesat60°C with SSC (0.15 M NaCl and0.015Msodiumcitrate) containing 0.5% sodiumdodecylsulfate, followed by2washes at600C withSSC containing2mMEDTA. The pieces were

then washed for two 5-min periods in SSC-2 mM EDTA andoncewith colddeionized water.A

100-pl

amount of 95%formamide-50 mM PIPES (pH 7.4)

wasadded,andincubationwascontinued at 60°C for

5 min. The formamide fraction was stored, and the filter pieces were incubated with 300 ,l of deionized waterfor2min at95°C. This fraction wascombined withthe 95% formamide fraction, E. colirRNA (15

,ug)

and NaCl (0.2 M) were added, and RNA was

precipitated after addition of2.5 volumes of ethanol

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(selectedRNA fraction). The RNA was washed with ethanol,dried, dissolved in water, and used for in vitro translation. In someexperiments, the selected RNA fraction was repurified on a small oligodeoxythymi-dylicacid-cellulosecolumn, as describedabove.

In vitro protein synthesis. RNA samples were translated in a micrococcal nuclease-treated fraction-atedreticulocyte system (31, 33, 35). In vitro-synthe-sized TK enzyme wasassayed as described previously

(34).

Labeling of infected cell polypeptides. BHK

celiswere pulse-labeled at 37°C for 15 min in phos-phate-buffered saline containing 500uCiof [3S]meth-ionine (The Radiochemical Centre, Amersham, Eng-land) per ml.Cell monolayers were then washed with ice-cold phosphate-bufferedsaline,harvested, and pre-pared for analysis by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (25).

Polypeptide nomenclature. As described previ-ously (25, 33) polypeptides synthesized in vitro and in vivo areclassified according to their apparent weights in thousands and areprefixed with VI or Vmw, re-spectively.

Biologicalcontainment. Allmanipulations with bacteriacontaining plasmid pTK1 or pTK6 or phage M13mp2am were conducted in a category II labora-tory, asrecommendedby GMAG.

RESULTS

Selection of mRNA's which

hybridize

to

pTKI

DNA. mRNA's

were

selected

by

incuba-tionof late RNA from infected BS-C-1 cellswith

nitrocellulose

filters

loaded with

plasmid DNA.

After the filters were thoroughly washed, hy-bridized RNA was eluted and translated. Fig. 1 shows that thisprocedure selected mRNA's

cod-ing for

a

major (VI43) and minor

(VI39) species

from

the array of late virus-induced

polypep-tides.

These mRNA's were not selected from

infected cell RNA

by pAT153

DNA filters or

from uninfected

cell RNA

by pTK1 DNA,

in

which

cases

only endogenous

reticulocyte bands

were

observed. The

specificity of the

procedure

was

further demonstrated

by

thefacts that V143

was not

the

most

abundantly represented

spe-cies in

translation

products.

of the

original

RNA

and VI39

was

undetetable before

mRNA

selec-tion.

To

investigate whether mRNA for active TK

enzyme was

also

enriched

by

hybridization

to

pTK1

DNA, RNA from BHK

cellsat5

h after

infection

at

31°C,

atime when TK mRNA levels

are

maximal,

wasused. Selected and

nonhybri-dized

RNAs were

examined for

the

ability

to

stimulate

incorporation

of

14C-amino

acid or to

direct

synthesis

of TK.

Fig.

2

(A

and

B)

shows

that

selected

RNAwasless active in

stimulating

protein

synthesis,

but had

marginally

greater

TK mRNA content.The measured

enrichment

of TK mRNA

(TK

activity

per unit of amino acid

incorporation

of selected

RNA

compared

_ _

a _

0 0

z s

F-C')

i

'U

- 'U

A A K. a 7

4c

z

FIG. 1. Selection of pTKI-specific mRNA.

Mock-infected (MI)orlateinfected(INF) BS-C-I cellRNA

washybridized tofilters containingpTKI DNA or

pAT153 DNA. Both nonselected and selected mRNA preparations were translated in vitro. RNA prepa-rationsused were the following: INFcellRNA not selected by pAT153 DNA (track 1), INFcellRNAnot

selectedbypTKIDNA(track 2),originalMIBS-C-I cellRNA (track 3), originalINFcellRNA(track4), INFcellRNA selected(SEL) bypTKI DNA (track 5),INF

cell

RNA selectedby pA

Ti

53DNA(track 6), MI cellRNA selectedbypTKIDNA(track7), andno

added RNA (track 8). Forreference, the molecular weights (in thousands) ofsomeprominent virus-in-ducedpolypeptidespresentareshown in track4(<J).

Selectedpolypeptides arelabeled in track 5

(4).

A

more thorough categorization oflate virus-induced

polypeptides synthesizedin vitro isgivenelsewhere

(32,34).

with

nonhybridized RNA) varied from

20- to

50-fold in

repeated experiments.

An accuratevalue

is

difficult

toobtain because the

stimulation

of

"C-amino

acid

incorporation by

selected RNA was so

small.

Translation of relevant RNA fractions

(Fig.

2C) revealed

that the

polypeptides

directed

by

selected mRNA were

VI43

and

VI39,

as found with late RNA

(Fig. 1).

Thegreater abundance

of

VI43 and

VI39

mRNA was

reflected

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[image:3.500.256.444.63.385.2]

596

PRESTON AND

B

ProteinSynthesis

0 1 2 3 4

C

IL

. 2 A I

RNA(pl)

FIG. 2. SelectionofTKmRNA. Earlyinfected (INF)BHKcellRNAwashybridizedtofilters containing pTK1 DNA. The original (0), nonselected (U), and selected (0) fractions were translated in vitro, and

productswereanalyzedeitherbyassayofTKenzyme(A)orincorporation of

["4Clamino

acidmixture into

protein (B).All RNApreparationswereat 1.2mg/ml,thisbeingachievedbyadditionofE. colirRNA to the selected RNApreparation. (C) Autoradiogram of ['5S]methionine-labeled polypeptidesdirectedbythe three

RNApreparations, togetherwithmock-infected (MI)BHK cell RNA andnoadded RNA.

greateryieldofselectedmRNA,asindicatedby thehigher intensities of these polypeptides, as compared with the majorendogenousband (mo-lecularweight,ca.50,000).

The selection experiments, therefore, show that themajor mRNA encoded by BamHIpat

both early and late times of infection specifies VI43. Another polypeptide,

VI39,

was also

syn-thesized, and TK mRNAwasenriched, strongly suggesting that TK activity resides in either VI43orVI39.

Hybrid-arrested translation with pTK1 DNA.AnalternativewayofmappingmRNA's

is by hybrid-arrested translation (16, 30). This methodcanbe used withsmallDNAfragments and might give more precise locations than mRNA selection, as it has been reported that DNAfragments shorter than300bp donotbind reproduciblytonitrocellulose filters (38). Figure

3 (left) shows translation of early RNA after incubation with pTK1 or pAT153 DNA under conditions which favor annealing of RNA to

DNA. With both plasmids, three polypeptide bands

(VI136, VI129,

and

VI88)

disappeared afterhybridization, andone(VI85) was synthe-sized in reduced amounts. These returned, to

someextent,after denaturation ofhybrids, but clearly their arrest cannot be attributed to

BamHIp sequences. The effect is presumably due tosomeunusual structural featuresofthe mRNA's, sinceitcanalsobeobtainedby mock-hybridization without addition of DNA (data

notshown).

Hybridization with pTK1 DNA,ascompared with that of pAT153 DNA, additionally pre-ventedsynthesis of VI43 and

VI39.

mRNA's for these polypeptides regained activity upon

de-naturation ofhybrids, thusdemonstrating that theyareencodedby BamHIp.Figure3 (right) shows that thetwo arrested bands correspond

toselected VI43 and VI39. It shouldbe noted, however, that VI39 comigrates withaminorcell

polypeptide, and that RNA preparations

con-taining high levels of its mRNA (i.e., early RNA)

must be used for efficientdetection in

hybrid-arrestexperiments.

Analysis ofsequences atthe 5' terminus ofTKmRNA. Initialexperimentswere aimed

atlocating those BamHIp sequenceswhichare represented in the mRNA's for VI43 and VI39. FragmentsofpTK1 generated by SmaIorAluI

were used in hybrid-arrested translation, and

A

TK

0 X

E

0. u

4

0 x

E

0. u

2

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[image:4.500.62.456.64.336.2]

z pAT pTK I

sr- IL

I Z H D H D

I 2 a A A a 7

43

39

Al

z MA

0) H D

.3

[image:5.500.111.398.76.364.2]

39

FIG. 3. Hybridarrestoftranslation by pTKI DNA. Earlyinfected(INF)BHKceURNA washybridized to

pATl53DNAorpTKIDNA anddirectly translated (tracks labeled H) or denatured before translation (tracks labeledD). RNA preparation usedwereno addedRNA (tracks I and 8), mock-infectedceURNA (track 2), early INFcellRNA (track 3), hybridized to pAT153 DNA (track 4), track 4 sampledenatured (track 5), hybridizedtopTK1 DNA (track 6), track 6 sample denatured (track 7). From a separate experiment, the productsofpTK1-selected RNA (track 9) werecompared (tracks 8 through 11) with those ofpTKI-arrested RNA(track10).(Track11showstrack 10sampledenatured.)Symbols: , known virus-inducedpolypeptides; (0) polypeptideswhosesynthesis is arrested byincubationwitheither pATl53 orpTK1 DNA.

these

experiments

showed that such

sequences

lay within the 2.02-kbp PvuII fragment

(0.50 to

2.52

kbp from the 5' BamHI site) previously

reported

to

possess

full

transforming activity

(7)

(data

not

shown).

Further definition

was

achieved

by

use

of small DNA

fragments

gen-erated

by double

digests

or

by recleavage of

larger

fragments followed by

a

second

cycle of

electrophoresis and purification. Figure

4

shows

hybrid-arrested translation in the region which

should contain the mRNA

5'

terminus and

un-translated

sequences

of the

TK gene

(42).

All

five BstNI

fragments,

derived

by

cleavage

of the

pTK1

BgII-SstI fragment (0.74

to

1.24),

ar-rested

translation

of

V143

and

VI39.

Fragments

C

(-0.38

to

0.61)

and

D

(0.61

to

0.74)

from the

EcoRI-BglII

double

digest failed

toarrest trans-lation

under standard

conditions,

whereas

frag-mentB

(0.74

to

3.0)

of thesame

digest

was

fully

active.This

experiment, therefore,

shows thata

fragment

as

small

as78

bp (the

estimated size of

BglII-SstI-BstNI fragment

E)

can

efficiently

ar-rest

translation.

Inspection of

Fig.

4,

tracks

7

through

16, shows

a

minor

polypeptide

of

an apparent

molecular

weight of 60,000 whose

synthesis

was

arrested

by

all

BglII-SstI-BstNI fragments.

This

species

has

not

been

resolved

so

clearly

in any

other

autoradiograms, and it

is

therefore

not

possible

to

determine whether the effect

is

due

to

BamHI

p

DNA

orto

the

self-arrest described above.

Nucleotide

sequences

of the

potentially

inter-esting

regions around the

BglII

site

were

deter-minedby the Maxam-Gilbert method (26) and

by

the use of

dideoxytriphosphates

in

copying

single-stranded fragments

cloned in

phage

M13mp2.

The

440-bp

region depicted

in

Fig.

5

combines

data from

fragments end-labeled

at

the

EcoRI

site

orthe

BglII

site and from

EcoRI

fragments

cloned

in

M13mp2.

Five

ATG

codons,

potential protein

synthesis

initiators,

can

be detected. The first

four of

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598 PRESTON AND McGEOCH

- C

BamHl

I

Pvull

0.5

EcoRI Bgl11

I D I B-.

Sstl

I BstNI sites C I E

I

D I

A/BI

A/B

1.0

EcoRlIBgI11

B C D

D H D H D

Bgl11ISstIl/ BstN I

E D C

D H D H D H

7 a a 44U11 12

B A U._{z i c}Z

D H D H - a

13 14 15 16 17 18 19

4

3

FIG. 4. Hybrid-arrested translation with TK mRNA 5'sequences. Fragments

of pTK1

from

an

EcoRI-BglIIdoubledigest(tracks1 through 6)or aBstNIdigest of theBglII-SstI fragment(tracks7through16)

wereused. RNAwastranslateddirectly(H)orafterdenaturation(D).Theorigins ofthefragmentsareshown

inthemap(BglII-SstI-BstNI fragmentsAand B havenotbeenordered). Track17,translationproducts of

the original RNA; track 18, BHKcell RNA; and track 19, endogenousproducts of the cell-free system.

Symbols:0,anadditionalpolypeptide (60) which is arrestedbyallBglII-SstI-BstNIfragments.

these, all upstreamfrom the BglII site, are in

closed reading frames since protein synthesis

termination codons follow closely in the same

phase, whereas the fifth, located 57-bp

down-streamfromtheBglII site, isin anopenframe

asfarastheanalysiscandetect.Provided

splic-ing doesnotoccurinthe region analyzed,this is the only candidate site for protein synthesis

initiation.

Upstream from the BgIII site, various

se-quences previously recognized in the initiation regions ofmanyeucaryoticgenes canbe

identi-fied (5, 6). Moststriking is the AT-richregion, whichis verysimilar toa Hogness box

(GTA-TAATAG), atpositions 197 through207. This

sequence is usually approximately 30 bp

up-stream from an mRNA 5' terminus, and the

sequence CGAATAC (224 through 230) in the

appropriate position resembles the consensus

sequences found at the mRNA 5' capping site (6). At5and12bpupstreamfromthe Hogness box are two GGTCCsequence, whichare very

similar totheGATCCconsensus (6). Twoother regions further upstream from the mRNA

ter-minusareatpositions 144through152 (the-70 to -80homology) and at positions 83 through 92,anAT-richsequencecommonly found130to

150bpupstreamfromthemRNA terminus(5). Consideration of theorganization ofsequences

therefore suggests that an mRNA is initiated

approximately 50 bp upstream from the BglII

site,aconclusionwhich iscompatible with

ini-tiation ofprotein synthesis 57 bp downstream

from theBglIIsite.

H 1

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[image:6.500.63.458.76.427.2]

SEQUENCES AT THE 5' TERMINUS OF THE HSV-1 TK GENE

PstI PvulI

5'GACTGC____AGCTTCAGGGA_TGGCG_

CTTCATC_CGTGGCCCGTTGCTCGCGV_TGCTGGCGGTGTCCrCGGGAA__TAAT_TGCQI5TCT

20 40 60 80 100

EcoRI Sau96I

TTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCG GAACACGCAGATGCAG

GGGGCGGCGGC

dCIACTTCGCA

120 146 GGYCAATCT 160 GATCC GATCC GTA 200

PstI Bgl II

TATTAAGfiTGACGCGTGTGG,CCTCGAATACCGAGCGACCC

eCGACCCGCTTAACAG,GTCAACAGCGTGCCGCAbA,TCTTGGTGGCGTGAAACTCC

TAATAG 220 YCATTCR MRNA 260

280

300

BstNI

CGCACCTCTTCGGCCAGCGiC,CTTGTAGAAGCGCGTATGGC,TTCGTACCCCTGCCATCAM0ACGCGTCTGCGTTCGAC

CTGCGCGTTCTCGCGGCCA

320 - 340 360 380 400

TAACAACCGACGTACGGCGT,1CGCCCTCG~CCGG4RCAA, 3'

420

440

FIG. 5. Sequenceof440bpatthe 5'terminus of the TK gene. Previously recognized consensus sequences

areshownatappropriatepositions, apartfromthe-140AT-richregion, which has a broken underline. The proposed initiation sitefor protein synthesis has a solid double underline; other ATG codons (all in a different, closedreadingframe)have broken double underlines. Restriction sites are given solid underlines, andtwohigh GC regions which may be important in interpreting hybrid-arrested translation are boxed. Y, Pyrimidine;R, purine.

In

view of the

implications from

study

of

the

TK

gene 5'sequences, the

effects

of the region upstream

from the

BglII site

onmRNA selection

and

hybrid-arrested translation

wasinvestigated

further, since

no arrest

could be obtained by

EcoRI-BglII fragments from this region (Fig.

4).

To

facilitate these

experiments, plasmid pTK6,

which contains

only the small

BamHI-BglII

fragment of pTK1 (0.0

to

0.74),

was

constructed

(see

Fig. 10). This

plasmid

DNA was

able

to

select mRNA for VI43 and VI39

apparently

as

efficiently

as

pTK1 DNA (Fig. 6, tracks

1

through 6), showing that

sequences

of

these

mRNA's extend

upstream

from the

BglII

site.

When

hybrid-arrested

translation

was

investi-gated further,

it was

found that reduction in

hybridization

temperature to

540C

and

a

fivefold

increase in

equivalent

DNA

concentration

al-lowed

detectable

arrest

of

VI43 and VI39

(Fig.

6,

tracks

7

through 14).

Presumably

the

require-ment

for lower

temperature

reflects

the

lower

GC

content

of the

region

immediately

upstream from

the

BglII

site.

A

further

interesting

point

is that

translation

was

arrested

more

efficiently

when

Sau96I-cleaved

pTK6 DNA,

as

opposed

toEcoRI-cleaved DNA,wasused. Theenzyme

Sau96I cleaves

adjacent

toavery

high

GC

region

that is17 to 34

bp

downstream from the EcoRI site. This

region

should reassociate

strongly

un-der the

hybridization

conditions,

and may

re-duce the

availability

of downstream sequences

for

hybridization

with mRNA.

The

conclusion

from these

experiments

isthat

V143

andVI39 mRNA sequences

extending

from

the

BglII

site

can

be detected

by

RNA

selection

and

hybrid-arrested translation under

appropri-ate

conditions. The altered characteristics of

hybrid-arrested translation

suggest

that the

mRNA

sequences

do

not

extend

entirely

through the

EcoRI-BglII

fragment

D

(0.61

to

0.74), since

BglII-SstI-BstNI fragment

E,

a

frag-ment

with

similar

overall

nucleotide

composi-tion

including

a

region of

very

high GC, inhibited

translation under standard conditions

(Fig. 4).

It

is

most

likely

that the mRNA

5'

terminus

is 20

to30

bp downstream from

the

Hogness

box and

that the

protein

synthesis initiation codon

is 57

bp downstream from the

BglII

site.

However,

from the results

presented here,

the

possibility

that

sequences between

the PvuII site

and the

very

high GC

region (0.50

to

0.63)

specify

a

short

5'

leader, which

is

spliced

onto

5'

noncoding

sequences, cannot

be

excluded.

Sequences

atTK

mRNA 3'

terminus.

Hy-brid-arrested translation with

fragments

derived

from

the

3'

end

of the TK gene

is

shown in

Fig.

7.

Three

fragments within SmaI

fragment

A

(0.25

to

1.91) inhibited

synthesis

of

V143 and

VI39.

Additionally,

truncated

polypeptides,

pre-sumably

resulting

from ribosome terminationat

the

duplexed site,

could be

detected,

and these

are

labeled

in tracks

2,

4 and 6 of

Fig.

7. The

intensities of these

bands,

as

compared

tothose ofVI43 after

denaturation,

werelower than

ex-pected

and

insufficient

for confident

alignment

of

restriction

sites with

coding

sequences. The

16,000-molecular-weight

species

formedanovel

band,

but the

29,000-

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[image:7.500.48.451.69.284.2]

600

PRESTON AND McGEOCH

Sau961

58 .54

pTKIpTK6 pAT -RNA

EcoRI

58 54

H D H D H D H D

2 3 4 5 6

3

FIG. 6. RNA selectionandhybrid-arrestedtranslation withpTK6DNA. Shownaretranslationproducts ofmRNA'sselectedby pTK1, pTK6, andpAT153DNAfilters (tracks 3, 4,and5, respectively); products of mock-infected (MI)BHKcellRNA(track1),infected (INF)cellRNA(track 2), endogenous proteins (track 6);

andhybrid-arrestedtranslationwithpTK6 DNA,cleaved with Sau96I(tracks7through 10)orEcoRI(tracks

11through 14),at54°Cor58°C.RNAwastranslateddirectly (H)orafterdenaturation (D).

weight bandswere onlydetectable as enhance-ments ofotherpolypeptides. Aproductof mo-lecularweight 35,000has beenproduced by

ar-restofpTK1-selectedmRNA with

SmaI-Hinfl-AvaI fragmentA(1.68to1.91) (datanotshown), and thereforeappearstobeagenuinetruncated

polypeptide.

The SmaI-AluI-DdeI fragment B (2.31 to

2.55) didnot arresttranslationof V143 and

VI39,

but fragmet A from this digest (1.91 to 2.31) partially inhibited synthesis of both polypep-tides(compare theintensities of actin andVI43 inFig. 7, track 8). This curious effect could bea real property ofsequences in SmaI-AluI-DdeI

fragment A, orcould be due to contamination by degradation products of SmaI fragment A

(0.25to1.91) during electrophoresis. Hybrid-ar-rested translationwastherefore carriedoutwith increasing concentrations ofa

BglII-SstI

frag-ment (0.74 to 1.24), SmaI fragment B (1.91 to

2.69) isolated from aSmaI digest of pTK1, or SmaIfragment B, isolated froma SmaI-BglII-SstI-HinfIdigestofpTK1, which reduced SmaI fragment A to fragments smaller than SmaI fragment B and should eliminate any possible

contamination. Figure 8 shows that maximum

inhibition ofVI43 synthesiswasachieved with

approximately 1 ug equivalent (i.e., derived from 1

,tg

ofpTK1 DNA) of each fragment, and that increasing the concentration ofSmaI fragment B afurthersixfold didnotgiveagreatereffect. Similar results were obtained with whichever digest wasused togenerate SmaIfragment B, and, therefore, the partialarrestisa character-istic of thisfragment.

The size of the35,000-molecular-weight trun-cated polypeptide produced by arrest with SmaI-Hinfl-AvaIfragment Asuggests thatthe codingsequencesofVI43 endatapproximately 230bp downstream, i.e.,veryclosetotheSmaI site, provided thereare nointrons in this region. Partial arrest with SmaI fragment B might therefore resultfrom hybridization with the 3' untranslatedregionorfromanoverlap into cod-ingsequencesproducing, atreduced efficiency, atruncatedpolypeptide whose reduced molec-ularweightcould notbedetected. Since it has been shown thathybridization of plasmid DNA

to the entire 3' noncoding region of 8-globin mRNA doesnotaffect translation in vitro (19),

U. z

1

1

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[image:8.500.111.401.73.354.2]

Sat

AIl

HinfI Aval

1 1

Alul

E I

Smai

A I

1.5

Ddel

A I

2.0

Pvull

Alul

8

I

Smai

2-5

Alul SmaiHinf I/AvaI

E a A

D H D H D H

1 2 3 4 5 6

SmaUAlul/DdeI

A B M

D H D H Z

[image:9.500.109.399.71.474.2]

7 8 9 10 11

FIG. 7. Hybrid-arrested translation with 3' fragments of TKgenesequences.Five fragmentsofpTKI DNA,

indicated in the map, were used in hybrid-arrested translation (tracks 1 through 10). RNA was either

translateddirectly(H)orafter denaturation(D). Shownaretranslationproducts of infected (INF)cellRNA

(track 11), mock-infected (MI) BHK ceU RNA (track 12) and endogenous proteins (track 13). The shorter

exposureonthelowerpaneldemonstratespartialinhibitionof VI43synthesis by SmaI-AluI-DdeI fragment

Amoreclearly(track 8). Symbols:<,putativetruncated

polypeptides.

the latter interpretation appears most likely. The 3' limit ofcoding sequences for VI43 and

V139 thereforeprobablylieswithin SmaI-AluI-DdeIfragmentA,nomorethan 30bp from the SmaI site.

Hybrid-arrestofTKsynthesis.Thecoding

sequencesforTKenzymewerealsoanalyzed by hybrid-arrested translation, and Table 1 shows thatfragmentswhich inhibitedsynthesisof TK

mapped in thesameregionasVI43andVI39.It

isinterestingtonotethat theputativetruncated

polypeptide generated by SmaI fragment B

re-tained TKactivity.

Synthesisinvivoofapolypeptide

equiv-alentto V139. CodingsequencesforVI43 and

V139 lie withinthe same 1,170 bp between the

BgM

(0.74) andSmaI (1.91) sites and presum-ablyhave thesamepolarity,sincethey behave inthesameaberrantwaytohybridarrestwith fragmentsfrom the mRNA termini. Sincethere

is insufficientcoding capacity for twoadjacent polypeptidesof thesesizes,theirsequencesmust

overlap. It wasconsidered importantto deter-mine whether both polypeptides were

synthe-_z

12 13

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602

PRESTON AND McGEOCH

o100

(A

~80

c60

d>

40*

&20-0 2 4 6

DNA(pg

equiv)

FIG. 8. Hybrid-arrestofVI43 with SmaIfragment

B and BglII-SstIDNA. Various concentrations of

[image:10.500.89.221.61.257.2]

fragments ofBglII-SstI (a), SmaIfragmentB iso-latedfrom aSmaIdigest (0), orSmaIfragmentB isolated from a SmaI-BglII-SstI-Hinfl digest (0) were used.Autoradiograms werescanned, andthe radioactivity in VI43wascompared with neighbor-ing,unaffected VI37.5.

TABLE 1. Hybrid-arrestofTKsynthesizedin vitro

[3H]thymidine phosphorylated DNAfragment used in Location (cpmx103)

hybrid-arrest (kbp)'

Hybrid- Dena-ized tured

pTK1 0.0 72.4

pAT153 78.9 59.4

SmaI-B 1.91-2.69 15.3 83.8

Hinfl-SmaI 1.50-1.91 1.1 91.9

AluI-E 1.24-1.58 1.0 52.3

BglII-SstI-BstNI-A| 0.0 68.2

BgIII-SstI-BstNI-B 1.01-1.24 0.0 67.6

BglII-SstI-BstNI-D 0.92-1.01 0.5 58.0

BglII-SstI-BstNI-C 0.82-0.92 0.0 66.4

BglII-SstI-BstNI-E 0.74-0.82 0.0 48.8

pTK6,54oCb 0.0-0.74 5.0 72.6

pTK6,58oCb 0.0-0.74 53.6 72.4

EcoRI-BglII-C -0.38-0.61 52.0 47.2 'Location offragmentsisgiveninkbp downstream from the BamHIsite.

bHybrid-arrestwithpTK6 used Sau96I-cleaved DNAat

fivefold higherconcentration at the temperatureindicated.

sized ininfected

cells

as

well

asin vitro. Figure 9 shows

pulse-labeled polypeptides

from BHK

cells

infected with

wild-type

HSV-1 or the

TK-mutant

dPyKV-7,

which fails to

synthesize

V143

(33).

Comparison

of tracks4, 5, 6, and 7 of Fig.

9

reveals

that a band

comigrating

with VI39and

of

approximately

thesamerelative

intensity

was

detected in

wild-type

HSV-1-infected

cells,

but notinmock-infected or

dPyKV-'-infected

cells.

The

probable

reason

why

the V139

equivalent

has not

previously

been

detected

in

infected cells

is

that the

intense band below it

is

rapidly

proc-essed

to a

slower

migrating form which obscures

it. A short

labeling

period (15

min was

used

to

obtain the results shown

in

Fig. 9) is

necessary

toprevent this masking effect.

DISCUSSION

Many

of the

techniques described here have

not

previously been applied

to

herpesviruses,

and it

is

therefore worthwhile

to

discuss them

with

reference

to

possible problems

arising from

the

high

GC

content

of HSV

DNA.

mRNA

selection

on

nitrocellulose

filters was

very

specific,

as

shown

in

Fig.

1

through

3.It was

possible

to

enrich BamHI

p-specific

RNAs with

no

detectable

contamination

from other

mRNA's. The

efficiency of hybridization

to

fil-ter-bound DNA is

relatively low,

with

only

5to

IN VITRO I IN VIVO

-RNA Ml SEL INF WT MI -7 WT -7

1 2 3 4 5 a 7 a a

FIG. 9. SynthesisofVI43 and VI39 invitro and in

vivo.Shownareendogenous products ofthe transla-tionsystem(track 1),polypeptidesdirected by

mock-infected (MI) BHK cellRNA, pTK1-selected (SEL) RNA, andinfected (INF) BHK cell RNA (tracks 2

through 4, respectively), polypeptides synthesizedin

BHK cellsat 5hafter infection at31°Cwith

wild-type(WT) HSV-1ordPyKj7(tracks 5, 7, 8, and 9),or MIBHKcells(track 6).

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10%

of the original mRNA activity recovered,

and high RNA concentrations

are

therefore

re-quired. The high GC

content

does

not

seriously

affect

hybridization of RNA

to

denatured,

im-mobilized pTK1 DNA, and

the

finding

that

pTK6 DNA

selects mRNA shows that only

a

small region of sequence homology

is

necessary.

The

washing

procedure described for adenovirus

mRNA's (39)

removes

nonspecifically bound

HSV mRNA's.

Hybrid-arrested

translation is

a

potentially

more

sensitive method of mRNA

mapping and

the

studies

presented

here have used the

small-est

fragments

yet

reported.

Since

both

DNA-DNA and DNA-DNA-RNA interactions

can occur,

temperature

control

to

favor DNA-RNA

reac-tions

mustcompensate

for

GC

content

(15, 43).

This

results

in

the

use

of relatively high

hybrid-ization temperatures for HSV mRNA's.

Hybrid-arrested translation could

not

detect

mRNA's

for minor

polypeptides

which

comigrate

with

major cellular

or

viral

bands,

but otherwise the

specificity

seems

equal

to

that of mRNA

selec-tion.

Some

mRNA's, however,

cannot

be

mapped

since

they

are

inactivated

by

mock-hy-bridization, emphasizing

the

importance of

in-cluding

appropriate

controls. DNA

fragments

as

small

as78

bp

efficiently

arrest

translation,

pro-vided they

hybridize entirely

to

mRNA

coding

regions. When the

area

of

overlap

includes

mRNA

ends, the results become

more

difficult

to

interpret and further

investigation

is

neces-sary.

Production of truncated

polypeptides

was

less

efficient than

recently

described for

influ-enza

mRNA's in the wheat germ

system

(20).

Possibly, ribosome release

at

the

abnormal site

is

inefficient, and the

resulting decreased

syn-thesis of

truncated

products

is

more

marked in

the

reticulocyte

lysate,

as

it

supports more

rounds of

translation

than the wheat

germ

sys-tem.

Hybrid-arrest and selection

experiments

with

pTK6 DNA

suggest

that sequences

represented

in VI43

and VI39 mRNA end within

90

bp

(the

site of the

very

high GC region)

upstream from

the

BglII site.

Analysis

of the nucleotide

se-quence in

this

region

shows

many

features

char-acteristic of

eucaryotic

mRNA

5'

termini.

The

positions of the

consensus sequences

found

in

other

genes

imply

that

an

mRNA initiation site

exists at

approximately

50

bp

from the

BgII

site.

Recently, S1 nuclease

mapping

has

identi-fied a

major

mRNA 5'

terminus

atthis

position

(48),

reinforcing

the

conclusions

made here. If

the AT-rich

region

80

bp

further upstream is necessary for

initiation

of

transcription,

the

fail-ure

of

EcoRI-cleaved

pTK1

DNAto

transform

TK-

cells

can

readily

be

interpreted.

Location

of

the 3' limit of

coding

sequences

close

to

the

SmaI

site (1.91) is based on the

properties of hybrid-arrest

with

SmaI

fragment

B and thesizes of

truncated

polypeptides

gen-erated

by

fragments hybridized

tothe 3' end of

VI43

mRNA.

The

finding

that the

putative

shortened polypeptide produced

by

hybridiza-tion with

SmaI

fragment

B has TK activity

might

explain the ability

of

SmaI-cleaved

plas-mids

to

transform

cells

(7), and it would be

interesting

toexamine TK mRNA in such

cells.

The experiments reported

here have derived

a

precise location

of

coding regions

for

VI43

and

VI39 and

have

therefore enabled

the

function

of

other sequences

necessary for TK gene

expres-sion

to

be understood.

Since

genomic

coding

sequences

for VI43 span

approximately

1,110 bp,

and assuming the location

ofprotein

synthesis

initiation

is

correct,

it is

clear

that

introns

of

significant

size

cannot

exist in this gene.

Fur-thermore,

the absence in adjacent sequences

of

other

mRNA's

with

similar

abundance

or

kinet-ics

of

synthesis indicates that the

TK gene is

controlled

as an

independent

unit

rather

than

one

of

a group

of coordinately expressed

prod-ucts.

Despite the high GC content

and

require-ment

for

a

viral

polypeptide

in

infected

cells (24,

35,

36), the TK

gene

exhibits all

the

postulated

signals

for RNA polymerase

binding and

initia-tion of

transcription.

An

important finding from mapping

BamHI

p-specific mRNA's

is

the identification of

two

polypeptides

whose

mRNA's

share coding

se-quences. Whether VI43 and VI39

are

produced

from

the

same

translational

reading frame

can-not

be

determined until further analysis of these

polypeptides

has been

performed.

Nevertheless,

mechanisms by which VI39 might be related

to

VI43

can

be

considered. The

possibility

of

pro-teolytic cleavage of VI43

was

eliminated by the

finding that the relative proportions of the

two

polypeptides

did

not

change during

a

chase of

up to 20h

in vitro

(data

not

shown).

Premature

termination of

protein synthesis

also

seems

un-likely, since SmaI

fragment

B

partially

inhibits

production

of both

V139

and

VI43,

even

though

it cannot

overlap sufficient

coding

sequences to

mask

a

pretermination

site. The other

possibili-ties

appear to

be

splicing

of

coding

sequences

from VI43 mRNA

or

false initiation of

protein

synthesis

at a

site

100 to 150

bp

downstream

from theVI43 initiation codon.

The

evidence

currently

available doesnot

sup-port the concept of

splicing

VI43 mRNA to

generate

VI39 mRNA. These mRNA's couldnot

be

separated

by

sucrose

density

gradient

cen-trifugation

or

polyacrylamide gel electrophoresis

under

denaturing

conditions

(data

not

shown),

and no band 100 to 150

bp

smaller

than

V143

mRNA

could

be

detected

when

pTK1

DNAwas

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604

AND McGEOCH

Ingiotion

TK 43

3p

1

:

mRNA

Hinf Alu

BmHlinf SaI I Eco BI

I

Term.

1K 29K 35K

Alu

Ss[t rif iU Snma

I1Ij

I I

IkbP

I I I2kbp I

PT

Kl

S

!AID!

pTI6L FT Bst NI AVGI Ddel

Sau961

FIG. 10. Summary ofmRNAmappingdata.Sequenceswhich arrest translation under standard conditions

areshown in solidlines;those whichrequirealtered conditionsorpartiallyarrestareshown in brokenlines. Points16,000, 29,000,and35,000molecularweightunitsfromtheproposed VI43initiation codonaremarked and should becomparedtotheAluI,HinflandAvaIsites whichgeneratedputativetruncatedpolypeptides ofthesesizes. Theproposedtermination sitefor VI43andVI39and initiation sitefor VI39arealsoindicated. Restriction sites above the linesarefrom completemapsofpTK1;those below have beenidentified onlyin

digests ofpTK1 subfragments.

used to probe fractionated RNA on Northern blots (J. B. Clementsand N. M.Wilkie, manu-scriptinpreparation). Such experiments might

not succeed because the expected mRNA size reduction is less thantheaveragelengthof the

heterogeneous polyadenylic acid tract. Any splice would be more than 270bp downstream from theBglII site,since removal of 100 to 150

bp in this region would prevent arrest ofVI39 synthesisbyone or moreof thesmall BglII-SstI-BstNI fragments C, D, or E. Alternatively, a situation in which ribosomes occasionallyread

through the usualinitiation codoniscompatible with the datapresentedhere, but rigorous dis-crimination between thepossibilitieswillrequire detailedpeptide mapping ofVI43 and the minor VI39 band. It should be noted that ribosome attachment for bothVI43andVI39occurs near the 5'terminus(18), since pTK6 DNAcanarrest

synthesisofbothpolypeptides.

Figure 10 summarizes the mapping of TK,

VI43,

and VI39 mRNA's, and indicates the

pu-tative initiation codon forVI39.

AttherecentColdSpring HarborHerpesvirus Workshop, wherethis workwaspresented, the complete sequence ofthe HSV-1 TKgene was reported by M. Wagner, J. Sharp, and W. C. Summers, and by S. McKnight. Analysis of strain CL101 shows that the SmaI-B overlaps coding sequences by 18 bp, and that the next ATG codon from theproposed VI43initiator is

130 bp downstream and in phase (M. Wagner, personalcommunication).

ACKNOWLEDGMENTS

Wethank J.H.Subak-Sharpe forhis interest in this work, and critical readingof themanuscript. M. Dunlop and A. Dolangaveskilledtechnicalassistance.

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