Activity of Herpes Simplex Virus Type 1 Latency-Associated

Vol. 64, No. 10

Activity of Herpes Simplex Virus Type 1 Latency-AssociatedTranscript (LAT) Promoter in Neuron-Derived Cells: Evidence for

Neuron Specificity and for a Large LAT TranscriptJOHN C. ZWAAGSTRA,1 HOMAYON GHIASI,"2 SUSAN M. SLANINA,1 ANTHONY B. NESBURN,"2S. 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, Los Angeles,California 90048-18691*; Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 900242;

and Department ofBiochemistry, University College and Middlesex School of Medicine,3and Sandoz Research Institute,4 London, WCIE 6BN United Kingdom

Received 4 August 1989/Accepted 17 July 1990

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here thatpromoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) hadneuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containingexcellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus

TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences

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, inneuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LATpromoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end ofthe previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern(RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected inour CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latentlyinfected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA boxsequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization ofRNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. Weconclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcriptionbegins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the firstavailable polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstableprecursor of the more stable 2- and 1.3-kb LATs.

Following primary infection in humans, herpes simplexvirus type 1 (HSV-1) establishes latent infections in sensoryneurons (14). During HSV-1 latency, detectable viral tran-scription is limited to an area within the genomic longrepeats in the vicinity of the immediate early gene ICP0 (20,21, 25). At least two abundant and stable latency-associatedtranscripts (LATs) (2 and 1.3 kilobases [kb]) that share their5' and 3' ends are derived by alternative splicing (30). TheseLATs partially overlap the 3' end of ICP0 and are antisense(complementary) to the ICP0 mRNA (20, 25, 29, 30). The 5'end of the stable 2-kb (and 1.3-kb) LAT in the internal longrepeat has been mapped to within a few bases (28-30). Thisposition corresponds to nucleotide 119462 of the genomicsequence (9, 15). A second copy of the LAT gene is presentin the corresponding location of the terminal long repeat.Recent reports with some LAT deletion mutants suggest thatLAT may play a role in reactivation of the virus from thelatent state (2, 7, 24). However, this is not supported by allLAT mutants (1, 6).Based on sequence analysis, we initially proposed that the

LAT promoter is located over 660 nucleotides upstream of

* Corresponding author.

the mapped 5' end of the stable LATs (30, 31), and later, byusing chloramphenicol acetyltransferase (CAT) assays, we

confirmed that in Vero cells, LAT promoter activity coin-cides with this region (32). This location for the LATpromoter is further supported by more recent studies inwhich viral mutants lacking the predicted promoter regionfailed to produce any LAT RNA during latent infections (2,7, 11, 24).

Since LAT is the only HSV-1 gene that is abundantlyexpressed during neuronal latency (20), the LAT promotermust be controlled differently from other HSV-1 gene pro-moters (22, 23). Furthermore, the LAT promoter is likely tohave neuronal specificity, since LAT is abundant in latentlyinfected neurons (20, 25) but is present at only low levelsduring acute tissue culture infection (22). We have thereforeextended our studies of the LAT promoter to neuron-derivedcells to confirm the location of the LAT promoter and to lookfor neuronal specificity.We show here that in neuron-derived cell lines, promoter

activity mapped to the same location as it did in Vero cells.Furthermore, we found that in nonneuronal cells, sequencesupstream of the TATA box decreased promoter activity incis, while in neuron-derived cells, inhibition by these cis-acting sequences was not observed. These upstream se-

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5020 ZWAAGSTRA ET AL.

quences may confer neuron specificity on the HSV-1 LATpromoter in vivo. We also show here that LAT transcriptionbegan near the promoter and appeared to continue forapproximately 8.3 kb, the location of the first-occurringconsensus polyadenylation site. A likely interpretation ofthis data is that this 8.3-kb RNA may be an unstable primaryLAT transcript that gives rise to the previously mapped,relatively abundant 2- and 1.3-kb LAT transcripts.

MATERIALS AND METHODS

Cells and virus. Plaque-purified herpes simplex virus type1 (HSV-1), strain McKrae, was grown as previously de-scribed (20) and was used for all infections. Cells were grownas monolayers in minimal essential medium, Dulbecco mod-ified Eagle medium, or F12 (GIBCO Laboratories, Inc.)supplemented with 10% fetal calf serum and antibiotics.Neuroblastoma cells (NB41A3; American Type Culture Col-lection CCL147) are of mouse origin. These neuroblastomacells retain several neuronal markers, including acetylcho-linesterase activity. In addition, they are nonpermissive forHSV-1 infection (27). Immortalized neurons were made byfusing H18TG2 cells, a 5-azaguanine-resistant mouse neuro-blastoma cell line, with neonatal rat dorsal root ganglianeurons. These cells are nonpermissive for lytic HSV-1infection and express LAT following HSV-1 infection (S. C.Wheatley, C. Dent, K. Lillycrop, L. M. Kemp, J. N. Wood,and D. S. Latchman, submitted for publication).

Plasmids. All restriction fragments were derived from theBamHI B restriction fragment from HSV-1 strain F (16). TheLAT gene from position -2592 to +663 relative to the 5' endof the stable 2-kb LAT was divided into five restrictionfragments. Fragments A to C and the details of their cloningin the proper orientation in front of the CAT gene withinplasmid pSVOCAT (4) have been previously described (32).Fragments A+ and A+ + were similarly cloned and consistof fragment A with additional upstream sequences as de-tailed in the legend to Fig. 2 and in Fig. 3. For someexperiments, similar constructs were made by using a dif-ferent promoterless CAT plasmid, p1O6CAT (3), in place ofpSVOCAT.CAT assays and quantitation procedures. CAT assays and

quantitation procedures have been previously described(32). Briefly, cell monolayers at approximately 60% conflu-ency, on 60-mm plates, were transfected with CAT con-structs by the calcium phosphate precipitation method (5).After 46 h, the cells were harvested and cell extracts wereprepared. Within an experiment, equal cell numbers wereused and, if necessary, corrections were made for theamount of protein present in the extracts. Acetylated formsof ['4C]chloramphenicol were detected by thin-layer chro-matography and subsequent autoradiography. The amountof acetylated and unacetylated chloramphenicol was quanti-tated by excising the spots from the thin-layer plates andcounting in a liquid scintillation counter. In some experi-ments, samples of cell extracts were analyzed by DNA dotblot hybridizations with a CAT-specific probe to determinethe relative amount of CAT DNA that had entered the cellsduring the transfection. Within a cell line, no differenceswere seen between the transfection efficiencies of fragmentsA+ +, A+, or A. Thus, differences between the CATactivities of these plasmids within a cell line were not a resultof differences in transfection efficiency. The differences intransfection efficiencies between cell lines was partiallycompensated for by using 10 ,ug of each plasmid in immor-talized neurons, BHK cells, and L cells; 5 ,ug of each

plasmid in neuroblastoma cells and CV-1 cells; and 2.5 ,ug ofeach plasmid in Vero cells.Primer extension. As modified from a procedure of P.

Krause and J. Ostrove (personal communication), 10 ,ug ofRNA from transfected Vero cells (isolated by the guanidin-ium-cesium chloride method [8]) was suspended in 10 RI of20 mM Tris-hydrochloride, pH 7.6-100 mM NaCl-0.1 mMEDTA. A 10-ng portion of 32P-end labeled CAT primer(approximately 5 x 105 cpm) was added. The sequence ofthe primer from 5' to 3' was GATGCCATTGGGATATATCAACGGT. This sequence is complementary to the CATmRNA sequence located between nucleotides 127 and 151downstream from the first T of the LAT TATA box in theCAT construct used for the transfection. This location wasconfirmed by partial sequencing of the plasmid and corre-sponds to CAT nucleotides 28 to 52 relative to the ATGcodon at which CAT translation initiates. The differencebetween these numbers is the result of a HindIll linker, amultiple cloning region, and noncoding CAT sequencesbetween the end of the LAT sequences and the start of thestructural CAT sequences in this plasmid. The reactionmixture was heat denatured for 3 min at 90°C, hybridized at55°C for 10 min, and slowly (approximately 1 h) cooled to30°C. Primer extension of the hybridized primer was donewith 20 U of cloned Moloney murine leukemia virus reversetranscriptase (Bethesda Research Laboratories, Inc.) at 37°Cfor 1 h according to the instructions of the manufacturer byusing 2 mM deoxynucleoside triphosphate, 100 ng of bovineserum albumin per Pd, and 20 U of RNasin (Promega Biotec)per 20-pdl reaction. The reaction was stopped by addingEDTA to 10 mM. The extended primer was precipitated with2 volumes of 95% ethanol, washed with 70% ethanol, sus-pended in formamide-dye loading buffer, heat denatured at95°C for 3 min, quick chilled on ice, and run on a 10%acrylamide sequencing gel containing 7 M urea. The gel wasthen processed for autoradiography. In one experiment thesame oligonucleotide used in the primer extension reactionwas used to prime a sequence reaction from the A-CATplasmid by using a Sequenase DNA sequence kit fromUnited States Biochemical Corporation. Products from thesequence reaction were run next to the primer-extendedproduct on a 10% sequencing gel containing 7 M urea.

Rabbits. New Zealand White male rabbits (approximately2 kg each) were used for all animal experiments. Theseanimals develop a primary and recurrent herpetic disease(13) which mimics HSV-1 keratitis in man.

Latent ganglionic HSV-1 infections. Latent ganglionicHSV-1 infections were done as previously described (20).Briefly, rabbits were bilaterally infected without cornealscarification by placing approximately 1 x 105 to 2 x 105PFU of virus into the conjunctival cul-de-sac, closing theeye, and rubbing gently for 30 s. Rabbits surviving after 4weeks were considered latently infected (12).

Rabbit trigeminal ganglia. Rabbit trigeminal ganglia weretaken from sacrificed rabbits and immediately placed inliquid 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 cuttingsections of trigeminal ganglia were done as described previ-ously (19). Hybridizations to identify RNA were done as wepreviously described (20, 30) by using 32P-labeled syntheticoligonucleotides as probes. Slides were exposed to photo-graphic emulsion for 2 to 3 days. Pretreatment of slides withRNase, but not DNase, eliminated hybridization. Three tofive 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 eachprobe. No hybridization with any of the probes resulted in anaccumulation of grains over any neurons from uninfectedrabbits. Positive probes showed an accumulation of grainsover the nuclei of some neurons from latently infectedrabbits compared with the amount of background grains onthe slide and compared with uninfected neurons (see Fig. 5).Positive probes detected positive neurons from at least twolatently infected rabbits, as judged by detection of at leastone positive neuron on at least two different slides from agiven rabbit.

Northern blot hybridizations. Total RNA was isolated fromuninfected or latently infected rabbit trigeminal ganglia fro-zen in liquid nitrogen or from uninfected or acutely infected(multiplicity of infection of 20; 18 h postinfection) CV-1 cellsas previously described (20). Northern blot hybridizationswere done as we previously described (20, 30).

Hybridization probes. Random-primed labeling with 32pwas done on linearized plasmids following the instructions ofthe manufacturer (Amersham Corp.). Synthetic oligonucle-otides (20-mers) were synthesized by using beta-cyanoethylphosphoramidite chemistry on a Pharmacia Gene Assem-bler. The sequences of the 20-mers were based on sequencesfor HSV-1 strains 17 syn+ (15) and F (31). End labeling ofoligonucleotides with [y-32P]ATP was done as describedpreviously (8).

RESULTS

CAT activity in neuron-derived cells. By using CAT assays,we previously showed that in Vero cells, LAT promoteractivity coincides with RNA polymerase II promoter con-sensus sequences (32) that are located over 660 nucleotidesupstream from the 5' end of the stable 2-kb LAT (30, 31). Todetermine whether LAT promoter activity in neuron-derivedcells mapped to the same unusual upstream region, weassessed the ability of portions of the LAT gene to consti-tutively function as a promoter in immortalized neurons andin neuroblastoma cells. DNA restriction fragments from theLAT gene were individually cloned into the plasmidpSVOCAT (4) in front of the CAT structural gene. Theproper orientation of each fragment was confirmed by re-striction enzyme analysis.

Cells were transfected with individual CAT constructs,and 46 h later CAT activity was measured. Representativeresults are shown in Fig. 1. The location of each fragmentrelative to the 5' end of the stable 2-kb (and 1.3-kb) LAT isshown at the bottom of the figure. Numbers above each laneindicate the percent conversion of [14C]chloramphenicol.Fragment A (-940 to -662) had CAT activity in immor-

talized neurons (Fig. la, lane A) and in neuroblastoma cells(Fig. lb, lane A) that was comparable to that ofpSV2CAT (astrong positive control containing CAT under the control ofthe simian virus 40 early promoter) (Fig. la and b, lanepSV2) in the same cells. This was indicative of significantpromoter activity by fragment A in these cells. Fragments Band C, which encompass the region from -662 to +663, hadno CAT activity in either neuron-derived cell line. Theseresults were identical to those we previously reported inVero cells (32). This mapped the in vitro promoter activityassociated with the LAT gene to the same location (i.e.,within fragment A) in two neuron-derived cell lines as inVero cells. As shown in the expanded view at the bottom ofFig. lb, fragment A contains several consensus sequencesnormally associated with an RNA polymerase II promoter,including a TATA box, a potential CAAT box, and several

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FIG. 1. CAT activity in neuron-derived cells of different DNAfragments derived from the LAT gene. Different portions of theLAT gene (32) cloned in front of the gene for CAT (in plasmidpSVOCAT [4]) were individually transfected into immortalized neu-rons (a) or neuroblastoma cells (b), and CAT activity was measuredas described in Materials and Methods (lanes A, B, and C). LanespSV2 are plasmid pSV2CAT (4), containing the simian virus 40 earlypromoter, as a positive control. Transfections were done with 10 ,ugof each plasmid in immortalized neurons and 5 j±g of each plasmid inneuroblastoma cells. Acetylated forms of [14C]chloramphenicolwere detected by thin-layer chromatography and subsequent auto-radiography. The LAT gene fragments are designated A to C (32)and are represented schematically at the bottom of panel b (also seeFig. 3). Nucleotide positions are relative to the 5' end of the stable2-kb LAT (nucleotide + 1) (28, 30), which corresponds to nucleotide119462 of the HSV-1 genome (9, 15). Numbers above each laneindicate the percent conversion of unacetylated [14C]chloramphen-icol to acetylated forms.

Spl sites (30, 31). This region also contains a sequencehaving significant homology to a Vmw65 binding site(TAATGARAT) (32).

Sequences upstream of fragment A decrease LAT promoteractivity in nonneuronal cells. To look for potential cis regu-lation by sequences upstream of the LAT promoter, twoadditional CAT constructs were made. Fragment A+ (-1271to -662) consisted of fragment A plus 331 upstream nucle-otides. Fragment A+ + (-2592 to -662) consisted of frag-ment A plus 1,652 upstream nucleotides. Fragments A+ +,A+, and A have a common 3' end. Fragments A+ and A+ +were cloned into plasmid pSVOCAT in the same manner asfragment A.The effect of these upstream sequences on promoter

activity in neuronal and nonneuronal cell lines was examined

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FIG. 2. The effect of upstream sequences on CAT activity driven by the LAT promoter in neuron- and nonneuron-derived cells. DNAfragments containing the LAT promoter region and different amounts of upstream sequences were cloned into pSVOCAT at the same locationused for the fragments in Fig. 1. Fragments A, A+, and A+ + have a common 3' end (nucleotide -662 relative to the 5' end of the stable 2-kbLAT). This is 26 nucleotides downstream from the first T in the TATA box sequence. Fragment A is 278 nucleotides long. Fragment A+ is609 nucleotides long. Fragment A+ + is 1,930 nucleotides long. These fragments are shown schematically in Fig. 3. Plasmids were individuallytransfected into neuron-derived cells (neuroblastoma or immortalized neurons) or nonneuron-derived cells (BHK, Vero, CV-1, or L).Representative results are shown. Immortalized neurons (lanes 4 to 6), BHK cells (lanes 7 to 9), and L cells (lanes 15 to 17) were transfectedwith 10 tg of each plasmid. Neuroblastoma cells (lanes 1 to 3) and CV-1 cells (lanes 12 and 13) were transfected with 5 ,ug of each plasmid,and Vero cells (lanes 10 and 11) were transfected with 2.5 ,ug of each plasmid. Numbers above the lanes indicate the percent conversion of[14C]chloramphenicol. Numbers in parentheses indicate the activity relative to fragment A for each cell line.

by CAT assays. Representative results are shown in Fig. 2.The percent conversion of ['4C]chloramphenicol is indicatedabove each lane in Fig. 2. The numbers in parenthesesindicate the percent activity of the other plasmids relative toA (100%) within each cell line. In this particular experiment,the addition of the upstream sequences contained in frag-ments A++ and A+ had little effect on the activity of theLAT promoter in neuron-derived cell lines (83 to 131% offragment A activity; lanes 1 to 6). In other experiments,these upstream sequences increased promoter activity up tothreefold (summarized in Fig. 3). In contrast, in BHK cells(lanes 7 to 9), the upstream sequences contained in fragmentA+ + and A+ reduced fragment A's promoter activity aboutthreefold. The effect of upstream sequences was furtherexamined in (nonneuronal) Vero and CV-1 cells, usingfragment A+ +. The upstream sequences caused a reductionin CAT activity of about 6- to 12-fold in these cells (lanes 10and 12).

Since both of the neuron-derived cell lines we used wereof mouse origin, it was important to compare these results tothose in nonneuronal cells of mouse origin. In L cells (acommonly used mouse cell line) there was a greater thanthreefold reduction in LAT promoter activity by upstreamsequences (Fig. 2, lanes 15 and 16). This was similar to BHKcells (Fig. 2, lanes 7 to 9) and confirmed that this phenome-non was related to the neuronal and not the species origin ofthe cells.To partially correct for transfection efficiency between

different cell lines, different amounts of plasmid were used indifferent cell lines (within a cell line, equal amounts of allplasmids were used). We used 2.5 ,ug of plasmid per plate forVero cells, 5 ,ug of plasmid per plate for CV-1 and neuro-blastoma cells, and 10 ,ug of plasmid per plate for immortal-ized neurons, L cells, and BHK cells. Within a given cellline, these concentrations gave similar CAT activity withfragment A and with the positive control plasmid pSV2CAT(Fig. la and b, lanes A and pSV2 for neuronal cells; Fig. 2,lanes 16 and 17 for L cells; not shown for other nonneuronalcells). Thus, in CAT assays, fragment A (containing theminimal LAT promoter) had promoter activity similar to that

of pSV2CAT and relative to pSV2CAT appeared to be anequally effective promoter in all cell types.To ensure against the unlikely possibility of a plasmid-

specific artifact, some experiments were also done withfragments cloned into a different CAT-containing plasmid(p106CAT [3]). Similar results were obtained (not shown).

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FIG. 3. LAT gene fragments used in CAT constructs. The upperportion of the figure shows the HSV-1 genomic organization in mapunits (mu). The expanded region indicates nucleotide positionsrelative to the entire HSV-1 DNA sequence (15) and relative to the5' end (+1) of the stable 2- and 1.3-kb LATs. The locations of theTATA box sequences and of the 2- and 1.3-kb LATs are shown forreference. The LAT gene fragments are shown as horizontal rec-tangles and are labeled A++, A+, and A to C, as in Fig. 1 and 2.Solid rectangles indicate CAT (promoter) activity comparable tothat of pSV2CAT in the same cells. Dotted rectangles indicatereduced activity. Open rectangles indicate no activity. Numbers inparentheses show the range of activity of A+ + and A+ relative toA in over 5 independent experiments.

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FIG. 4. Primer extension mapping of the transcriptional start site of the LAT promoter-CAT plasmid. Primer extension was done asdescribed in Materials and Methods by using a 32P-end-labeled 25-nucleotide primer complementary to CAT mRNA, and the gel wasprocessed for autoradiography. All lanes represent the result of primer extension with RNA from transfected Vero cells. (a) Lane 1, p106CAT(promoterless CAT); lane 2, plasmid A (p1O6CAT with fragment A); lane 3, untransfected control. Marker lane is 32P-end-labeled Hinfl-cutijx174 DNA. The size of the primer extension product is approximately 123 nucleotides, corresponding to a transcriptional start siteapproximately 28 nucleotides from the first T of the TATA box sequence. (b) The same oligonucleotide used for primer extension experimentswas used as a primer in a set of sequencing reactions (see Materials and Methods). This generated the sequence shown in lanes 1 to 4, whichis complementary to the RNA produced by this plasmid with the LAT TATA box region as a promoter. The predicted sequence of the regionfrom the TATA box to the beginning of the Hindlll linker used to clone the LAT sequences into the plasmid is shown to the left of the gel.Lane 5 shows the result of a primer extension experiment similar to that in panel a. The extended primer comigrates with the first T in theHindlIl linker. This is 28 nucleotides from the first T in the TATA box.

The unlikely possibility that the decrease in promoter activ-ity seen with A++ and A+ could be due to a decrease intransfection efficiency (compared with A) of these plasmidsin nonneuronal compared with neuronal cells was addressedas follows. In some experiments, a portion of the cell extractused for the CAT assay was monitored for transfectionefficiency by DNA dot blots by using a CAT-specific 32P-labeled DNA probe. As expected, although transfectionefficiencies differed between cell lines, within a given cell nodifferences in transfection efficiency that could account forthe decreased promoter activity of A+ + or A+ comparedwith A were detected (not shown).

In vitro LAT transcriptional start site. To determine theapproximate location of the start of transcription in the CATconstructs, primer extension experiments were done asdescribed in Materials and Methods. RNA was preparedfrom transfected or untransfected cells and hybridized with a32P-end-labeled 25-nucleotide-long synthetic oligonucleotideprimer. The primer was complementary to CAT mRNAsequences located 127 to 151 nucleotides downstream fromthe first T of the LAT TATA box (see Materials andMethods). The primer was extended with reverse tran-scriptase, denatured, and sized on a polyacrylamide gel (Fig.4a). Lane 1 shows the result of primer extension with RNAprepared from cells transfected with p1O6CAT (promoterlessCAT, without fragment A). Lane 3 shows a primer extensionreaction with RNA from cells that were not transfected. Noproduct is seen in either control lane. Lane 2 shows theresult of a primer extension reaction with RNA from cellstransfected with p1O6CAT containing fragment A. We esti-mated the size of the major extended product as about 123nucleotides. Smaller faint bands are probably the result ofpremature termination of the primer extension reaction.Since the primer was complementary to nucleotides 127 to

151 relative to the first T of the TATA box, a size of 123nucleotides placed the start of transcription at about 28nucleotides from the first T of the TATA box (151 minus123). This would be HSV-1 nucleotide 118802 (9) or nucleo-tide -660 relative to the 5' end of the stable LATs. The LATsequence in this region is "...GCCGATCGCGG...," withthe underlined T being the approximate transcriptional startsite.To further confirm the transcriptional start site, the result

of a similar primer extension reaction was sized relative tothe sequence of the CAT plasmid on a sequencing gel (Fig.4b). The CAT plasmid sequence was obtained by using thesame oligonucleotide primer to prime a sequence reactionfrom the A-CAT plasmid (see Materials and Methods). Theextended primer comigrated with the underlined T in thesequence "...CGGCGITCGAA..." on this gel. The corre-sponding LAT sense strand sequence is "...GCCGCAAGCTT.." This is the same location relative to the TATA box(28 nucleotides from the first T) as estimated from Fig. 4a. Inthis case, this base is an A, not the T found at this locationin the LAT sequence. This A is the second base in theHindIlI linker used to clone the LAT A fragment in this CATconstruct.Our finding that in vitro transcription started approxi-

mately 28 nucleotides downstream from the LAT TATA boxstrongly supports the notion that CAT transcription in theseplasmids is under the control of the LAT sequences infragment A. Furthermore it demonstrates that the LATsequences near the TATA box are capable of functioning asa typical promoter in vitro. This supports the notion thatfragment A contains the LAT promoter and makes it likelythat during neuronal latency in vivo, LAT transcription maybegin at or near the same position (i.e., -660).

In situ hybridization near the LAT promoter. The CAT

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FIG. 5. In situ hybridization of trigeminal ganglia from latently infected rabbits. Trigeminal ganglia from latently infected or uninfectedrabbits were removed, sectioned, and processed for in situ hybridization as described in Materials and Methods. All probes were syntheticoligonucleotides (20-mers) end labeled with [32P]ATP. (A) Section from a latently infected rabbit hybridized to a probe corresponding to (thecomplement of) nucleotides +1292 to +1311 from within the stable 2-kb LAT. (B) Uninfected rabbit with the same probe as in panel A. (C)Latently infected rabbit with a probe from the region between the TATA box and the 5' 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 the 5' end of the stable 2-kb LAT (probe 11from Fig. 6). The arrow points to a faintly positive cell.

assays and the primer extension results reported above bothstrongly suggest that the LAT promoter is located over 660nucleotides upstream of the start of the stable LATs. To lookfor small amounts of LAT RNA between the LAT TATAbox and the 5' end of the stable LATs, we carefully analyzeda series of in situ hybridizations to sections from trigeminalganglia of rabbits latently infected with HSV-1.

Figure 5 shows representative in situ hybridization resultsin which 32P-end-labeled synthetic oligonucleotides wereused as probes. These 20-mers were based on the DNAsequence of this region (15, 31) and were constructed so thatRNA detected with these probes would be the same sense asLAT RNA. To eliminate possible false positives, computeranalysis was done to ensure that no 20-mer would havehomology to any other portion of the LAT region (30).Several 20-mers were also rejected because they hybridizedto neurons from uninfected rabbits. Panel A shows thestrong hybridization typical of a probe corresponding to aregion within the stable 2-kb LAT to a section from the

trigeminal ganglia of a rabbit latently infected with HSV-1.Panel B shows lack of hybridization with the same probe toa section from an uninfected rabbit. Panels C and D showfaint hybridization to latently infected sections using probesbetween the LAT promoter and the 5' end of the stable 2-kbLAT. The number of grains in these positive cells is greatlyreduced compared with panel A, indicating much less LATRNA in the positive cell. In addition, the number of cellsshowing faint hybridization was only about 10% of that seenwith probes hybridizing to the 2-kb LAT.The faint hybridization seen with the probes in panels C

and D (Fig. 5) was at the limit of detectability in our latentlyinfected rabbit system. Under normal circumstances, inwhich one expects to see numerous, strong hybridizationsignals, these probes might have been scored as negative.However, it was clear that these probes were not simply"negative." In this analysis we therefore considered a probepositive if at least one neuron was faintly positive (as in Fig.SC or D) on at least two sections from each of at least two

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

TATA

Probes 1 2 3

BatnHI EooRv LAT- RNA M

5'

4 5 6 78910H E S

Start 1.3 & 2 kb LAT

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 situ hybridization.Results from the experiment partially shown in Fig. 5 are shown schematically. In situ hybridization with each 32P-end labeled 20-mer wasperformed on 3 to 5 sections of trigeminal ganglia from each of four rabbits latently infected with HSV-1. The 20-mers were based on thesequence of this region and were of complementary sense to LAT RNA. The approximate locations of the probes relative to the 5' end ofthe stable 2-kb LAT (+1) are shown at the top. Exact locations 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 of hybridization similar to Fig. SC and D, detected in at least twoof the four latently infected rabbits (one or more neurons in at least two sections per rabbit). Open boxes indicate that no hybridization wasdetected. The dark rectangle indicates the start of intense hybridization (as shown in Fig. 5A) at the start of the stable 2- and 1.3-kb LATs(30). The large open rectangle (note break) represents our previous results (29, 30), showing no hybridization to a BamHI-EcoRV restrictionfragment (-6141 to -824). The direction of LAT transcription is from left to right, as indicated by the large arrow.

latently infected rabbits. Each probe was hybridized to 3 to5 sections of trigeminal ganglia from each of four rabbitslatently infected with HSV-1. On this basis, at least 9 of the12 probes tested between the TATA box and the 5' end ofthe stable 2-kb LAT hybridized faintly to sections of trigem-inal ganglia from latently infected rabbits (Fig. 6). The lackof detected hybridization with the remaining three probeswas probably due to technical limitations related to detectingextremely small amounts of RNA. None of these probeshybridized to sections from uninfected rabbits (not shown).Similar results were also obtained with sections of humantrigeminal ganglia (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 of the TATA box,and probe 2 (-707 to -688), immediately to the left of theTATA box (at -688 to -684), were negative (Fig. 6, probes1 and 2). In addition, we have previously shown that arestriction fragment probe from -6140 to -824 (BamHI-EcoRV) does not hybridize to latently infected ganglia fromrabbits or humans (29, 30) (Fig. 6). Probe 3 (-658 to -639),the first probe to the right of the TATA box, was positive.This placed the start of transcription between probes 2 and 3or within 30 nucleotides of the first T of the TATA box. Thisis in excellent agreement with our in vitro primer extensiondata, which placed the start of LAT transcription 28 nucle-otides from the TATA box. These results strongly suggestthat this TATA box sequence is the functional TATA box ofthe LAT promoter. The greatly reduced amount of detect-able RNA between the promoter and the stable LATssuggests that the RNA from this region was relativelyunstable.

Northern blot hybridization of RNA from latently infectedrabbits. Northern blot hybridization ofRNA from trigeminalganglia of latently infected rabbits did not reveal any smallbands in the 500- to 700-base range that might represent theregion between the TATA box and the 5' end of the 2-kbLAT. However, by using probe 8 from Fig. 6, which islocated between the TATA box and the 5' end of the 2-kbLAT, a very faint band with an estimated size of 8 to 9 kbwas detected (Fig. 7, lane 2; designated 8.3 kb). For com-parison, an equal amount of the same RNA preparationhybridized with a probe that was specific for the 2-kb LATregion is shown in lane 1. This probe was the same size andspecific activity as the probe in lane 2.Lanes 1 and 2 (Fig. 7) show a typical exposure for the

detection and analysis of the stable LATs. (The 1.3-kb LAT

is not seen in lane 1 because this probe is entirely within theintron of the 1.3-kb LAT.) The band designated 8.3 kb isalmost indistinguishable in lane 2 and would not normally beconsidered meaningful. However, by overexposing the au-toradiogram, the 8.3-kb band was detected above the back-ground (lane 3). The detection of this band in RNA fromlatently infected rabbits was a rare event. This band was notdetected by other laboratories (2, 11) nor was it detected inthe majority of our RNA preparations (not shown). Sincethis RNA was barely detectable with the much more sensi-tive in situ hybridization assay, the inability of Northernblots to detect this RNA consistently was not unexpected.The simplest explanation for the 8.3-kb band was that it

represented an RNA initiating near the LAT promoter and(because of its size) extending through and past the 2-kbLAT. Examination of the published HSV-1 sequence (15)revealed that the first consensus polyadenylation signal(AATAAA) was approximately 8.3 kb downstream from theLAT promoter, just 34 nucleotides prior to the ICP4 polya-denylation signal on the opposite DNA strand (see Fig. 8).We detected a similar 8.3-kb band during acute tissue cultureinfection and were able to map it to the region between theTATA box and the potential polyadenylation site by usingoligonucleotide probes flanking these regions (results notshown). A similar result with RNA from acutely infectedtissue culture cells was recently reported (2), while thismanuscript was in preparation. Thus, the faint 8.3-kb bandwe detected in latently infected neurons may be an unstable,possibly polyadenylated, primary LAT transcript fromwhich the 2- and 1.3-kb LATs are derived.

DISCUSSION

When we first proposed that the LAT promoter waslocated over 660 nucleotides upstream of the 2-kb LAT, wealso raised the possibility that LAT transcription might startnear this promoter region (30, 31). This was based on the factthat in ganglia from latently infected rabbits, we occasionallydetected what appeared to be very faint in situ hybridizationwith probes upstream of the 2-kb LAT (30). Since then otherlabs have reported in situ hybridization in this region inganglia from latently infected mice (2, 10). However, theseexperiments were done by using large, nonoverlapping re-striction fragments as probes and the start of transcriptioncould not be precisely determined. In this report, the use ofshort oligonucleotide (20-mers) probes enabled us to deter-mine that during neuronal latency in rabbits, transcription of

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-8.3 kb* 28S

* 18S

FIG. 7. Northern blot hybridization of RNA from ganglia oflatently infected rabbits. RNA was isolated from trigeminal gangliaof latently infected rabbits, and Northern blot hybridizations were

done as described in Materials and Methods by using 32P-end-labeled 20-mers as probes. Each lane contains approximately 20 Fgof total RNA. Lane 1 shows hybridization with an oligonucleotidecorresponding to (the complement of) nucleotides +261 to +280.This region is within the 1.3-kb LAT intron and therefore hybridizesto the 2-kb LAT but not the 1.3-kb LAT (30). Lane 2 showshybridization with oligonucleotide number 8 from Fig. 6, which ismidway between the TATA box sequence and the 5' end of thestable 2-kb LAT. These probes were labeled to the same specificactivity, and the same amount of radioactivity was used to probelanes 1 and 2. Lanes 1 and 2 represent an autoradiogram exposure of12 h and are from the same gel. Lane 3 is an 8-day overexposedautoradiogram of lane 2. The point marked 2 kb indicates thelocation of the major 2-kb LAT. The point 8.3 kb marks the largeband visible in lane 2 and 3. The apparent size of this band is 8 to 9kb, 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-kbdesignation is consistent with this apparent size and corresponds tothe distance from the TATA box sequence to the first consensus

polyadenylation sequence. None of these probes hybridized to RNAfrom uninfected rabbits (30).

the faint LAT RNA began immediately downstream from theputative promoter, within 30 nucleotides of the first T in theTATA box sequence. This is the first report in which thestart of "upstream" LAT transcription in latently infectedneurons has been fine mapped. This result strongly supportsthe notion that this TATA box sequence is part of the LATpromoter.We also reported here the detection by Northern blot

analysis of a minor 8.3-kb LAT transcript in ganglia oflatently infected rabbits. This is the first visualization byNorthern blot of this 8.3-kb LAT during neuronal latency. Asimilar large LAT band has been detected by Northern blotsof RNA from acutely infected cells and mapped to approx-imately the region between the LAT promoter and thenearest potential polyadenylation sequence approximately8.3 kb downstream (not shown) (2). Based on the sequence

of strain 17 syn+ (9, 15), the distance from the TATA box(nucleotide 118774) to the start of the polyadenylation signal(nucleotide 127143) is 8,369 nucleotides. This distance couldvary by several hundred bases in different strains because ofdifferences in the repeated regions near the junction. Thelocation of this faint, large LAT is consistent with our

TRL UL IRLIRSUTRS

NucleoUdepositon 118,00 125,000 132:000

CPO

TATA

&3kbLAT (unstable)

ICP4

PolyA

-

I? ?

) \~~'72kbLAT (gable)

i? 2 kb LATremoved ?

1.3kbLAT (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 at the top. Theexpanded region indicates the nucleotide positions relative to theentire HSV-1 DNA sequence. The approximate locations of theICPO and ICP4 genes are shown for reference. Transcription of theLAT gene in the internal long repeat begins approximately 28nucleotides from the first T of the TATA box (118774) approxi-mately at nucleotide 118802 and extends to near the polyadenylationsignal located at nucleotide 127143. The derivation of the stable 2-and 1.3-kb LATs from the unstable 8.3-kb transcript is likely butremains to be demonstrated. The series of small arrows at the end ofthe 8.3-kb LAT indicates that termination may be inefficient, sincein occasional Northern analyses ofRNA from acutely infected cells,using probes that correspond to the 5' end of LAT, we detectedLAT bands with apparent sizes of 12 to 14 kb (not shown). If the2-kb LAT is an intron, the 6.3-kb LAT shown may be a possibleresult.

earliest results that detected weak in situ hybridizationsignals with probes derived from BamHI SP, which isdownstream from the 2-kb LAT (20).We originally proposed that the LAT promoter was lo-

cated over 660 nucleotides upstream from the 5' end of the2-kb LAT (30, 31), based on sequence analysis showing thatthis area contained an excellent TATA box consensus se-

quence as well as other RNA polymerase II consensussequences. Subsequently, we published the first report thatthis upstream region was capable of promoter activity intransient CAT assays in Vero cells (32). This report demon-strates that this region is also capable of promoter activity intransient assays in neuron-derived cells, as would be ex-

pected for the LAT promoter. Furthermore, we found thatthis promoter activity had neuron specificity. These resultsagain support the notion that this region contains the LATpromoter. The authenticity of this putative LAT promoter isfurther supported by recent reports (2, 7, 11, 24) indicatingthat virus mutants with deletions encompassing the TATAbox region do not express any LAT during neuronal latency.To ensure that our transient CAT assays reflected tran-

scription initiating from a position consistent with its beingdirected by the TATA box consensus sequences, primerextension experiments were done with RNA from cellstransfected with the A-promoter region (nucleotides -940 to-662). We found that transcription began about 28 nucleo-tides from the first T of the TATA box sequence. Thisconfirmed that in vitro this region can function as a pro-moter, further supporting the notion of this region as theLAT promoter. The start of transcription that we determinedby primer extension was in agreement with our in situ


12 h 12 h1 2

........

2kb

8day exposure

.3

....

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hybridization data in latently infected neurons which placedthe start of transcription within 30 nucleotides of the TATAbox. This position is also in agreement with that determinedfor the start of LAT transcription during acute tissue cultureinfection with an RNA protection assay (2).The immortalized neuron cell line and the neuroblastoma

cell line used for transient assays in this report are bothnonpermissive for lytic HSV-1 infection. Furthermore, fol-lowing HSV-1 infection, LAT is expressed in the immortal-ized neuron cell line (Wheatley et al., submitted). Thesecharacteristics mimic neurons during in vivo latency andmake these cells particularly relevant for these experiments.By using these neuron-derived cell lines, we found that thepresence of sequences upstream of the LAT promoter haddifferential effects on promoter activity in neuron- comparedto nonneuron-derived cells. In Vero, CV-1, BHK, and Lcells, upstream sequences decreased promoter activity by 3-to 12-fold. This apparent down regulation was not observedin immortalized neurons or neuroblastoma cells. In fact, insome experiments in neuron-derived cells, addition of theseupstream sequences enhanced promoter activity as much as300% relative to the A fragment (Fig. 3). It is possible thatdown regulation in nonneuronal cells was caused by nonneu-ronal trans-acting cellular factors interacting with these cisupstream sequences. This notion is supported by prelimi-nary observations that cotransfection of nonneuronal cellswith exogenous upstream sequences appeared to competeout down regulation by the upstream sequences in fragmentA+. In addition, transfection with large amounts of the A+or A+ + CAT constructs eliminated down regulation, againsuggesting a competitive effect (data not shown).

In summary, we have (i) used transient CAT assays inneuron-derived cells to map LAT-associated promoter ac-tivity to a location over 660 nucleotides upstream from the 5'end of the stable 2-kb LAT, (ii) shown by primer extensionthat in CAT constructs, transcription began about 28 nucle-otides from the first T of a consensus TATA box sequencelocated at nucleotide 118774 (-688), (iii) shown by a combi-nation of Northern blots and in situ hybridization of latentlyinfected neurons that transcription of a large 8.3-kb LATbegan within 30 nucleotides of the first T of this TATA boxsequence at about nucleotide 118802 (-660), and (iv) shownthat the LAT promoter had neuronal specificity.Although we have not proven that the 2- and 1.3-kb LATs

are derived from the 8.3-kb LAT, this appears to be thesimplest and the most likely hypothesis, since mutantslacking only the immediate region around the LAT promoterdid not produce either the 8.3 kb LAT or the 2- and 1.3-kbLATs (2, 11). The likely derivation of the 2- and 1.3-kb LATsfrom a primary 8.3-kb LAT raises the question of theprocessing events involved. The 1.3-kb LAT can be derivedfrom the 2-kb LAT by a simple splice (30). Likewise, the2-kb LAT could be derived by a simple splice from the8.3-kb LAT. However, if the 2-kb LAT is derived by a singlesplice, either the 2-kb LAT or the 5' end of the 8.3-kb LATwould have to be an intron. We have not been able to findany example of a 5' intron in the literature. Therefore, it islikely that either (i) the 5' end of the 2-kb LAT contains an asyet undetected small exon from the 5' end of the 8.3-kb LATor (ii) the 2-kb LAT is an intron. In the latter case, thebiologically active LAT might be an approximately 6.3-kbpolyadenylated RNA resulting from the removal of a 2-kbintron from the 8.3-kb primary transcript (Fig. 8). Althoughwe cannot point to a specific 6.3-kb LAT band, we havefound that Northern blots from acutely infected cells usuallydetect regions of LAT-specific RNAs between the 2- and

8.3-kb LATs (data not shown). Some of this material couldrepresent such a 6.3-kb RNA. If the 2-kb LAT is an intron,the coding region for a potential LAT protein would besomewhere in the putative 6.3-kb LAT. This might explainwhy a LAT protein coded by the 2-kb LAT has not beendetected (28, 31).

It is also possible that the 2- and 1.3-kb LATs are notderived by splicing but rather are stable regions (perhaps dueto secondary structure) that are left intact after degradationof the less stable 8.3-kb primary transcript. In this case,predictions of the biologically active region of the LAT genewould be more difficult.One intriguing feature of the 8.3-kb transcript is that it

spans the HSV-1 junction. Thus, during acute infection, thecopy of the LAT gene in the internal repeat produces aprimary transcript of 8.3 kb that extends approximately 800bases into the short repeat. Since the HSV-1 genome iscircularized shortly after acute infection and also appears tobe circularized during latency (17, 18) the LAT gene in theterminal repeat would make an identical 8.3-kb transcript.However, if at any time the HSV-1 genome were in a linearform, the copy of the LAT gene in the terminal repeat wouldproduce a transcript that is truncated at about 7.5 kb byrunning off the end of the HSV-1 genome. Recent resultswith some (2, 7, 24) but not all (1, 6) LAT mutants suggestthat LAT may be involved in HSV-1 reactivation. It istherefore tempting to speculate that reactivation may beassociated with a short period of linearization of the genomeand that some difference between the complete 8.3-kb tran-script and a truncated runoff transcript might play a role inthe switch from latency to reactivation. Alternatively, onecould also speculate that LAT may play some role incircularization (or linearization) of the genome.The results presented here suggest that the LAT promoter

is controlled, in part, by upstream sequences that result inneuronal specificity. Exactly what these sequences are, whatcellular and viral factors they interact with, and how theyregulate LAT expression remain to be determined.

ACKNOWLEDGMENTS

This work was partially supported by the Discovery Fund for EyeResearch, the Factor Family Foundation, and Public Health Servicegrants EY07566 and EY05939. J.C.Z. is a Factor Family Founda-tion Scholar. K.P. is an Iris and B. Gerald Cantor Scholar.We thank Anita Avery and Richard Lit for technical assistance

and John Ong and Don Brown for suggestions and critical reading ofthe manuscript.

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11. Mitchell, W. J., I. Steiner, M. S. Brown, A. R. MacLean, J. H.Subak-Sharpe, and N. W. Fraser. 1990. A herpes simplex virustype 1 variant, deleted in the promoter region of the latency-associated transcripts does not produce any detectable minorRNA species during latency in the mouse trigeminal ganglion. J.Gen. Virol. 71:953-957.

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22. Spivack, J. G., and N. W. Fraser. 1988. Expression of herpessimplex virus type 1 (HSV-1) latency-associated transcripts andtranscripts affected by the deletion in avirulent mutant HFEM:evidence for a new class of HSV-1 genes. J. Virol. 62:3281-3287.

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24. Steiner, I., J. G. Spivack, R. P. Lirette, S. M. Brown, A. R.MacLean, J. H. Subak-Sharpe, and N. W. Fraser. 1989. Herpessimplex virus type 1 latency associated transcripts are evidentlynot essential for latent infection. EMBO J. 8:505-511.

25. Stevens, J. G., E. K. Wagner, G. B. Devi-Rao, M. L. Cook, andL. T. Feldman. 1987. RNA complementary to a herpesvirusalpha gene mRNA is prominant in latently infected neurons.Science 235:1056-1059.

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30. Wechsler, S. L., A. B. Nesburn, R. Watson, S. M. Slanina, andH. Ghiasi. 1988. Fine mapping of the latency-related gene ofherpes simplex virus type 1: alternative splicing produces dis-tinct latency-related RNAs containing open reading frames. J.Virol. 62:4051-4058.

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32. Zwaagstra, J. C., H. Ghiasi, A. B. Nesburn, and S. L. Wechsler.1989. In vitro promoter activity associated with the latencyassociated transcript gene of herpes simplex virus type 1. J.Gen. Virol. 70:2163-2169.

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