Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript.

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Vol. 64, No. 10

Activity of Herpes Simplex Virus

Type 1

Latency-Associated

Transcript

(LAT) Promoter in

Neuron-Derived

Cells: Evidence for

Neuron

Specificity and for

a

Large

LAT

Transcript

JOHN C. ZWAAGSTRA,1 HOMAYON GHIASI,"2 SUSAN M. SLANINA,1 ANTHONY B. NESBURN,"2

S. C. WHEATLEY,3 K. LILLYCROP,3 JOHN WOOD,4 DAVID S. LATCHMAN,3 KAMALESH PATEL,'

AND STEVEN L. WECHSLER'2*

Ophthalmology Research, Cedars-Sinai Medical Center, Halper Building 111, 8700 Beverly Boulevard, LosAngeles, California 90048-18691*;Department ofOphthalmology, UCLA School of Medicine, LosAngeles, California 900242;

andDepartment of Biochemistry, University College and Middlesex School of Medicine,3

andSandozResearch Institute,4 London, WCIE6BNUnited Kingdom

Received 4 August 1989/Accepted 17 July 1990

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that

promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had

neuronalspecificity. Promoteractivityin these transient CATassayscoincided withaDNAregion containing

excellent RNApolymerase II promoterconsensus sequences. Primerextension analysis ina LAT

promoter-CATplasmidconstructplacedthestartoftranscription about28 nucleotidesfrom thefirstT in theconsensus

TATAboxsequence. Neuronalspecificityof thispromoterwassuggested byexamining theeffectofsequences

upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells,

promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in

neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT

promoterpredicted byourtransientCATassayswaslocatedover660 nucleotidesupstreamfromthe5'endof

thepreviously mapped 2-kilobase (kb) LAT. This unusual location wasexplained by in situ and Northern

(RNA) blot hybridization analysesthat suggested that LATtranscriptionbegannearthepromoterdetectedin

ourCATassays,ratherthannearthe5'endofthe2-kb LAT. In situhybridizationwithneuronsfrom latently

infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box

sequence. Thissuggested that LATtranscription begannearthisTATA box. Northern blothybridization of

RNA from ganglia of latently infected rabbits revealed a faint8.3-kb band ofthesame sense as LAT. We

conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660

nucleotides upstreamofthe5' endofthepreviously characterized stable 2-kb LAT, (iii) LAT transcription

beginsabout 28 nucleotidesfromthe first ToftheconsensusTATA boxsequenceand extendstonearthefirst

available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable

precursorof themorestable 2- and 1.3-kb LATs.

Following primary infection in humans, herpes simplex

virustype1(HSV-1) establishes latent infections insensory

neurons (14). During HSV-1 latency, detectable viral

tran-scription is limited to an area within the genomic long

repeatsinthevicinityof theimmediateearlygeneICP0(20, 21, 25).Atleasttwoabundantandstablelatency-associated transcripts (LATs) (2 and 1.3 kilobases[kb])thatsharetheir

5' and3' endsarederivedbyalternativesplicing (30).These

LATspartially overlapthe3' endof ICP0andareantisense

(complementary)totheICP0mRNA(20, 25, 29, 30).The 5'

endof the stable 2-kb(and 1.3-kb)LATin the internallong

repeathasbeenmapped towithinafew bases (28-30). This

position corresponds to nucleotide 119462 of the genomic sequence(9, 15).Asecondcopyof the LATgeneispresent in the corresponding location ofthe terminal long repeat.

RecentreportswithsomeLATdeletionmutantssuggestthat

LAT may play arole in reactivation of the virus from the

latent state (2, 7, 24).However, this is notsupported byall

LATmutants (1, 6).

Basedonsequenceanalysis,weinitiallyproposedthat the

LATpromoteris located over660 nucleotidesupstream of

*Correspondingauthor.

themapped5'end of the stable LATs (30, 31),andlater, by using chloramphenicol acetyltransferase (CAT) assays, we

confirmed that in Vero cells, LAT promoter activity

coin-cides with this region (32). This location for the LAT

promoter is further supported by more recent studies in

which viral mutants lacking the predicted promoter region

failed toproduceany LATRNAduringlatentinfections (2,

7, 11, 24).

Since LAT is the only HSV-1 gene that is abundantly

expressed during neuronallatency (20), the LAT promoter

must becontrolled differently fromother HSV-1gene

pro-moters(22, 23). Furthermore,the LAT promoter is likelyto

have neuronalspecificity, since LAT is abundantinlatently

infected neurons (20, 25) but is present at onlylow levels

duringacutetissueculture infection(22).We have therefore

extendedourstudies of the LAT promotertoneuron-derived

cellstoconfirmthelocationofthe LATpromoterandtolook

for neuronal specificity.

We show here thatinneuron-derived celllines, promoter

activity mappedto thesamelocationasit did inVero cells. Furthermore,wefound that in nonneuronalcells,sequences

upstreamof the TATA box decreased promoter activity in

cis, while in neuron-derived cells, inhibition by these

cis-acting sequences was not observed. These upstream

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quences may confer neuron specificity on the HSV-1 LAT

promoter invivo. Wealso show here that LAT transcription began near the promoter and appeared to continue for approximately 8.3 kb, the location of the first-occurring consensus polyadenylation site. A likely interpretation of

thisdata isthat this 8.3-kb RNA may beanunstable primary LAT transcript that gives rise to the previously mapped, relatively abundant 2- and 1.3-kb LAT transcripts.

MATERIALS AND METHODS

Cellsandvirus. Plaque-purified herpes simplex virustype 1 (HSV-1), strain McKrae, was grown as previously

de-scribed(20) and was usedforallinfections.Cellsweregrown

asmonolayers in minimal essentialmedium, Dulbecco mod-ified Eagle medium, or F12 (GIBCO Laboratories, Inc.) supplemented with 10% fetal calf serum and antibiotics. Neuroblastomacells(NB41A3;American TypeCulture

Col-lection CCL147)are ofmouseorigin. Theseneuroblastoma cells retain several neuronal markers, including acetylcho-linesterase activity. Inaddition, they arenonpermissive for HSV-1 infection (27). Immortalized neurons weremade by fusing

H18TG2

cells, a5-azaguanine-resistantmouse

neuro-blastoma cell line, with neonatal rat dorsal root ganglia

neurons. These cells are nonpermissive for lytic HSV-1

infectionand express LATfollowingHSV-1

infection

(S. C.

Wheatley,C.Dent,K.Lillycrop,L. M. Kemp,J. N.Wood,

and D. S.Latchman, submittedforpublication).

Plasmids. Allrestrictionfragmentswere derivedfromthe BamHI B restrictionfragmentfrom HSV-1 strain F(16).The LAT genefromposition -2592to+663relativetothe 5'end of the stable 2-kb LAT was divided into five restriction fragments. FragmentsAtoC and thedetailsof theircloning

in the proper orientation in front of the CAT gene within

plasmid pSVOCAT(4) have beenpreviously described(32). Fragments A+ andA++ weresimilarly cloned and consist

of

fragment

A with additional upstream sequences as

de-tailed in the legend to Fig. 2 and in

Fig.

3. For some

experiments, similar constructs were made by using a

dif-ferent promoterless CATplasmid,

p1O6CAT

(3),in place of pSVOCAT.

CAT assays andquantitation procedures. CAT assays and

quantitation procedures have been previously described

(32). Briefly, cell monolayers atapproximately 60% conflu-ency, on 60-mm plates, were transfected with CAT

con-structs by thecalcium phosphate precipitation method (5).

After46 h, the cells were harvested and cellextracts were

prepared. Within an experiment, equal cell numbers were

used and, if necessary, corrections were made for the

amount ofproteinpresentin the extracts. Acetylatedforms

of

['4C]chloramphenicol

were detected by thin-layer chro-matography and subsequent autoradiography. The amount

ofacetylated andunacetylated chloramphenicol was

quanti-tated by excising the spots from the thin-layer plates and

counting in a liquid scintillation counter. In some

experi-ments, samples ofcell extracts were analyzed by DNA dot blothybridizations with aCAT-specific probe to determine the relative amount of CAT DNA that had entered the cells

during the transfection. Within a cell line, no differences

were seenbetween the transfection efficiencies of fragments

A+ +, A+, or A. Thus, differences between the CAT

activitiesof these plasmids withinacell line were not a result ofdifferences in transfection efficiency. The differences in

transfection efficiencies between cell lines was partially compensated for by using 10 ,ug of each plasmid in

immor-talized neurons, BHK cells, and L cells; 5 ,ug of each

plasmidin neuroblastoma cells and CV-1cells;and 2.5,ugof eachplasmid in Verocells.

Primer extension. As modified from a procedure of P. Krause and J. Ostrove (personal communication), 10 ,ug of RNAfrom transfected Vero cells(isolated bythe

guanidin-ium-cesium chloride method [8])was suspendedin 10

RI

of 20 mMTris-hydrochloride, pH 7.6-100 mM NaCl-0.1 mM EDTA. A 10-ng portion of 32P-end labeled CAT primer (approximately 5 x

105

cpm) was added. The sequence of theprimer from 5' to 3' wasGATGCCATTGGGATATAT CAACGGT. This sequence is complementary to the CAT mRNA sequence located between nucleotides 127 and 151 downstream from the first T of the LAT TATA box in the

CAT constructusedfor the transfection. This location was

confirmed by partial sequencing of the plasmid and

corre-sponds to CAT nucleotides 28 to 52 relative to the ATG codon at which CAT translation initiates. The difference between these numbers is the result ofaHindIll linker, a

multiple cloning region, and noncoding CAT sequences between the end of the LAT sequences and the startofthe structural CAT sequences in this plasmid. The reaction mixture was heat denatured for 3 minat90°C, hybridizedat

55°C for 10 min, and slowly (approximately 1 h) cooled to

30°C. Primer extension of the hybridized primer was done with 20 U of cloned Moloney murineleukemia virus reverse transcriptase(Bethesda ResearchLaboratories, Inc.) at 37°C for 1 haccordingtotheinstructions of the manufacturerby

using 2 mM deoxynucleoside triphosphate, 100 ng of bovine

serumalbumin per Pd, and 20U of RNasin(PromegaBiotec)

per 20-pdl reaction. The reaction was stopped by adding

EDTA to 10 mM. The extendedprimer wasprecipitated with 2 volumes of 95% ethanol, washed with 70% ethanol,

sus-pended in formamide-dye loading buffer, heat denatured at

95°C for 3 min, quick chilled on ice, and run on a 10%

acrylamidesequencing gel containing 7 Murea.Thegelwas

thenprocessed for autoradiography. In one experiment the same oligonucleotide used in the primer extension reaction was used to prime a sequence reaction from the A-CAT plasmid by using a Sequenase DNA sequence kit from United States Biochemical Corporation. Products from the sequence reaction were run next to the primer-extended product on a10% sequencing gel containing 7 M urea.

Rabbits.New Zealand White male rabbits(approximately

2 kg each) were used for all animal experiments. These animals develop a primary and recurrent herpetic disease (13) which mimics HSV-1 keratitis in man.

Latent ganglionic HSV-1 infections. Latent ganglionic

HSV-1 infections were done as previously described (20). Briefly, rabbits were bilaterally infected without corneal scarification by placing approximately 1 x 105 to 2 x

105

PFU of virus into the conjunctival cul-de-sac, closing the eye, and rubbing gently for 30 s. Rabbits surviving after 4 weeks were considered latently infected (12).

Rabbittrigeminal ganglia. Rabbit trigeminal ganglia were taken from sacrificed rabbits and immediately placed in liquid nitrogen for RNA extractions for Northern (RNA) blots or into the preservative periodate-lysine-paraformalde-hyde (26)for sectioning prior to in situ hybridizations.

In situ hybridizations. Fixing, embedding, and cutting sections of trigeminal ganglia were done as described previ-ously (19). Hybridizations to identify RNA were done aswe previously described (20, 30) by using 32P-labeled synthetic oligonucleotides as probes. Slides were exposed to photo-graphic emulsion for 2 to 3 days. Pretreatment ofslides with RNase, but not DNase, eliminated hybridization. Three to five sections of ganglia from each of four latently infected

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HSV-1 LAT PROMOTER IN NEURONAL CELLS 5021

rabbits and two uninfected rabbits were examined with each probe. No hybridization with any of the probes resulted in an accumulation of grains over any neurons from uninfected rabbits. Positive probes showed an accumulation ofgrains

over the nuclei of some neurons from latently infected

rabbits compared with the amount of background grains on

the slide and compared with uninfected neurons (see Fig. 5). Positive probes detected positive neurons from at leasttwo

latently infected rabbits, as judged by detection of at least one positive neuron on at least two different slides from a

givenrabbit.

Northernblot hybridizations. Total RNA was isolated from

uninfected orlatently infected rabbit trigeminal ganglia fro-zeninliquidnitrogen orfrom uninfected or acutely infected (multiplicity ofinfection of 20; 18 h postinfection) CV-1 cells as previously described (20). Northern blot hybridizations were done as we previously described (20, 30).

Hybridization probes. Random-primed labeling with 32p

wasdone on linearized plasmids following the instructions of the manufacturer (Amersham Corp.). Synthetic

oligonucle-otides (20-mers) were synthesized by using beta-cyanoethyl

phosphoramidite chemistry on a Pharmacia Gene

Assem-bler.The sequencesof the 20-mers were based on sequences

for HSV-1 strains 17 syn+ (15) and F (31). End labeling of

oligonucleotides with

[y-32P]ATP

was done as described previously (8).

RESULTS

CAT activityinneuron-derivedcells.Byusing CAT assays, we previously showed that in Vero cells, LAT promoter

activity coincides with RNA polymerase II promoter con-sensus sequences (32)that arelocatedover 660nucleotides upstreamfromthe 5' endofthe stable 2-kb LAT (30, 31). To

determinewhether LAT promoteractivity in neuron-derived cells mapped to the same unusual upstream region, we

assessed the ability of portions ofthe LAT gene to

consti-tutively functionas apromoterinimmortalizedneuronsand in neuroblastomacells. DNArestrictionfragments from the LAT gene were individually cloned into the plasmid

pSVOCAT (4) in front of the CAT structural gene. The proper orientation of each fragment was confirmed by

re-striction enzymeanalysis.

Cells were transfected with individual CAT constructs, and 46 h later CATactivity was measured. Representative

results are shown in Fig. 1. Thelocation ofeach fragment relative tothe 5' endofthe stable 2-kb(and 1.3-kb) LAT is

shownatthe bottom of thefigure.Numbers above each lane indicate the percent conversion of

[14C]chloramphenicol.

Fragment A (-940 to -662) had CAT activity in immor-talizedneurons (Fig. la, lane A) and in neuroblastoma cells

(Fig. lb,laneA)thatwascomparabletothatofpSV2CAT (a

strongpositivecontrolcontaining CAT under the control of the simian virus 40 early promoter) (Fig. la and b, lane

pSV2) in the same cells. This was indicative of

significant

promoter

activity

by

fragment

Ain these cells.Fragments B andC, which encompass theregionfrom-662 to+663, had

no CAT activity in either neuron-derived cell line. These results were identical to those we previously reported in Vero cells (32). This mapped the in vitropromoteractivity

associated with the LAT gene to the same location (i.e.,

within fragment A) in two neuron-derived cell lines as in Verocells. As shown in theexpandedviewatthe bottom of

Fig.

lb,

fragment A contains several consensus sequences

normally associated withanRNApolymerase II promoter,

including aTATAbox, a potentialCAATbox, and several

a

rmtnortahzed Neurons

b

NEtArobtlasI(m

Iekls

139 <05 <05 12.4 %Conversion

* Acetylated

** * * ~~Unacetylated

A B C p)SV2

1 <05 <0.5 9.7 %.,Conversion

* Acertylated

* * * Unacelylaled

A B C pSV2

.94(:1 (iP92 151 +43

HarIII Pviitl t1FfllT Hvin * 2kbstabhe LAT SpiSpi CAMT .TATA

TAATGAIAT1688 1l 6ii

FIG. 1. CATactivity in neuron-derived cells ofdifferent DNA

fragments derived from the LAT gene. Different portions ofthe

LAT gene (32) cloned in front ofthe gene for CAT (in plasmid

pSVOCAT [4])wereindividually transfected into immortalized

neu-rons(a) or neuroblastoma cells(b), and CATactivitywasmeasured

asdescribed inMaterials and Methods(lanesA, B, andC). Lanes

pSV2areplasmid pSV2CAT (4), containing the simian virus40early

promoter, as apositivecontrol.Transfectionsweredonewith 10 ,ug

of eachplasmid in immortalizedneuronsand 5j±g of eachplasmid in

neuroblastoma cells. Acetylated forms of [14C]chloramphenicol

weredetectedbythin-layer chromatography andsubsequent

auto-radiography. TheLATgenefragments aredesignatedAtoC (32)

and arerepresentedschematicallyatthe bottom ofpanelb(alsosee

Fig. 3). Nucleotidepositionsarerelativetothe 5'end ofthestable

2-kbLAT(nucleotide+1) (28, 30), whichcorrespondstonucleotide

119462 ofthe HSV-1 genome (9, 15). Numbers above each lane

indicatethe percentconversion ofunacetylated

[14C]chloramphen-icoltoacetylated forms.

Spl sites (30, 31). This region also contains a sequence

having significant homology to a Vmw65 binding site

(TAATGARAT) (32).

Sequencesupstream offragmentAdecrease LAT promoter

activity in nonneuronal cells. To look forpotential cis regu-lation by sequences upstream of the LAT promoter, two

additionalCAT constructs were made.FragmentA+(-1271

to -662) consisted offragmentAplus 331 upstream

nucle-otides. Fragment A+ + (-2592 to -662) consisted of

frag-mentAplus 1,652 upstreamnucleotides.

Fragments

A+ +, A+, andAhaveacommon3'end.FragmentsA+ and A+ +

were cloned into plasmidpSVOCAT in thesame manneras fragment A.

The effect of these upstream sequences on promoter

activityinneuronal and nonneuronal cell lineswasexamined

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5022 ZWAAGSTRA ET AL.

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859 93 7 12 2 I3 3 5 : 65 8 3062t7.4

.e

4 1 _i4' . {

-i;,.

ofrvL

acetvi1ated

Nont

a--etya DNwA _a-e

FIG. 2. Theeffect ofupstream sequences onCATactivity drivenbythe LAT promoterinneuron-and nonneuron-derivedcells. DNA

fragments containingthe LAT promoterregion and differentamountsof upstream sequenceswerecloned intopSVOCATatthesamelocation

usedfor the fragments in Fig.1.FragmentsA, A+,and A++havea common3' end(nucleotide-662relativetothe 5'endof the stable 2-kb

LAT). This is26nucleotides downstream from the firstTintheTATAboxsequence.FragmentAis 278 nucleotideslong. Fragment A+ is

609nucleotideslong. FragmentA++is 1,930 nucleotideslong. ThesefragmentsareshownschematicallyinFig.3.Plasmidswereindividually

transfected into neuron-derived cells (neuroblastoma or immortalized neurons) or nonneuron-derived cells (BHK, Vero, CV-1, or L).

Representative resultsareshown.Immortalizedneurons(lanes4to6),BHKcells(lanes7to9),andLcells(lanes15to17)weretransfected

with10 tg of eachplasmid. Neuroblastoma cells(lanes1 to3)and CV-1 cells(lanes12and13)weretransfectedwith 5 ,ugofeachplasmid,

andVerocells(lanes10 and11)weretransfected with2.5 ,ugof eachplasmid. Numbers above the lanes indicate thepercentconversion of

[14C]chloramphenicol. Numbers inparentheses indicatetheactivity relativetofragmentAfor each cellline.

byCAT assays. Representativeresultsareshown inFig. 2. The percentconversionof['4C]chloramphenicolis indicated

above each lane in Fig. 2. The numbers in parentheses

indicatethe percentactivity of the otherplasmids relativeto A(100%) within each cell line. In this particular experiment, the addition of the upstream sequences contained in frag-ments A++ and A+ had little effecton the activity of the LAT promoterin neuron-derived cell lines (83 to 131% of

fragment A activity; lanes 1 to 6). In other experiments, these upstream sequencesincreasedpromoteractivityupto

threefold (summarizedinFig. 3). In contrast, in BHK cells

(lanes7 to9),theupstream sequencescontained in fragment

A++and A+reducedfragmentA's promoteractivityabout

threefold. The effect of upstream sequences was further

examined in (nonneuronal) Vero and CV-1 cells, using fragment A+ +. The upstream sequences caused a reduction inCATactivity of about 6- to 12-fold in these cells (lanes 10 and 12).

Sincebothof the neuron-derived cell lines we used were

ofmouseorigin, itwasimportant to compare these results to

those in nonneuronal cells of mouse origin. In L cells (a

commonly used mouse cell line) there was a greater than

threefold reduction in LAT promoter activity by upstream sequences(Fig.2,lanes 15 and 16).Thiswassimilar to BHK

cells(Fig. 2,lanes 7to9) and confirmedthatthis

phenome-non wasrelatedtotheneuronal andnotthespeciesorigin of

the cells.

To partially correct for transfection efficiency between

differentcelllines,different amountsof plasmid were used in

different cell lines (within a cell line, equal amounts of all

plasmidswereused). We used 2.5 ,ug of plasmid per plate for Vero cells, 5 ,ug of plasmid per plate for CV-1 and

neuro-blastomacells, and 10 ,ug of plasmid per plate for

immortal-ized neurons, L cells, and BHK cells. Within a given cell

line,

these concentrations gave similar CAT activity with

fragmentAand with the positive control plasmid pSV2CAT

(Fig. laand b, lanes A and pSV2 for neuronal cells; Fig. 2, lanes 16 and 17 for L cells; not shown for other nonneuronal

cells). Thus, in CAT assays, fragment A (containing the

minimalLATpromoter) had promoter activity similar to that

ofpSV2CAT and relative topSV2CAT appeared to be an

equally effectivepromoterin all celltypes.

To ensure against the unlikely possibility of a

plasmid-specific artifact, some experiments were also done with fragments cloned into a different CAT-containing plasmid

(p106CAT [3]). Similar results were obtained (not shown).

1RL UL IRL IRSUS w

GenomeI1RL|~

Ger r

Nuctkdso 116,000 117,000 11,000 119,000 120,000 121,000 12Z000

Po6skin

-3000 -2000 -1000 +1 +1000 +2000

Poskionrebbve

toS' o 2kbLAT

2kbstableLAT 2kbloL3T

CATactiviy

inreuronal derh,scells

TATAbox 1.3 kb LAT A++ _ 8 (83-190%)

A+ (89-306%)

A _(100%) B

C CATactivityin A++ (S30%)

ron4Wuronal A+ i_ (1035%)

ceil A _ (100%)

Be

cc

FIG. 3. LAT genefragmentsusedinCATconstructs.The upper

portion of the figureshowsthe HSV-1genomicorganization inmap

units (mu). The expanded region indicates nucleotide positions

relativetotheentire HSV-1DNAsequence(15)and relative tothe

5'end (+1)of thestable 2-and 1.3-kb LATs. The locationsofthe TATAbox sequences and of the 2- and1.3-kbLATs areshownfor

reference. The LAT genefragments are shown as horizontal

rec-tanglesand arelabeledA++, A+,andA toC, as in Fig. 1 and 2.

Solid rectangles indicate CAT (promoter) activity comparable to

that of pSV2CAT in the same cells. Dotted rectangles indicate

reducedactivity. Openrectanglesindicatenoactivity. Numbers in

parenthesesshow the range ofactivity ofA++andA+relativeto

Ainover5independent experiments.

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a.

b.

LATsense ge

23

4 3:

200

- 23cx

-23

* oo

j82

.6 _JA7

(P,asrnd,,

3 4 5

c-FIG. 4. Primer extension mapping of the transcriptional start site of the LAT promoter-CAT plasmid. Primer extension was done as

described in Materials and Methods by using a 32P-end-labeled 25-nucleotide primer complementary to CAT mRNA, and the gel was

processedforautoradiography. All lanes represent the result of primer extension with RNA from transfected Vero cells. (a) Lane 1,p106CAT (promoterless CAT); lane 2, plasmid A(p1O6CATwithfragment A); lane 3,untransfected control. Marker lane is32P-end-labeledHinfl-cut

ijx174 DNA. The size of the primer extension product is approximately 123 nucleotides, corresponding to a transcriptional start site

approximately28nucleotidesfrom thefirst T of the TATA box sequence. (b) The same oligonucleotide used for primer extension experiments

was used as a primer in a set of sequencing reactions (see Materials and Methods). This generated the sequence shown in lanes 1 to 4, which

is complementaryto the RNAproduced by this plasmid with the LAT TATA box region as a promoter. The predicted sequence of theregion

fromthe TATA box to the beginning of theHindllllinker used to clone the LAT sequences into the plasmid is shown to the left of the gel.

Lane5 shows theresultof aprimerextension experiment similar to that in panel a. The extended primer comigrates with the first T in the

HindlIl linker. Thisis 28 nucleotides from the first T in the TATA box.

Theunlikely possibilitythatthe decrease in promoter

activ-ity seen with A++ and A+ could be due to a decrease in

transfection efficiency (compared with A)of these plasmids innonneuronal compared with neuronal cells was addressed asfollows.In someexperiments,aportionof the cell extract used for the CAT assay was monitored for transfection

efficiency by DNA dot blots by using a CAT-specific

32P-labeled DNA probe. As expected, although transfection

efficiencies differedbetween celllines,withinagiven cell no

differences in transfection efficiency that could account for the decreased promoteractivity of A++ or A+ compared with A were detected (not shown).

In vitro LAT transcriptional start site. To determine the

approximatelocationofthestartoftranscriptionin the CAT constructs, primer extension experiments were done as

described in Materials and Methods. RNA was prepared from transfectedoruntransfected cells andhybridizedwitha

32P-end-labeled25-nucleotide-longsynthetic oligonucleotide primer. The primer was complementary to CAT mRNA sequences located 127to 151 nucleotides downstreamfrom

the first T of the LAT TATA box (see Materials and

Methods). The primer was extended with reverse

tran-scriptase,denatured,andsizedon apolyacrylamidegel (Fig. 4a). Lane 1shows the result of

primer

extension withRNA preparedfrom cells transfected withp1O6CAT(promoterless

CAT,withoutfragment A).Lane3showsaprimerextension

reactionwith RNA from cells thatwere nottransfected. No

product is seen in either control lane. Lane 2 shows the result ofaprimer extensionreaction with RNA from cells transfected with

p1O6CAT

containing fragment A. We esti-mated the size of themajor extendedproduct asabout 123 nucleotides. Smaller faint bands are probably the result of premature termination of the primer extension reaction.

Since the primerwas complementary to nucleotides 127to

151 relative to the first T of the TATA box, a size of 123

nucleotides placed the start of transcription at about 28

nucleotides from the first T of the TATA box (151 minus 123). This would be HSV-1 nucleotide 118802(9) or nucleo-tide-660 relativetothe 5'end of the stable LATs. The LAT sequence in this region is "...GCCGATCGCGG...," with theunderlined Tbeing theapproximatetranscriptional start site.

To further confirm thetranscriptional startsite, the result ofa similarprimerextension reactionwas sizedrelative to

the sequenceof the CATplasmid on asequencing gel (Fig.

4b). The CATplasmid sequencewasobtainedbyusing the

same oligonucleotide primer to prime a sequence reaction

from the A-CATplasmid (seeMaterials andMethods). The

extended primer comigrated with the underlined T in the sequence "...CGGCGITCGAA..." onthis gel. The

corre-sponding LAT sense strand sequence is "...GCCGCAAG CTT.." This is the samelocation relativetothe TATA box

(28 nucleotides fromthefirstT)asestimated fromFig.4a. In this case, this base isanA, notthe T foundatthis location in the LAT sequence. This A is the second base in the

HindIlI linker usedtoclone the LAT Afragmentin this CAT

construct.

Our finding that in vitro transcription started approxi-mately28nucleotides downstream from theLAT TATAbox

strongly supportsthe notion that CAT

transcription

in these plasmids is under the control of the LAT sequences in

fragment A. Furthermore it demonstrates that the LAT sequences neartheTATA boxare

capable

of

functioning

as a typical promoter in vitro. This supports the notion that

fragmentAcontains the LAT promoter and makes it

likely

thatduringneuronallatencyin

vivo,

LAT

transcription

may

begin at or nearthe sameposition (i.e., -660).

In situ hybridization near the LAT promoter. The CAT

VOL.64, 1990

I

A--A

A

A-

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FIG. 5. Insituhybridization of trigeminal ganglia fromlatentlyinfected rabbits. Trigeminalgangliafrom latently infectedoruninfected

rabbitswereremoved, sectioned, and processed for in situ hybridization as described inMaterialsand Methods. Allprobes weresynthetic

oligonucleotides(20-mers) end labeled with[32P]ATP.(A)Section fromalatentlyinfected rabbit hybridizedto aprobecorrespondingto(the

complement of) nucleotides +1292 to +1311from within the stable 2-kb LAT. (B) Uninfectedrabbitwith the same probe as inpanelA.(C)

Latently infected rabbitwith aprobe fromtheregionbetween the TATA box and the5' end of the stable 2-kb LAT (probe 8 from Fig. 6).

(D) Latently infected rabbit with a different probe from the region between the TATA box and the5'end of the stable 2-kb LAT (probe 11

from Fig.6). The arrow points to a faintly positive cell.

assaysand the primer extension results reported above both

stronglysuggestthat the LAT promoter is located over 660 nucleotides upstream of the start of the stable LATs. To look for small amounts of LAT RNA between the LAT TATA

box and the5'end ofthestableLATs,wecarefully analyzed

a series of in situ hybridizations to sections from trigeminal

gangliaofrabbits latently infected with HSV-1.

Figure5shows representative in situ hybridization results in which 32P-end-labeled synthetic oligonucleotides were used as probes. These 20-mers were based on the DNA sequenceof this region (15, 31) and were constructed so that RNAdetected with these probes would be the same sense as LAT RNA. To eliminate possible false positives, computer

analysis was done to ensure that no 20-mer would have

homology to any other portion of the LAT region (30). Several 20-mers were also rejected because they hybridized

to neurons from uninfected rabbits. Panel A shows the

strong hybridization typical of a probe corresponding to a

region within the stable 2-kb LAT to a section from the

trigeminal gangliaof a rabbitlatently infected withHSV-1. Panel B shows lack ofhybridization with thesameprobeto

a section from an uninfected rabbit. Panels C and D show

fainthybridizationtolatentlyinfected sectionsusing probes

betweenthe LATpromoterand the5' end of the stable2-kb

LAT.The numberof grainsin these positive cellsisgreatly reducedcompared with panel A, indicating muchless LAT RNA in the positive cell. In addition, the number ofcells showingfainthybridizationwasonlyabout 10% of that seen

withprobes hybridizing to the 2-kb LAT.

The faint hybridization seenwiththe probes in panels C andD(Fig. 5)was atthe limitof detectability in our latently infected rabbit system. Under normal circumstances, in which one expects to see numerous, strong hybridization signals, these probes might have been scored as negative. However, it was clear that these probes were not simply "negative."Inthisanalysis we therefore consideredaprobe

positiveifatleastone neuron wasfaintly positive (asinFig.

SC orD) on atleasttwo sections from each of at leasttwo

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-6100 // -900 400 -700 -00 -500 -400 -300 -200 -100 +1 100

II/ 'I

TATA

Probes 1 2 3

BatnHI EooRv LAT- RNA M

5'

4 5 6 78910

H ES

Start 1.3& 2 kbLAT

11 2b>

3,

FIG. 6. Detection of low-abundance RNA between the TATA box sequence and the 5' end of the stable 2-kb LAT by in situhybridization. Results from the experiment partially shown in Fig. 5are shown schematically. In situ hybridization with each32P-endlabeled20-mer was

performedon 3 to5 sections of trigeminal ganglia from each of four rabbits latently infected with HSV-1. The 20-mers were based on the

sequenceofthis region and were of complementary sense to LAT RNA. The approximate locations of the probes relative to the 5' end of thestable 2-kb LAT (+1) are shown at the top.Exactlocations are: 1, -789 to -770; 2, -707 to -688; 3, -658 to-639;4, -569 to -550;

5,-470 to -451; 6, -440 to-421; 7, -359 to -340; 8, -338 to -319; 9, -319 to -300; 10, -299 to -280; 11, -140 to -121; 12, -70 to-51;

13,-39 to -20; and 14, -20 to -1. Shaded boxes correspond to low levels ofhybridizationsimilar to Fig.SCand D,detected in at least two ofthefourlatently infected rabbits (one or more neurons in at least two sections per rabbit). Open boxes indicate that no hybridization was

detected.The dark rectangle indicates the start of intense hybridization (as shown in Fig. 5A)at thestart of the stable 2- and 1.3-kb LATs

(30). Thelargeopen rectangle (note break) represents our previous results (29, 30), showing no hybridization to a BamHI-EcoRV restriction

fragment(-6141 to -824). The direction of LATtranscription is from left to right, as indicated by the large arrow.

latently infected rabbits. Each probe was hybridized to 3 to 5 sections of trigeminal ganglia from each of four rabbits

latentlyinfected with HSV-1. On this basis, at least 9 of the 12probes tested between the TATA box and the 5' end of thestable 2-kb LAT hybridized faintly to sections of

trigem-inal gangliafrom latently infected rabbits (Fig. 6). The lack ofdetected hybridization with the remaining three probes wasprobably due to technical limitations related to detecting

extremely small amounts of RNA. None of these probes

hybridizedtosections from uninfected rabbits (not shown).

Similar results were also obtained with sections of human

trigeminalganglia (not shown).

The start of transcription appeared to be close to the

TATA box consensus sequence. Probe 1 (-791 to -772),

approximately 85 nucleotides upstream ofthe TATA box, andprobe 2 (-707 to -688), immediately to the left of the TATAbox(at -688 to -684), werenegative (Fig. 6, probes 1 and 2). In addition, we have previously shown that a

restriction fragment probe from -6140 to -824

(BamHI-EcoRV) doesnothybridizetolatently infected ganglia from

rabbitsorhumans (29, 30)(Fig. 6). Probe 3 (-658 to -639), thefirstprobe totheright ofthe TATA box, waspositive.

Thisplacedthestartoftranscriptionbetweenprobes 2and 3

orwithin 30nucleotides ofthefirstTof the TATA box. This

isin excellent agreement with our invitroprimer extension

data,whichplaced thestart ofLATtranscription 28 nucle-otides from the TATAbox. These results strongly suggest thatthisTATAboxsequenceisthefunctionalTATAboxof the LAT promoter. The greatly reducedamountof detect-able RNA between the promoter and the stdetect-able LATs suggests that the RNA from this region was relatively unstable.

Northern blothybridization ofRNAfromlatently infected rabbits.Northern blothybridizationof RNA fromtrigeminal ganglia oflatently infected rabbits did not reveal any small

bandsin the 500-to700-base range that mightrepresent the

region between the TATA box and the 5' end of the 2-kb

LAT. However, by using probe 8 from Fig. 6, which is located between the TATA box and the 5' end of the 2-kb LAT, a veryfaint band with an estimated size of 8 to 9 kb

was detected (Fig. 7, lane 2; designated 8.3 kb). For

com-parison,

an equal amount of the same RNA

preparation

hybridizedwithaprobethat was specificfor the 2-kb LAT

regionis shown in lane 1. Thisprobewasthesame size and

specificactivity astheprobe in lane 2.

Lanes 1 and 2 (Fig. 7) show a typical exposure for the

detectionandanalysisof the stable LATs.

(The

1.3-kbLAT

isnotseenin lane 1 because this probe is entirely within the intron of the 1.3-kb LAT.) The band designated 8.3 kb is almostindistinguishable in lane 2 and would notnormally be considered meaningful. However, by overexposingthe au-toradiogram, the 8.3-kb band was detected above the back-ground (lane 3). The detection of this band in RNA from latently infected rabbits wasarare event.Thisband was not detectedby other laboratories (2, 11) nor was it detected in the majority of our RNA preparations (not shown). Since this RNAwasbarely detectable withthe much more

sensi-tive in situ hybridization assay, the inability of Northern

blots to detect this RNAconsistentlywas notunexpected. The simplest explanation for the 8.3-kb band was that it represented anRNA initiating near the LAT promoter and

(because of its size) extending through and past the 2-kb LAT. Examination of the published HSV-1 sequence (15) revealed that the first consensus polyadenylation signal (AATAAA)wasapproximately8.3 kb downstreamfrom the LATpromoter,just 34 nucleotidespriortothe ICP4 polya-denylation signal on the opposite DNA strand (see Fig. 8). Wedetectedasimilar8.3-kb bandduringacutetissue culture infection and wereabletomapittothe regionbetween the TATA box and the potential polyadenylation site byusing oligonucleotide probes flanking these regions (results not shown). A similar result with RNA from acutely infected

tissue culture cells was recently reported (2), while this

manuscript was inpreparation. Thus, the faint 8.3-kb band wedetectedinlatently infectedneuronsmaybe anunstable,

possibly polyadenylated, primary LAT transcript from which the 2- and 1.3-kb LATs arederived.

DISCUSSION

When we first proposed that the LAT promoter was

locatedover660 nucleotides upstream of the2-kbLAT, we

also raised thepossibilitythat LATtranscription mightstart nearthis promoterregion(30, 31).This was basedonthe fact

that ingangliafromlatentlyinfectedrabbits,weoccasionally detected whatappearedtobeveryfaint in situhybridization withprobes upstream of the 2-kbLAT(30).Since then other labs have reported in situ hybridization in this region in

ganglia from latently infected mice (2, 10). However, these

experiments were doneby using large, nonoverlapping

re-striction fragments as

probes

and the start oftranscription

couldnotbe

precisely

determined. In this report, theuseof short

oligonucleotide (20-mers) probes

enabledus to deter-mine thatduringneuronal

latency

in

rabbits, transcription

of

VOL.64, 1990

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-8.3 kb

*28S

[image:8.612.319.552.69.247.2]

*18S

FIG. 7. Northern blot hybridization of RNA from ganglia of latentlyinfected rabbits. RNAwasisolated from trigeminal ganglia oflatently infected rabbits, and Northern blot hybridizationswere

done as described in Materials and Methods by using

32P-end-labeled 20-mers asprobes. Each lane containsapproximately 20 Fg of totalRNA. Lane 1 showshybridizationwithanoligonucleotide

correspondingto (the complement of) nucleotides +261 to +280.

This region is within the 1.3-kb LAT intron and therefore hybridizes

to the 2-kb LAT but not the 1.3-kb LAT (30). Lane 2 shows hybridization with oligonucleotide number 8 from Fig. 6,which is

midway between the TATA box sequence and the 5' end ofthe

stable 2-kb LAT. These probes were labeled tothe same specific activity, and thesame amountofradioactivity wasused toprobe lanes 1 and 2. Lanes 1 and 2representanautoradiogramexposureof

12 h andarefrom the samegel. Lane 3 isan 8-day overexposed autoradiogram of lane 2. The point marked 2 kb indicates the

location of the major 2-kb LAT. The point 8.3 kb marks the large bandvisible in lane 2and 3. Theapparentsize of this band is 8to9 kb, based on the mobilities of the 18S and 28S ribosomal RNAs (indicated by asterisks) and the 2- and 1.3-kb LATs. The 8.3-kb designation is consistent with thisapparentsize and correspondsto

the distance from the TATA box sequence tothe first consensus

polyadenylationsequence.None of theseprobeshybridizedtoRNA fromuninfected rabbits (30).

the faintLAT RNA beganimmediately downstream fromthe

putativepromoter,within 30nucleotides of the first T in the

TATAbox sequence. This is the first report in which the

start of"upstream" LAT transcription in latently infected

neuronshas been fine mapped. This result stronglysupports

thenotion that this TATA boxsequenceispartof the LAT

promoter.

We also reported here the detection by Northern blot

analysis of a minor 8.3-kb LAT transcript in ganglia of

latently infected rabbits. This is the first visualization by

Northernblot of this 8.3-kb LAT duringneuronal latency. A

similarlarge LATband has been detected byNorthern blots

of RNAfromacutelyinfectedcells and mapped to

approx-imately the region between the LAT promoter and the nearest potential polyadenylation sequence approximately

8.3 kbdownstream (not shown) (2). Basedon thesequence

of strain 17 syn+ (9, 15), the distance from the TATAbox

(nucleotide 118774)tothestartof thepolyadenylation signal (nucleotide 127143) is8,369nucleotides. Thisdistance could varyby several hundred bases in different strainsbecause of

differences in the repeated regions near the junction. The

location of this faint, large LAT is consistent with our

TRL UL

IRLIRS

UTRS

NucleoUdepositon 118,00 125,000 132:000

CPO

TATA

&3kbLAT (unstable)

ICP4 PolyA

-I? ?

) \~~'7

2kb LAT(gable)

i? 2kb LATremoved ?

1.3kb LAT(stable)

6.3 kb LAT ?(unstable)?

bobgicslyactiveLAT?

FIG. 8. Proposed structure of the LAT gene. The HSV-1

ge-nomic organization in map units (mu) is shown atthe top. The

expanded region indicates the nucleotide positions relativeto the

entire HSV-1 DNA sequence. The approximate locations of the

ICPO and ICP4genesareshown forreference.Transcriptionofthe

LAT gene in the internal long repeat begins approximately 28

nucleotides from the first T of the TATA box (118774) approxi-matelyatnucleotide 118802 and extendstonearthepolyadenylation signal locatedatnucleotide 127143.The derivationofthe stable

2-and 1.3-kb LATsfromthe unstable 8.3-kb transcriptis likelybut

remainstobedemonstrated.The seriesof smallarrowsatthe end of

the8.3-kb LAT indicates that terminationmaybeinefficient, since

inoccasional Northern analyses of RNA from acutelyinfectedcells, using probes that correspond to the 5' end ofLAT, wedetected

LAT bands withapparentsizes of 12to 14 kb(not shown). If the

2-kb LAT is an intron, the 6.3-kb LAT shown maybe apossible result.

earliest results that detected weak in situ hybridization

signals with probes derived from BamHI SP, which is

downstream from the 2-kb LAT(20).

We originally proposed that the LAT promoter was

lo-catedover660 nucleotides upstream fromthe 5' end ofthe

2-kb LAT(30, 31),basedonsequenceanalysis showingthat

this area contained an excellent TATA box consensus

se-quence as well as other RNA polymerase II consensus sequences. Subsequently, wepublishedthe first report that

this upstream region was capable of promoter activity in

transientCATassaysinVerocells(32). This report

demon-stratesthat thisregionisalsocapableof promoteractivityin

transient assays in neuron-derived cells, as would be

ex-pectedfor the LAT promoter. Furthermore, wefound that

this promoteractivity hadneuron specificity. These results

again support the notion that this regioncontains the LAT

promoter.Theauthenticityof thisputativeLAT promoteris

furthersupported by recentreports (2, 7, 11, 24) indicating

that virus mutantswith deletions encompassing the TATA

boxregiondonotexpress anyLATduringneuronallatency.

To ensure that our transient CAT assays reflected

tran-scription initiatingfroma position consistent with itsbeing

directed by the TATA box consensus sequences, primer

extension experiments were done with RNA from cells

transfected with theA-promoter region(nucleotides -940to

-662). We found thattranscription began about 28

nucleo-tides from the first T of the TATA box sequence. This

confirmed that in vitro this region can function as a

pro-moter, further supporting the notion of this region as the LATpromoter.Thestartoftranscriptionthatwedetermined

by primer extension was in agreement with our in situ

12h 12 h

[image:8.612.113.242.73.264.2]

1

2

...

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8day exposure .3

....

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hybridization data in latently infectedneuronswhich placed

thestartoftranscriptionwithin30nucleotides of theTATA

box. Thisposition is also inagreementwiththatdetermined

for thestartof LATtranscription duringacutetissue culture

infection withan RNAprotection assay (2).

Theimmortalized neuroncell line and the neuroblastoma

cell line used for transient assays in this report are both nonpermissive for lytic HSV-1 infection. Furthermore, fol-lowingHSV-1 infection,LATisexpressed inthe

immortal-ized neuron cell line (Wheatley et al., submitted). These

characteristics mimic neurons during in vivo latency and make these cellsparticularlyrelevantfortheseexperiments. By usingtheseneuron-derived celllines, wefoundthat the presence of sequences upstream of the LAT promoter had

differential effectsonpromoteractivityin neuron-compared

to nonneuron-derived cells. In Vero, CV-1, BHK, and L

cells,upstream sequencesdecreasedpromoteractivity by

3-to 12-fold.This apparentdownregulationwas notobserved

inimmortalized neurons or neuroblastomacells. Infact, in

some

experiments

inneuron-derivedcells, addition ofthese upstream sequencesenhancedpromoteractivityasmuchas

300%relative to the Afragment (Fig. 3). Itis possible that

down

regulation

innonneuronal cellswascausedby

nonneu-ronal trans-acting cellularfactors interacting withthesecis

upstream sequences. This notion is supported by

prelimi-nary observations that cotransfection of nonneuronal cells

with exogenous upstream sequences appeared to compete

outdownregulation by theupstream sequencesinfragment

A+. Inaddition, transfection with largeamountsofthe A+

orA++ CATconstructs eliminated downregulation, again

suggesting

a

competitive

effect(datanotshown).

In summary, we have (i) used transient CAT assays in neuron-derived cells to map LAT-associated promoter

ac-tivity

to alocationover660nucleotides upstreamfromthe 5'

end ofthe stable 2-kb LAT,

(ii)

shownby primer extension

that in CAT constructs,transcription beganabout 28

nucle-otides from the firstT ofa consensus TATA box sequence

locatedatnucleotide 118774(-688), (iii) shownbya combi-nation ofNorthern blots and in situhybridization of latently infected neurons that transcription of a large 8.3-kb LAT

began

within30nucleotidesof the firstTof thisTATAbox sequenceataboutnucleotide118802(-660),and(iv) shown

that the LAT promoterhad neuronal

specificity.

Although

wehavenotproventhat the 2- and 1.3-kbLATs

are derived from the 8.3-kb LAT, this appears to be the

simplest

and the most

likely hypothesis,

since mutants

lacking only

the immediate

region

around the LAT promoter

did not

produce

eitherthe 8.3 kb LATorthe 2- and 1.3-kb LATs(2, 11).The

likely

derivation ofthe 2- and 1.3-kb LATs

from a

primary

8.3-kb LAT raises the

question

of the

processing

eventsinvolved.The1.3-kbLAT canbe derived from the 2-kb LAT by a simple splice (30). Likewise, the

2-kb LAT could be derived

by

a

simple splice

from the 8.3-kb LAT.

However,

if the 2-kb LAT is derived

by

a

single

splice,

either the 2-kb LATorthe 5' endofthe8.3-kb LAT

would have to be an intron. We havenotbeen able to find

anyexample ofa5' intron in the literature.

Therefore,

it is

likely

thateither

(i)

the 5' endof the 2-kb LAT containsan as

yetundetected smallexonfromthe 5'endofthe8.3-kbLAT or

(ii)

the 2-kb LAT is an intron. In the latter case, the

biologically

active LAT

might

be an

approximately

6.3-kb

polyadenylated

RNA

resulting

from the removal ofa 2-kb

intron fromthe 8.3-kb

primary transcript (Fig.

8).

Although

we cannot point to a

specific

6.3-kb LAT

band,

we have

foundthat Northernblotsfrom

acutely

infected cells

usually

detect

regions

of

LAT-specific

RNAs between the 2- and

8.3-kb LATs(data not shown). Some of this material could representsuch a6.3-kb RNA. If the 2-kb LAT is an intron, the coding region for a potential LAT protein would be somewhere in the putative 6.3-kb LAT. This might explain why a LAT protein coded by the 2-kb LAT has not been detected(28, 31).

It is also possible that the 2- and 1.3-kb LATs are not derivedby splicing but ratherarestableregions (perhaps due

tosecondary structure)thatareleft intact afterdegradation

of the less stable 8.3-kb primary transcript. In this case,

predictions ofthebiologically active regionof the LAT gene would be more difficult.

One intriguing feature of the 8.3-kb transcript is that it spanstheHSV-1junction. Thus, duringacuteinfection,the copy of the LAT gene in the internal repeat produces a

primary transcript of 8.3 kb that extends approximately 800 bases into the short repeat. Since the HSV-1 genome is

circularizedshortlyafteracuteinfection andalsoappearsto

be circularizedduring latency (17, 18) the LAT geneinthe

terminal repeat would make an identical 8.3-kb transcript.

However, ifatanytimetheHSV-1genomewereinalinear form,the copyofthe LAT genein theterminalrepeatwould produce a transcript that is truncated at about 7.5 kb by

running off the end of the HSV-1 genome. Recent results with some (2, 7,24) but not all (1, 6) LAT mutants suggest that LAT may be involved in HSV-1 reactivation. It is therefore tempting to speculate that reactivation may be

associatedwithashortperiodoflinearization ofthe genome and thatsomedifference betweenthe complete8.3-kb

tran-scriptand atruncated runofftranscript might play arolein the switch from latency to reactivation. Alternatively, one could also speculate that LAT may play some role in circularization(orlinearization) ofthe genome.

The resultspresentedhere suggest that the LAT promoter iscontrolled, in part, by upstream sequences that resultin

neuronalspecificity. Exactlywhat these sequences are, what cellular and viralfactors they interact with, and how they

regulateLATexpression remain to be determined.

ACKNOWLEDGMENTS

Thisworkwaspartially supported bytheDiscoveryFund forEye

Research,the FactorFamily Foundation,andPublic Health Service

grantsEY07566andEY05939.J.C.Z. isaFactorFamily

Founda-tion Scholar. K.P.isanIris andB. Gerald Cantor Scholar.

Wethank Anita AveryandRichard Lit for technical assistance

and JohnOng andDon Brownforsuggestionsandcriticalreadingof

themanuscript.

LITERATURE CITED

1. Block, T. M., J. G. Spivack, I. Steiner, S. Deshmane, M. T.

McIntosh, R. P. Lirette, and N. W. Fraser. 1990. A herpes

simplex virustype 1latency-associated transcript mutant

reac-tivates with normal kinetics from latent infection. J. Virol.

64:3417-3426.

2. Dobson,A.T.,F.Sederati,G.Devi-Rao,W. M.Flanagan,M.J.

Farrell, J. G.Stevens,E. K. Wagner,and L. T.Feldman.1989.

Identification of the latency-associated transcriptpromoterby

expressionof rabbitbeta-globinmRNAinmousesensorynerve

ganglia latently infected with a recombinant herpes simplex

virus. J. Virol. 63:3844-3851.

3. Gilman, M. Z., R. N. Wilson, and R. A. Weinberg. 1986.

Multiple protein-bindingsites in the5'-flankingregion regulate

c-fosexpression. Mol. Cell Biol. 6:4305-4316.

4. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.

Recombinant genomes which expresschloramphenicol

acetyl-transferase inmammaliancells. Mol.CellBiol. 2:1044-1051.

5. Graham,F.L.,andA.J.Van Der Eb.1973. Anewtechniquefor

the assayofinfectivityof humanadenovirus5 DNA. Virology VOL. 64,1990

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

52:456-467.

6. Ho, D. Y., and E. S. Mocarski. 1989. Herpes simplex virus latent RNA (LAT) is not required for latent infection in the mouse. Proc.Natl. Acad. Sci. USA 86:7596-7600.

7. Leib, D. A., C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks, D. M. Coen, D. M. Knipe, and P. A. Schaffer. 1989. Adeletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced

frequency. J. Virol. 63:2893-2900.

8. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: alaboratory manual.Cold Spring Harbor Laboratory,

ColdSpringHarbor, N.Y.

9. McGeoch, D. J., M. B. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J.Perry, J. E. Scott, and P. Taylor. 1988.The complete DNA sequence of the long uniqueregion in the genome of herpes simplex virus type 1. J. Gen. Virol. 69:1531-1574.

10. Mitchell,W. J., R. P.Lirette,and N. W. Fraser. 1990.Mapping

of low abundance latency-associated RNA in the trigeminal

gangliaofmice latently infectedwithherpes simplexvirustype

1.J. Gen. Virol. 71:125-132.

11. Mitchell, W. J.,I. Steiner, M. S. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser. 1990. A herpessimplex virus type 1 variant,deleted in the promoter region ofthe

latency-associated transcripts does notproduce any detectable minor

RNAspecies during latency inthe mousetrigeminalganglion.J.

Gen. Virol.71:953-957.

12. Nesburn, A. B., M. L. Cook,andJ. G. Stevens. 1972. Isolation

of herpes simplex virus: isolation from rabbit trigeminalganglia

betweenepisodes ofrecurrentocular infection. Arch.

Ophthal-mol.88:412-417.

13. Nesburn, A. B., J. M. Elliott, and H. M. Leibowitz. 1967.

Spontaneousrecurrenceof experimental herpes simplex

kerati-tis inrabbits. Arch. Ophthalmol. Vis. Sci. 78:523-529.

14. Paine, T. F., Jr. 1964. Latent herpessimplexinfectioninman. Bacteriol. Rev. 28:472-479.

15. Perry, L. J., and D. J. McGeoch. 1988. The DNAsequences of thelong repeat region and adjoining parts of the long unique

region in thegenome ofherpes simplex virus type 1. J. Gen.

Virol.69:2831-2846.

16. Post, L. E., A. J. Conley, E. S.Mocarski, and B. Roizman. 1980.

Cloning of reiteratedandnonreiterated herpes simplex virus1

sequences as BamHI fragments. Proc. Natl. Acad. Sci. USA 77:4201-4205.

17. Rock, D. L., and N. W. Fraser. 1983. Detection of HSV-1 genome in central nervous system of latently infected mice. Nature(London) 302:523-525.

18. Rock, D. L., and N. W. Fraser. 1985. Latentherpes simplex

virustype 1 DNAcontains twocopies ofthevirionDNAjoint

region.J. Virol. 55:849-852.

19. Rock, D. L., W. A. Hagemoser, F. A. Osorio, and D. E. Reed.

1986.Detectionof bovineherpesvirustype 1 RNAintrigeminal

ganglia of latently infected rabbits by in situ hybridization. J.

Gen. Virol. 67:2515-2520.

20. Rock, D. L., A. B. Nesburn, H. Ghiasi, J. Ong, T. L.Lewis, J. R. Lokensgard, and S. L. Wechsler. 1987. Detection of latency

related viral RNAs in trigeminal ganglia of rabbits latently

infected with herpes simplex virus type 1. J. Virol. 61:3820-3826.

21. Spivack, J. G., and N. W. Fraser. 1987. Detection of herpes

simplexvirustype 1transcriptsduringlatentinfectioninmice. J.Virol. 61:3841-3847.

22. Spivack, J. G., and N. W. Fraser. 1988. Expressionofherpes

simplexvirus type1(HSV-1) latency-associated transcripts and

transcriptsaffected bythedeletioninavirulentmutantHFEM:

evidence for anew class of HSV-1 genes. J. Virol.

62:3281-3287.

23. Spivack, J. G., and N. W. Fraser. 1988. Expression of herpes

simplexvirustype 1latency-associated transcriptsin the

trigem-inalganglia of mice duringacuteinfection and reactivation of

latentinfection.J. Virol. 62:1479-1485.

24. Steiner, I., J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean,J. H.Subak-Sharpe, and N. W. Fraser. 1989. Herpes

simplexvirustype 1latencyassociatedtranscripts are evidently

notessentialfor latent infection. EMBO J. 8:505-511.

25. Stevens, J. G., E. K. Wagner, G. B.Devi-Rao,M. L.Cook, and L. T. Feldman. 1987. RNA complementary to aherpesvirus

alpha gene mRNA is prominant in latentlyinfected neurons.

Science 235:1056-1059.

26. Stroop, W. G., D. L. Rock, and N. W. Fraser. 1984.Localization

of herpessimplex virus inthetrigeminalandolfactorysystems

of the mouse central nervous systemduring acute andlatent

infectionsbyin situhybridization. Lab.Invest.51:27-38.

27. Vahlne, A., and E. Lyke.1978.Herpessimplex virus infection of

in vitro cultured neuronal cells (mouse neuroblastoma C1300

cells).J.Gen. Virol. 39:321-332.

28. Wagner, E. K., G. Devi-Rao, L. T. Feldman, A. T. Dobson, Y. Zhang, W. M. Flanagan, and J. G. Stevens. 1988. Physical

characterizationof theherpes simplexviruslatency-associated

transcriptin neurons. J.Virol. 62:1194-1202.

29. Wechsler, S. L., A. B. Nesburn, R. J. Watson, S. Slanina, andH.

Ghiasi. 1988.Finemappingof themajor latency-relatedRNA of

herpessimplexvirus type 1inhumans. J.Gen. Virol.

69:3101-3106.

30. Wechsler, S.L., A. B. Nesburn, R. Watson, S. M. Slanina, and H. Ghiasi. 1988. Fine mapping of the latency-related gene of

herpes simplex virustype 1: alternative splicing produces

dis-tinct latency-related RNAscontainingopen reading frames. J.

Virol. 62:4051-4058.

31. Wechsler, S. L., A. B. Nesburn, J. C. Zwaagstra, and H. Ghiasi.

1989. Sequence ofthe latencyrelated geneof herpes simplex

virustype 1. Virology168:168-172.

32. Zwaagstra, J. C., H. Ghiasi, A. B. Nesburn, and S. L. Wechsler.

1989. In vitro promoter activity associated with the latency

associated transcriptgene of herpes simplex virus type 1. J.

Gen. Virol.70:2163-2169.