Vol. 64, No. 10

JOURNAL OF VIROLOGY, Oct. 1990, p. 5019-5028

0022-538X/90/105019-10$02.00/0 Copyright © 1990, American Society for Microbiology

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, Los Angeles, California 90048-18691*; Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 900242; and Department of Biochemistry, University College and Middlesex School of Medicine,3 and 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 that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoterCAT 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, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the 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 in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor

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stable 2- and 1.3-kb LATs. the mapped 5' end of the stable LATs (30, 31), and later, by using chloramphenicol acetyltransferase (CAT) assays, we confirmed that in Vero cells, LAT promoter activity coincides 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 to produce any LAT RNA during latent infections (2, 7, 11, 24). Since LAT is the only HSV-1 gene that is abundantly expressed during neuronal latency (20), the LAT promoter must be controlled differently from other HSV-1 gene promoters (22, 23). Furthermore, the LAT promoter is likely to have neuronal specificity, since LAT is abundant in latently infected neurons (20, 25) but is present at only low levels during acute tissue culture infection (22). We have therefore extended our studies of the LAT promoter to neuron-derived cells to confirm the location of the LAT promoter and to look for 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, sequences upstream of the TATA box decreased promoter activity in cis, while in neuron-derived cells, inhibition by these cisacting sequences was not observed. These upstream se-

Following primary infection in humans, herpes simplex virus type 1 (HSV-1) establishes latent infections in sensory neurons (14). During HSV-1 latency, detectable viral transcription is limited to an area within the genomic long repeats in the vicinity of the immediate early gene ICP0 (20, 21, 25). At least two abundant and stable latency-associated transcripts (LATs) (2 and 1.3 kilobases [kb]) that share their 5' and 3' ends are derived by alternative splicing (30). These LATs 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 long repeat has been mapped to within a few bases (28-30). This position corresponds to nucleotide 119462 of the genomic sequence (9, 15). A second copy of the LAT gene is present in the corresponding location of the terminal long repeat. Recent reports with some LAT deletion mutants suggest that LAT may play a role in reactivation of the virus from the latent state (2, 7, 24). However, this is not supported by all LAT mutants (1, 6). Based on sequence analysis, we initially proposed that the LAT promoter is located over 660 nucleotides upstream of

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Corresponding author. 5019

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ZWAAGSTRA ET AL.

quences may confer neuron specificity on the HSV-1 LAT promoter in vivo. We also 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 this data is that this 8.3-kb RNA may be an unstable primary LAT 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 type 1 (HSV-1), strain McKrae, was grown as previously described (20) and was used for all infections. Cells were grown as monolayers in minimal essential medium, Dulbecco modified Eagle medium, or F12 (GIBCO Laboratories, Inc.) supplemented with 10% fetal calf serum and antibiotics. Neuroblastoma cells (NB41A3; American Type Culture Collection CCL147) are of mouse origin. These neuroblastoma cells retain several neuronal markers, including acetylcholinesterase activity. In addition, they are nonpermissive for HSV-1 infection (27). Immortalized neurons were made by fusing H18TG2 cells, a 5-azaguanine-resistant mouse neuroblastoma cell line, with neonatal rat dorsal root ganglia neurons. These cells are nonpermissive for lytic HSV-1 infection 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 the BamHI B restriction fragment from HSV-1 strain F (16). The LAT gene from position -2592 to +663 relative to the 5' end of the stable 2-kb LAT was divided into five restriction fragments. Fragments A to C and the details of their cloning in the proper orientation in front of the CAT gene within plasmid pSVOCAT (4) have been previously described (32). Fragments A+ and A+ + were similarly cloned and consist of fragment A with additional upstream sequences as detailed in the legend to Fig. 2 and in Fig. 3. For some experiments, similar constructs were made by using a different promoterless CAT plasmid, p1O6CAT (3), in place of

pSVOCAT. CAT assays and quantitation procedures. CAT assays and

quantitation procedures have been previously described (32). Briefly, cell monolayers at approximately 60% confluency, on 60-mm plates, were transfected with CAT constructs by the calcium phosphate precipitation method (5). After 46 h, the cells were harvested and cell extracts were prepared. Within an experiment, equal cell numbers were used and, if necessary, corrections were made for the amount of protein present in the extracts. Acetylated forms of ['4C]chloramphenicol were detected by thin-layer chromatography and subsequent autoradiography. The amount of acetylated and unacetylated chloramphenicol was quantitated by excising the spots from the thin-layer plates and counting in a liquid scintillation counter. In some experiments, samples of cell extracts were analyzed by DNA dot blot hybridizations with a CAT-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 seen between the transfection efficiencies of fragments A+ +, A+, or A. Thus, differences between the CAT activities of these plasmids within a cell line were not a result of differences in transfection efficiency. The differences in transfection efficiencies between cell lines was partially compensated for by using 10 ,ug of each plasmid in immortalized neurons, BHK cells, and L cells; 5 ,ug of each

J. VIROL.

plasmid in neuroblastoma cells and CV-1 cells; and 2.5 ,ug of each plasmid in Vero cells. Primer extension. As modified from a procedure of P. Krause and J. Ostrove (personal communication), 10 ,ug of RNA from transfected Vero cells (isolated by the guanidinium-cesium chloride method [8]) was suspended in 10 RI of 20 mM Tris-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 the primer from 5' to 3' was GATGCCATTGGGATATAT 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 construct used for the transfection. This location was confirmed by partial sequencing of the plasmid and corresponds to CAT nucleotides 28 to 52 relative to the ATG codon at which CAT translation initiates. The difference between these numbers is the result of a HindIll linker, a multiple cloning region, and noncoding CAT sequences between the end of the LAT sequences and the start of the structural CAT sequences in this plasmid. The reaction mixture was heat denatured for 3 min at 90°C, hybridized at 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 murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Inc.) at 37°C for 1 h according to the instructions of the manufacturer by using 2 mM deoxynucleoside triphosphate, 100 ng of bovine serum albumin per Pd, and 20 U of RNasin (Promega Biotec) per 20-pdl reaction. The reaction was stopped by adding EDTA to 10 mM. The extended primer was precipitated with 2 volumes of 95% ethanol, washed with 70% ethanol, suspended in formamide-dye loading buffer, heat denatured at 95°C for 3 min, quick chilled on ice, and run on a 10% acrylamide sequencing gel containing 7 M urea. The gel was then processed 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 a 10% 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). Rabbit trigeminal 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-paraformaldehyde (26) for sectioning prior to in situ hybridizations. In situ hybridizations. Fixing, embedding, and cutting sections of trigeminal ganglia were done as described previously (19). Hybridizations to identify RNA were done as we previously described (20, 30) by using 32P-labeled synthetic oligonucleotides as probes. Slides were exposed to photographic emulsion for 2 to 3 days. Pretreatment of slides with RNase, but not DNase, eliminated hybridization. Three to five sections of ganglia from each of four latently infected

VOL. 64, 1990

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 of grains 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 least two latently infected rabbits, as judged by detection of at least one positive neuron on at least two different slides from a given rabbit. Northern blot hybridizations. Total RNA was isolated from uninfected or latently infected rabbit trigeminal ganglia frozen in liquid nitrogen or from uninfected or acutely infected (multiplicity of infection 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 was done on linearized plasmids following the instructions of the manufacturer (Amersham Corp.). Synthetic oligonucleotides (20-mers) were synthesized by using beta-cyanoethyl phosphoramidite chemistry on a Pharmacia Gene Assembler. The sequences of 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).

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