This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maillet, S.
Right arrow Articles by Labetoulle, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maillet, S.
Right arrow Articles by Labetoulle, M.

 Previous Article  |  Next Article 

Journal of Virology, September 2006, p. 9310-9321, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.02615-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Herpes Simplex Virus Type 1 Latently Infected Neurons Differentially Express Latency-Associated and ICP0 Transcripts

Séverine Maillet,1 Thierry Naas,2 Sophie Crepin,1 Anne-Marie Roque-Afonso,3 Florence Lafay,1 Stacey Efstathiou,4 and Marc Labetoulle1,5*

Laboratoire de Virologie Moléculaire et Structurale, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France,1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 K.-Bicêtre, France,2 Service de Microbiologie, Hôpital Paul Brousse, Assistance Publique-Hôpitaux de Paris, 94800 Villejuif, France,3 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom,4 Service d'Ophtalmologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, 94275 Le Kremlin-Bicêtre, France5

Received 15 December 2005/ Accepted 28 June 2006


arrow
ABSTRACT
 
During the latent phase of herpes simplex virus type 1 (HSV-1) infection, the latency-associated transcripts (LATs) are the most abundant viral transcripts present in neurons, but some immediate-early viral transcripts, such as those encoding ICP0, have also been reported to be transcribed in latently infected mouse trigeminal ganglia (TG). A murine oro-ocular model of herpetic infection was used to study ICP0 gene expression in the major anatomical sites of HSV-1 latency, including the TG, superior cervical ganglion, spinal cord, and hypothalamus. An HSV-1 recombinant strain, SC16 110LacZ, revealed ICP0 promoter activity in several neurons in latently infected ganglia, and following infection with wild-type HSV-1 strain SC16, in situ hybridization analyses identified ICP0 transcripts in the nuclei of neurons at times consistent with the establishment of latency. Reverse transcription (RT)-PCR assays performed on RNA extracted from latently infected tissues indicated that ICP0 transcripts were detected in all anatomical sites of viral latency. Furthermore, quantitative real-time RT-PCR showed that neurons differentially expressed the LATs and ICP0 transcripts, with splicing of ICP0 transcripts being dependent on the anatomical location of latency. Finally, TG neurons were characterized by high-level expression of LATs and detection of abundant unspliced ICP0 transcripts, a pattern markedly different from those of other anatomical sites of HSV-1 latency. These results suggest that LATs might be involved in the maintenance of HSV-1 latency through the posttranscriptional regulation of ICP0 in order to inhibit expression of this potent activator of gene expression during latency.


arrow
INTRODUCTION
 
Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus, which establishes lifelong latent infection. During the acute phase of infection, viral replication is characterized by the expression of immediate early (IE), early, and late genes in an ordered cascade that leads to the release of infectious particles that can attach to nerve endings. Following axonal transport to neuronal cell bodies, HSV-1 can either replicate or persist in a latent state in the infected host (62).

During latency infectious virus cannot be detected in neuronal tissues (62). The viral genome is circularized, complexed with histones, and maintained in a repressed state with the exception of the latency-associated transcripts (LATs) that are encoded within the repeats flanking the unique long region of the HSV-1 genome (12, 33, 60). The LATs (2 kb and 1.5 kb in size) are stable unpolyadenylated introns that are retained within the nuclei of latently infected neurons (reviewed in references 33 and 56) and are spliced from a primary 8.3-kb transcript (19). Their unusual stability has been related to modifications in the consensus sequences of splicing (84), resulting in an unstable 6.3-kb spliced exon (13, 85) and a stable 2-kb LAT intron that is expressed during both acute and latent infections (73, 84). During latent neuronal infection, the 2-kb LAT may undergo subsequent splicing that results in a 1.5-kb LAT (1, 46, 70). The functions of LATs are not yet clearly understood, but mutational analyses and transfection assays of LATs strongly suggest that they have a key role in establishment of latency and/or viral reactivation (29, 31, 53, 64, 71, 77), besides their antiapoptotic, anti-interferon, and proneuronal survival properties (24, 32, 52, 54, 55, 78).

Several studies failed to detect lytic viral gene expression during HSV-1 latency (20, 33). However, some studies reported the detection of transcripts from genes encoding the immediate-early viral protein ICP4 and the early viral protein thymidine kinase (TK) in latently infected trigeminal ganglion (TG) neurons (9, 34). This observation was initially interpreted as a possible result of spontaneous reactivation (33). Since the gene encoding the ICP0 protein of HSV-1 is contained entirely within the region of the HSV-1 genome encoding LATs (reviewed in reference 3), it might be transcribed during latency, as recently reported by Chen et al. for latently infected TG neurons (10). ICP0 is the first viral protein produced during the productive cycle and is thus classically considered a relevant indicator of early-stage reactivation (18, 59, 65). It nonspecifically transactivates both viral and cellular promoters (18, 25, 42) and plays a crucial role in the initiation of productive infection following low-multiplicity infections of cells in culture (7, 8, 17, 63). ICP0 is able to initiate both viral gene expression from quiescent genomes in cells in culture and viral reactivation from latently infected sensory neurons in vivo (6, 26, 27, 42). Furthermore, a HSV-1 strain with the gene(s) for ICP0 expression deleted may mimic latency in nonneuronal cells in culture (28, 58). This protein might thus play a key role in the balance between latency and viral reactivation (15, 18, 56).

Since most data concerning LAT and ICP0 gene expression during HSV-1 latency is derived from the analysis of sensory neurons, especially those of TG, we used the oro-ocular model of HSV-1 infection (37, 39), which allowed comparison of LAT and ICP0 transcription in various types of latently infected neurons.

After inoculation of HSV-1 into the mouse upper lip, the virus rapidly spreads to the nervous system through sensory, autonomic, and motor neuronal pathways and finally becomes latent, not only in the TG, but also in numerous other structures that are connected to both oral and ocular tissues, such as the superior cervical ganglia (SCG), spinal cord, and hypothalamic nuclei. Previous studies had shown that inoculation of HSV-1 into the lower lip (80), the snout (14, 66), or the anterior chamber of the eye or cornea of mice (61) or rabbits (49, 68) leads to latent infection in TG and SCG. These results are in agreement with the observation that HSV-1 latency in humans occurs within the TG, the central, and the autonomic nervous pathways, including the SCG (21, 21, 23, 45, 82). However, we observed larger numbers of neurons expressing LATs in the TG than in the other latently infected structures despite a similar level of productive infection (39). These data suggested that fundamentally different patterns of HSV-1 latency may occur in distinct neuronal cell types.

Using the oro-ocular HSV-1 infection model and three complementary methods of detecting gene expression during latency, i.e., a reporter gene detection assay using a recombinant strain of HSV-1, in situ hybridization (ISH), and reverse transcription (RT)-PCR, we investigated transcription of the ICP0 gene in several latently infected structures, including the TG and the SCG. Using quantitative real-time RT-PCR, we quantified ICP0 transcripts and 2-kb LATs in anatomically distinct sites of HSV-1 latency and showed that the numbers of transcripts differed with the type of latently infected structure, i.e., sensory, autonomous, or central. In contrast to other latently infected structures, ICP0 transcripts were preferentially unspliced in the TG, where large amounts of LATs were found, suggesting a potential mechanism of regulation between latency and reactivation mediated by these two viral transcripts.


arrow
MATERIALS AND METHODS
 
Virus strains and cells. Wild-type HSV-1 strain SC16 (30) and the recombinant strain SC16 110LacZ (41), which contains an ICP0 promoter-driven lacZ inserted into the nonessential US5 gene of HSV-1, were used. Viruses were propagated and titrated as previously described (37-39).

Mice, virus inoculation, and euthanasia. Six-week-old inbred female BALB/c mice (Janvier Breeding, Le Genest Saint Ile, France), receiving unrestricted access to food and water, were used. The procedures involving experimental animals conformed to ethical standards such as those of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in research and were approved by a local ethical committee.

A 1-µl suspension of SC16 or SC16 110LacZ containing 106 plaque-forming units was inoculated into the left upper lip of the mice, and the mice were observed daily for clinical signs of ocular infection from days 0 to 28 postinoculation (dpi), as previously described (37, 39). Mice that showed clinical signs of ocular disease such as blepharitis, conjunctivitis, keratitis, or iritis (about 70% of the mice) between 6 and 10 dpi were randomly sacrificed at either 6 dpi for the acute infection study or 28 dpi, when clinical signs were resolved, for the latent infection study. As controls, four mock-infected mice were sacrificed at 6 and 28 dpi. To confirm some of the results observed at 28 dpi, an independent experiment comparing mice sacrificed at either 28 or 90 dpi was conducted. Before tissue dissection, mice were perfused intracardially with 1x phosphate-buffered saline (PBS) for RNA extraction or successively with 1x PBS, 4% paraformaldehyde, and 20% sucrose for tissue cryosection (37).

Cryostat sections and RNA isolation. The spinal cord and the whole head, containing the SCG, were prepared as previously described, and 10-µm frontal cryosections were collected in three parallel series (37).

Tissues (spinal cord, TG, SCG, and hypothalamus) were quickly dissected (14 min per mouse on average), incubated overnight at 4°C in RNA Later (QIAGEN, Les Ulis, France), and stored at –80°C. Total RNA was extracted, using the RNeasy Mini extraction kit (QIAGEN), combined to an RNase-free DNase (QIAGEN) treatment for 15 min, eluted in 30 µl of RNase-free water, and stored at –80°C.

Assessment of ß-galactosidase activity and HSV-1 antigens on cryosections. Every third cryosection of SC16 110LacZ-infected mice was thawed, rehydrated in PBS, and incubated in a solution containing 0.33 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.1% Triton X-100 for 3 h at 37°C.

HSV-1 antigens were detected on SC16- and SC16 110LacZ-infected cryosections by a peroxidase antiperoxidase assay, using a polyclonal anti-HSV-1 antibody (38).

ICP0 protein was detected on some sections adjacent to those positive for ICP0 transcripts with ISH, using an immunofluorescence assay with a rabbit polyclonal anti-ICP0 antibody (kindly provided by Roger Everett, Glasgow, United Kingdom) diluted to 1/100, and revealed with swine fluorescent anti-rabbit antibodies (Dako, Trappes, France) diluted to 1/50.

Detection of ICP0 transcripts by ISH. ISH was performed as follows. (39, 40) (i) A digoxigenin-labeled riboprobe was produced from EcoRI digestion of the pSLAT5 plasmid, provided by Jane Arthur (2), and was in vitro transcribed with T3 RNA polymerase, using the RNA Dig transcription kit, (Roche, Meylan, France). (ii) The riboprobe (100 ng) was hybridized on tissue sections overnight at 64°C (39). A stringent wash in 0.1x SSC (15 mM Na, 1.5 mM sodium citrate) plus 30% deionized formamide and 10 mM Tris-HCL (pH 7.5) was carried out at 64°C for 30 min. The probe was detected by (i) sheep anti-DIG antibody diluted to 1/500 (Roche) and (ii) rabbit anti-sheep HRP antibody diluted to 1/500 (Dako). The antibody solutions contained 1% blocking reagent (Roche). The signal was amplified with a GenPoint tyramide-biotin system (item no. k0620; Dako). The slides were counterstained with Giemsa blue stain, washed, dried, and mounted with Entellan (Merck, Fontenay-sous-bois, France) (38).

To ensure that the ISH signal was not due to hybridization with HSV-1 DNA, some slides were treated for 30 min at 37°C with 1 µg of RQ1 RNase-free DNase, (Promega, Charbonnières-les-bains, France) before hybridization. The conditions for DNase treatment were verified by incubating viral DNA with 1 µg of DNase and checking for complete degradation after 30 min of incubation.

All the sections adjacent to those used for the ISH experiments were tested for HSV-1 antigens, using an immunochemical detection method with specific polyclonal anti-HSV-1 antibodies (Dako), as previously described (37, 38).

RT-PCR on acutely or latently infected tissues. The One-Step RT-PCR kit (QIAGEN) was used for RNA amplification. Primers used for spliced ICP0 transcripts were described by Chen et al. (10) (annealing temperature, 62°C). For amplification of the ICP0 transcripts, only the O2 reverse primer (5'-CCCAGACATCCGGGGCGGGCT-3') was used for RT, and the O1 sense primer (5'-AGCGAGTACCCGCCGGCCTG-3') was added for the amplification step (PCR). The products were separated by agarose electrophoresis.

The primers used for TK amplification were 5'-CTTAACAGCGTCAACAGCGTGCCG-3' and 5'-GTGGCCCTGGGTTCGCGCGA-3' (annealing temperature, 55°C), and those used for UL18 amplification were 5'-AGCGAATTCTTAGGGATAGCGTATAACGGGG-3' and 5'-ATCGGATCCATGCTGGCGGACGGCTTT-3' (annealing temperature, 62°C).

Analysis of PCR product specificity by Southern blot hybridization. Following transfer to a nylon membrane (Hybond; Roche), the DNA was UV cross-linked to the membrane prior to overnight hybridization with 0.5 pmol/ml of digoxigenin-labeled probe produced with either the Dig Oligonucleotide Tailing kit (Roche) for IE110 probe O3 (described in reference 10) or the Dig High Prime DNA Labeling and Detection Starter Kit II (Roche) for TK and UL18 probes.

Mapping the ICPO transcription start site. Reverse transcription and rapid amplification of cDNA ends (RACE) were performed with the 5'RACE system, version 2.0 (Invitrogen, Cergy Pontoise, France). Total RNAs extracted with the RNeasy Mini kit (QIAGEN) from a TG and a SCG of a latently infected mouse at 28 dpi (QIAGEN) were used to determine the ICPO transcription initiation site. After a reverse transcription step with gene-specific primer GSP1 (5'-TCACGCCCACTATCAGGTAC-3') and reverse transcriptase, the cDNA was tailed with terminal deoxynucleotidyltransferase and was subsequently amplified with another gene-specific primer, O2, previously described for spliced ICPO transcript amplification, or with the primer GPP2 (5'-GTCACATGGCGACCCCCAAC-3') combined with an oligo(dT) adapter primer provided with the kit. This PCR product was used as a template for a nested PCR assay with another adapter and GSP3 (5'-CAGGCCGGCGGGTACTCGCA-3') primers. The PCR product obtained was directly sequenced. The transcription initiation site was determined as the first nucleotide following the sequence of the adapter primer.

Production of RNA standards for quantification. The 2-kb LAT, GAPDH, and intron-containing ICP0 transcripts were RT-PCR amplified, and cDNA was cloned, using the TOPO TA cloning kit (Invitrogen). Spliced ICP0 PCR products were cloned into the pCRscript (Stratagene, Paris, France) vector as recommended by the manufacturer. The vectors were EcoRV or SacII (Promega) digested and in vitro transcribed using SP6 or T7 RNA polymerase (Roche), respectively. RNA quantities were evaluated by optical density at 260 nm.

Real-time RT-PCR. Transcript quantitation was performed by TaqMan technology on a Light Cycler 1.0 instrument (Roche). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were quantified, using 5 µl of RNA diluted to 1/10 and 0.5 µM of GAPDH primers (described in reference 11) at an annealing temperature of 50°C with the Light Cycler RNA Master SYBR Green I kit (Roche).

Spliced or intron-containing ICP0 transcripts were quantified from 5 µl of RNA, using the Sensiscript reverse transcription kit (QIAGEN) for 1 h at 42°C with 0.6 µM of the primer O2. The cDNA was dialyzed on 0.45-µm-diameter dialysis membranes (Millipore, Saint-Quentin-en-Yvelines, France) and amplified with 0.5 µM of either primer O1 or O4 (5'-AACTCGTGGGTGCTGATTGAC-3') and the probe O3 (5'6-6-carboxyfluorescein, 3'6-carboxytetramethylrhodamine), using the PCR Master kit (Roche).

For 2-kb LAT transcripts, 5 µl of RNA, 0.3 µM of primers (described in reference 11) at an annealing temperature of 55°C, and 0.2 µM of the LAT3 probe (5'-6-6-carboxyfluorescein-CCCACCCCGCCTGTGTTTTTGTG-3' 6-carboxytetramethylrhodamine) were amplified with the Quantitect Probe RT-PCR kit (QIAGEN).

Quantification of spliced and intron-containing ICP0 transcripts and 2-kb LATs was normalized against the number of GAPDH transcripts in the same RNA extracts. Standards for each transcript gave a linear amplification curve over at least 4 logs of template concentrations. The thresholds for ICP0 transcripts, 2-kb LATs, and GAPDH transcripts were reached between 50 to 100, 5,000, and 10,000 copies per reaction, respectively. Quantitation was reproducible for each type of tissue at the same stage of infection (see Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Quantitation of spliced and intron-containing ICP0 transcripts and 2-kb LATs based on real-time RT-PCR


arrow
RESULTS
 
Two-thirds of HSV-1-infected animals developed clinical signs of disease, typified by ruffling of fur, abnormal gait, weight loss, and ocular disease, between 6 and 10 dpi. From 10 dpi, the clinical signs resolved, leading to latent infections (39). Four mice (A1 to A4) were sacrificed at 6 dpi for RT-PCR assays, and 13 mice (L1 to L13) were sacrificed at 28 dpi for analyses of ICP0 promoter activity, ISH, and RT-PCR assays.

Pathogenesis of wild-type SC16 and recombinant SC16 110LacZ. After inoculation of either the wild-type strain SC16 (21 and 31 mice in two independent studies) or the recombinant strain SC16 110LacZ (51 and 61 mice), we observed a difference in survival rates (65% and 84%, respectively; {chi}2 test, P < 0.01), but the proportion of mice that developed ocular disease among those surviving primary infections was similar for the wild-type and recombinant virus (40% and 35%, respectively; {chi}2 test, P > 0.10) (Fig. 1). The mice developed comparable clinical signs of herpetic disease between 6 and 10 dpi. Immunohistochemical staining for HSV-1 antigens in cryosections of brain, trigeminal and superior ganglia, and spinal cord from acutely infected mice revealed that the recombinant and wild-type viruses had similar spreads (data not shown).


Figure 1
View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. Survival rates of mice infected with either the recombinant SC16 110LacZ or the wild-type SC16 strain of HSV-1. Four independent experiments were performed: 21 and 31 mice were inoculated with 1 µl of a suspension containing 106 PFU of SC16, and 61 and 51 mice were infected with SC16 110LacZ.

Detection of ICP0 promoter activity and ICP0 transcripts on histological sections by ISH. During the acute phase of infection with SC16 110LacZ, ICP0 promoter-driven ß-galactosidase activity and HSV-1 antigens were detected in the same cells and in the same neurological structures as previously described for wild-type HSV-1 strain SC16 (39).

Following the establishment of latency with SC16 110LacZ, ICP0 promoter activity was revealed by ß-galactosidase detection in four mice (L1 to L4) in only a few cells (one to three) of the TG, the SCG, and the spinal cord (Fig. 2) in the absence of viral antigens, but no positive cells were detected in the brain or hypothalamic nuclei. All cryosections were negative for HSV-1 antigens, indicating that HSV-1 did not reactivate.


Figure 2
View larger version (67K):
[in this window]
[in a new window]
  		FIG. 2. Detection of ß-galactosidase activity on latently infected mice. Animals were infected with SC16 110LacZ (A, B, and C) or mock infected (D, E, and F) and sacrificed at 28 dpi. ICP0 promoter activity was detected using the ß-galactosidase detection assay (blue staining within the cytoplasms). (A and D) SCG; (B and E) TG; (C and F) spinal cord; (A) two positive cells in the superior cervical ganglion (arrows); (B) one positive cell in the trigeminal ganglion (arrow); (C) one positive cell in the spinal cord (arrow).

The riboprobe used for the detection of ICP0 transcripts overlaps exon 2 and intron 2 of the ICP0 gene. This riboprobe may thus theoretically hybridize both spliced and intron-containing ICP0 transcripts (see Materials and Methods for details). ISH analyses were then used to examine peripheral and central nervous system tissues from four mice latently infected with wild-type SC16 (L5 to L8). Several ICP0 transcript-positive neurons were detected in all the mice following signal amplification with a biotinyl-tyramide-streptavidin-HRP system (Dako Genpoint). ICP0 transcripts were detected mainly within the nuclei of neurons of the TG (180 positive cells on average) and the SCG (on average, 2 positive cells on the left side and 33 on the right side) (Fig. 3 and Table 1), but no positive neurons were detected in the spinal cord or the hypothalamus. Since the numbers of neurons in the TG and SCG have been estimated to be 20,000 to 50,000 and 10,000 to 30,000, respectively (51), the percentage of ICP0-positive neurons in the TG and SCG were 0.4 to 0.9% and 0.03 to 0.1%, respectively. Previous ISH analysis for LATs showed 265 positive neurons per TG, 9 per spinal cord, and no positive cells in SCG or the hypothalamus (39). No positive hybridization signal was observed on sections from mock-infected mice, suggesting that the positive signals were not due to cross-hybridization with cellular DNA or RNA. DNase treatment before hybridization provided similar results, indicating that the positive signal was not due to viral genome hybridization and thus was specific to RNA hybridization. In addition, no antigen-positive neurons, as assessed by immunoassay using either polyclonal anti-HSV-1 antibodies or anti-ICP0 antibodies (data not shown), were observed in histological sections adjacent to those positive for ICP0 transcripts at 28 dpi, suggesting that HSV-1 did not reactivate in the ISH-positive tissues.


Figure 3
View larger version (156K):
[in this window]
[in a new window]
  		FIG. 3. Detection of ICP0 transcripts by ISH. SC16 (A, C, and E)- or mock (B, D, and F)-infected mice were sacrificed when latency was established at 28 dpi. Cells positive for ICP0 transcripts, revealed by dark brown staining, are located predominantly within the nuclei. (A and B) SCG at 28 dpi; (A) two positive cells; (C and D) TG at 28 dpi; (C) numerous positive cells; (E and F) TG at 90 dpi; (E) numerous positive cells. Arrows indicate the nuclei of positive cells.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Number of cells positive for LAT and ICP0 transcripts detected by ISH

Detection and quantitation of ICP0 transcripts and LATs. ICP0 transcripts were amplified in RNA extracted from all acutely infected structures, including the TG, SCG, spinal cord, and hypothalamus of the acutely infected mouse A1 (6 dpi) (Fig. 4). ICP0 transcripts were also detected in RNA extracted from the same structures of latently infected mouse L9 (28 dpi), but the amplification signal was lower than for acutely infected tissues.


Figure 4
View larger version (34K):
[in this window]
[in a new window]
  		FIG. 4. Amplification of ICP0 transcripts using RT-PCR. (A) RT-PCR products were separated on an agarose gel. (B) Southern blot of the amplification products hybridized with the internal probe O3. Lanes 1, 2, and 3, spinal cord; lanes 4, 5 and 6, SCG; lanes 7, 8 and 9, TG; lanes 10, 11, and 12, hypothalamus; lane 13, negative control without the reverse transcriptase step; lanes 1, 4, 7, and 10, mock-infected mice; lanes 2, 5, 8, and 11, infected mice sacrificed at 6 dpi (acute phase); lanes 3, 6, 9, and 12, infected mice sacrificed at 28 dpi (latent phase of infection). An 88-base pair amplified fragment was observed for acutely and latently infected tissues (middle and right wells) but not for mock-infected tissues. The signal specificity was confirmed by Southern blotting.

In order to verify that the amplification products corresponded to ICP0 transcripts, two control experiments were performed. Southern blot analysis showed that the amplified product hybridized to an ICP0-specific DNA probe (Fig. 4B), and 5'RACE analysis of RNA revealed that ICP0 transcripts started at their cognate transcription start sites (data not shown). Furthermore, no amplification product was found in the absence of reverse transcriptase or RNA in the reaction mix or in RNA obtained from mock-infected tissues.

Quantitation of HSV-1 transcripts during acute infection using real-time RT-PCR. Spliced and intron-containing ICP0 transcripts and 2-kb LATs were quantified in each neurological structure of three mice acutely infected with SC16 (A2 to A4), with the exception of one right SCG that was below the threshold of detection for 2-kb LATs (5,000 copies per assay). On average, 107 to 108 copies of ICP0 transcripts were found in the spinal cord, whereas 104 to 107 copies were found in the TG, the SCG, and the hypothalamus (Table 2). Normalization to GAPDH confirmed a maximal expression of viral transcripts in the spinal cord (Table 2). For ICP0 transcripts, splicing efficiency was about 90% in each type of tissue (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Percentage of ICP0 transcripts spliced at 6 and 28 dpi

Quantitation of HSV-1 transcripts during the latent phase of infection. Spliced and intron-containing ICP0 transcripts and 2-kb LATs were quantified in RNA extracted from neurological structures of four latently infected mice (L10 to L13). Despite wide mouse-to-mouse variability, the average transcript copy number was calculated in order to compare the expression of viral transcripts in the various latently infected structures.

The total amount of ICP0 transcripts varied from 1.8 x 106 copies in the left TG to 2.3 x 102 in the right SCG (Table 3). Normalization with GAPDH showed tissue-dependent differences in ICP0 gene transcription during HSV-1 latency (Table 2).

Spliced ICP0 transcripts were found in all the structures. However, some tissues in some mice were negative or under the threshold of detection (50 copies per assay), but no animal was entirely negative. In the positive extracts, amounts varied from 7.1 x 103 copies in the hypothalamus and the left TG to 71 copies in the right TG, i.e., very close to the threshold of detection (Table 2). Normalization to GAPDH showed that expression of spliced ICP0 RNA varied depending on the tissue type; it was more abundant in the SCG, TG, and the spinal cord (from 2.5 x 103 to 2.9 x 104 copies of transcripts per 109 copies of GAPDH) than in the hypothalamus, where only 53 spliced ICP0 transcripts were found per 109 copies of GAPDH (Table 2). These results also showed a dramatic decrease in the amounts of spliced ICP0 transcripts detected during the transition from the acute to the latent phase of infection in the spinal cord and the hypothalamus (about 50,000-fold), compared to those for the SCG (about 1,000-fold) and the TG (from 320- to 2,000-fold) (Table 2).

Intron-containing ICP0 transcripts were found in each type of neurological structure during latency with the exception of the left SCG (Table 2). Normalization to GAPDH showed that intron-containing ICP0 transcripts were differentially expressed within various latently infected structures. The TGs were the structures with maximal expression (7.7 x 105 and 5.0 x 104 copies per 109 GAPDH copies for left and right sides, respectively). Amounts were lower in the other structures: from 3.9 x 104 copies per 109 GAPDH copies in the spinal cord to 50 copies per 109 GAPDH copies in the hypothalamus (Table 2). We thus observed a dramatic decrease of intron-containing ICP0 transcripts in the transition from acute to latent infection in the hypothalamus (about 10,000-fold) and in the other structures (between 20- and 2,000-fold), with the exception of the left TG, where the number of transcripts remained nearly the same (Table 2 and Fig. 5). Furthermore, ICP0-splicing efficiencies varied depending on the tissue type: from 94% to 100% in the SCG to 0.3% of spliced ICP0 transcripts in the left TG (Table 3).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Bar graphs comparing HSV-1 transcripts at 6 dpi and 28 dpi. (A) Spliced ICP0 transcripts; (B) intron-containing ICP0 transcripts; (C) 2-kb LAT transcripts.

The numbers of 2-kb LATs varied from 108 copies in the TG to 104 in the SCG (Table 2). Normalization to GAPDH showed that 2-kb LATs were differentially expressed depending on the latently infected structure, being more abundant in the TG (5.0 x 107 to 2.0 x 108 copies per 109 GAPDH copies) than in the other structures (from 7.4 x 106 copies per 109 GAPDH copies in the right SCG to 2.1 x 104 copies per 109 GAPDH copies in the hypothalamus) (Table 2). The numbers of 2-kb LATs decreased from the acute to the latent infection, except in the TG, where the quantities remained constant in the right side and increased fivefold in the left side (Table 2 and Fig. 5).

Comparison of LATs and ICP0 transcripts showed striking differences between infected tissues (Table 4). At 6 dpi, LATs were about 10-fold more abundant than spliced ICP0 transcripts in the TG, while they were produced equally in the SCG, the spinal cord, and the hypothalamus. During the latent stage of HSV-1 infection, LATs were about 100,000-fold more abundant than spliced ICP0 transcripts in the TG, about 1,000-fold more in the spinal cord and the hypothalamus, and 100-fold more in the SCG. The LATs were far more numerous than ICP0 transcripts (either spliced or intron-containing) in all the structures during latent infection.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Ratios between numbers of LAT and ICP0 transcripts

Using RT-PCR detection of TK and UL18 transcripts with a 10-copy detection threshold, none of the latently infected tissues from mice L10 to L13 expressed TK or UL18 genes, which represent early and late genes of HSV-1. These data suggest that HSV-1 gene expression in these tissues is unlikely to be a consequence of viral reactivation. In contrast, tissues from acutely infected mice A2 to A4 were positive for these two lytic-cycle transcripts (Fig. 6).


Figure 6
View larger version (89K):
[in this window]
[in a new window]
  		FIG. 6. Amplification of TK and UL18 transcripts, using RT-PCR from spinal cord. RNA extracted from mock (lane 1)-, latently (lanes 2 to 5), and acutely (lanes 6 to 8) infected tissues, corresponding to mice L10 to L13 and A2 to A4, respectively. Panels A and B show agarose gels for TK and UL18 amplification products, respectively. No amplification product could be detected in mock- and latently infected RNA extracts. (C) Southern blot using a digoxigenin-labeled probe specific to the UL18 amplification product. Equivalent results were obtained for UL18 transcripts in TG, SCG, and hypothalamus and for TK transcripts in TG, SCG, spinal cord, and hypothalamus. Panels D and E show semiquantitative evaluations of the RT-PCR sensitivity for TK and UL18 transcripts, respectively, using increasing amounts of corresponding transcripts (from wells left to right, 0 to 106 copies).

To test if the large amounts of LATs and intron-containing ICP0 transcripts within the TG at 28 dpi were remnants of transcripts produced during the acute phase, eight additional mice were sacrificed in an independent experiment (L14 to L16 sacrificed at 28 dpi and L17 to L21 sacrificed at 90 dpi, among which L17 to L19 were used for RT-PCR and L20 and L21 were used for ISH). We observed that LATs and ICP0 transcripts, either spliced or intron containing, were present in similar or slightly increased numbers in the TG of mice sacrificed at 90 dpi than in those at 28 dpi (Table 5).


View this table:
[in this window]
[in a new window]
  		TABLE 5. Comparison of the numbers of spliced and intron-containing ICP0 transcripts and 2-kb LATs in the left TG at 28 and 90 dpi


arrow
DISCUSSION
 
The pattern of HSV-1 gene expression during the latent phase of infection has been debated for many years, and many questions remain unanswered. Since the discovery of LATs in the 1980s (reviewed in 56, 62), viral lytic genes were commonly considered to be silenced during HSV-1 latency. In the 1990s, some studies reported the expression of productive-cycle genes of HSV-1, such as those encoding the immediate-early protein ICP4 and the early protein TK (9, 34), during the latent phase of infection. Concerning the gene encoding ICP0, which has a key role in the balance between latency and reactivation of HSV-1 (17), Kubat et al. showed in latently infected mouse TG that the ICP0 promoter was hypoacetylated, in an heterochromatin-like state, suggesting silencing of the ICP0 promoter during HSV-1 latency (35, 36). The observation that the ICP0 promoter is hypoacetylated is probably not in conflict with our results, since it was made from whole ganglia and is population weighted. Thus, it is likely that some individual neurons (or even genomes within neurons) are in a different transcriptional or chromatin state. Indeed, Liacono et al. showed that the ICP0 promoter may be activated in neurons in the absence of viral regulatory proteins in transgenic mice expressing the ICP0 promoter-driven lacZ gene (44), suggesting that these neurons express the transcription factors required for ICP0 promoter activation. Additionally, Chen et al. detected ICP0 transcripts in the TG of latently infected mice (10).

Our study reports the detection of ICP0 transcripts during latency not only in the TG, which is the structure generally investigated for HSV-1 latency, but also in several other anatomical sites of HSV-1 latency, including the spinal cord, hypothalamus, and SCG, after inoculation of virus into the upper lips of mice. Quantitative RT-PCR showed differential expression of ICP0 transcripts, with a splicing efficiency varying according to the latently infected structure. Expression of LATs also varied among the several anatomical sites of HSV-1 latency, suggesting that the expression of LATs and ICP0 transcripts may be involved in the regulation of the balance between latency and reactivation. This conclusion is consistent with earlier observations suggesting a role for LATs in promoting the accumulation of intron-containing ICP0 transcripts during latency (10).

The ICP0 promoter is activated in several latently infected structures. We used the SC16 110LacZ recombinant strain of HSV-1 that contains the ICP0 promoter-driven lacZ gene inserted within the nonessential US5 gene. This virus allows both expression of the native ICP0 protein and detection of ICP0 promoter activity, as revealed by ß-galactosidase assays on histological sections (41). Despite a slight difference in survival rates for the wild-type SC16 and the recombinant SC16 110LacZ strains of HSV-1, as previously described (75), no difference in ocular and neurological pathogenesis was observed. ICP0 promoter activity was observed in all the neurological sites of HSV-1 replication during the acute phase of infection in a manner consistent with the results of Shimeld et al. (65). The same recombinant virus strain had also been used as a marker of HSV-1 latency in sensory neurons (75), and using the genetic background of the 17+syn strain of HSV-1, the IE110-driven lacZ gene was shown to reliably report the expression of native ICP0 during the acute infection (79). Thus, the recombinant HSV-1 strain SC16 110LacZ could also be a convenient tool to study ICP0 promoter activity during latency.

Reproducible ICP0 promoter activity was found on sections from latently infected TG, the SCG, and the spinal cord, which were also known to be sites of LAT promoter activity during HSV-1 latency in the animal model used (39). For each histological section, only one to three positive cells could be observed. Other structures expressing HSV-1 latency in this model, such as the hypothalamus, the Edinger-Westphal nucleus, and the facial motor nerve nucleus (39), were negative in this assay. However, ICP0 promoter activity might be underestimated, since the reporter cassette was located in the ectopic US5 region of the HSV-1 genome, which may correspond to a region of the HSV-1 genome that is more highly repressed than the long repeat regions that encode LAT and ICP0 (12, 33, 60).

ICP0 gene transcription in several latently infected structures. In order to determine whether the native ICP0 promoter is active during latency, ICP0 gene transcription was examined in latently infected mice, using ISH and RT-PCR.

The hybridization signal was observed only when amplified with the GenPoint tyramide-biotin complex (Dako), suggesting a very low level of ICP0 transcription during latency. Positive cells were observed only within neurons of the SCG and the TG, and the ISH signal was localized mainly within the nuclei of infected neurons. Since the riboprobe was complementary to a region overlapping exon 2 and intron 2 of the ICP0 transcripts, the nuclear signal could reflect the predominant hybridization of unspliced ICP0 transcripts within neuronal nuclei during HSV-1 latency, which is consistent with the results of Ellison et al., which show that intron-containing ICP0 transcripts are retained in the nuclei of acutely infected cells (16). Similarly, 2-kb LATs are also localized mainly into the nuclei of latently infected neurons (39, 73), and given the similarity in the numbers of LAT-positive and ICP0-positive neurons in the TG (Table 1), these observations suggest a potential mechanism of regulation involving both LATs and ICP0 transcripts within the nuclei of latently infected neurons. Furthermore, our inability to detect ICP0 protein suggests that ICP0 transcripts detected during latent infections may not yield significant amounts of ICP0 protein.

RT-PCR analysis of latently infected tissues at 28 dpi detected ICP0 transcripts not only in the TG and the SCG, which were positive by ISH, but also in other latently infected structures such as the spinal cord and the hypothalamus. Although we cannot formally rule out the possibility that the detection of ICP0 transcripts is due to the slow degradation of a stable ICP0 transcript, a similar pattern of ICP0 transcription, as revealed by ISH and RT-PCR experiments, was found in the TG at 90 dpi (Table 5), suggesting long-term persistence and/or continuous production of ICP0 RNA during latency in the absence of virus reactivation. Additionally, two other mice sacrificed at 90 dpi were found positive for ICP0 ISH in the TG neurons, with a pattern similar to that observed at 28 dpi (Fig. 3E and F). Regardless of whether these ICP0 transcripts are a consequence of latent phase ICP0 transcription or persistence of stable transcripts, it is clear that their abundance and patterns of processing differ in different anatomical sites of latency. Furthermore, detection of ICP0-specific RNA in latently infected ganglia occurs in the absence of detectable reactivation.

While ICP0 transcripts in latently infected TG at 28 dpi have previously been shown by Chen et al. (10), further evidence is provided here that ICP0 transcripts are present in the main neurological structures where HSV-1 establishes a latent infection. In contrast to the result of Chen et al., no TK transcripts were detected during the latent phase of infection in our model, even though the TK detection threshold of our assay was very low. This discrepancy may be explained by differences in the animal models and/or in the viruses used. The oro-ocular model used here, which is based on inoculation into the upper lip, results in minimal trauma compared to corneal scarification, which results in a persistent inflammatory response in the TG at 30 dpi, as revealed by Shimeld et al. (67). Corneal trauma induces up-regulation of transcriptional factors such as oct-1, c-jun, and c-fos (81) that have binding sites in the promoter regions of several IE and early genes of HSV-1 (33, 50, 72) and may explain the low level of TK expression observed following corneal scarification. With the oro-ocular model, no signs of inflammation were seen 28 dpi at the site of inoculation, and the mice had clear and normal corneas, suggesting that nerve stimulation is unlikely to persist in the TG neurons at 28 dpi, thus explaining the inability to detect TK transcripts in our experiments. Alternatively, it could be that the biological patterns during latent infection of SC16 (used in our study) and HSV-1(KOS) (used by Chen et al.) are different. Indeed, it has been shown that the HSV-1(SC16) strain is more neurovirulent than the KOS strain (38, 76).

Differential accumulation of LATs and splicing of ICP0 transcripts during HSV-1 latency. ICP0 transcripts were found in large amounts in all the neuronal tissues from mice sacrificed at 6 dpi, with maximal production in the spinal cord. ICP0 transcripts were also found in all latently infected tissues, especially in the TG, but in lower amounts (102- to 105-fold reduction) than during the acute infection. Since the sites of maximal transcription of ICP0 were different in acutely and latently infected tissues, ICP0 gene transcription during HSV-1 latency seems to be regulated at the level of the neuron itself rather than dependent on the severity of the acute infection.

About 90% of ICP0 transcripts were spliced during acute infection in all tissues examined, which presumably reflects that correct splicing and translation of ICP0 protein is mandatory for productive HSV-1 infection (18). In latently infected tissues, the splicing efficiency of ICP0 transcripts was strikingly different and varied depending on the structure: 60 to 100% of ICP0 transcripts in the SCG and the hypothalamus were spliced versus 18% in the right TG, 6% in the spinal cord, and only 0.3% in the left TG. The marked accumulation of intron-containing ICP0 transcription in the TG is in keeping with the results of Chen et al. (10). This accumulation of unspliced ICP0 transcripts within the TG and the spinal cord could reflect a regulatory mechanism of HSV-1 latency maintenance in these structures.

Expression of 2-kb LATs during latency also differed among tissues, with maximal and the most-stable expression in the TG, in contrast to the other HSV-1-latently infected structures (Fig. 5C). These differences between the TG and the other latently infected tissues in the stable expression of LATs over time are consistent with previous ISH results (39, 61).

Taken together, these results showed a peculiar behavior of the TG to express and accumulate LATs and unspliced ICP0 transcripts during the latent stage of HSV-1 infection. The presence of immediate-early transcripts (ICP0) with no early (tk) or late (UL18) gene expression suggests that at least in a proportion of neurons HSV-1 latency could result from a blockade between immediate-early and early gene expression, as previously suggested by the results of Chen et al. (10) and debated in several reviews (33, 56). However, three points remain to be elucidated. (i) Do the patterns of ICP0 transcription observed occur uniformly in all latently infected cells or do they reflect events occurring in a smaller LAT-positive subset of neurons? (ii) Is the regulation of ICP0 splicing mediated by LATs, and (iii) is the inability to fully splice ICP0 RNA resulting in a block in reactivation?

A hypothesis for the control of HSV-1 latency. This study provides evidence for differences in the abilities of neurons to express HSV-1 transcripts during latency. The TG, which contains peripheral sensory neurons, can be distinguished from other HSV-1 latently infected structures as follows: (i) both ICP0 transcripts and 2-kb LAT transcripts are more abundant than in the other latently infected structures; (ii) the splicing of ICP0 transcripts is relatively inefficient; and (iii) herpetic recurrences are more frequent in tissues connected to the TG (e.g., the lip or the cornea) than in tissues connected to other structures, including the iris, which is connected to the SCG, and the retina, which is connected to the hypothalamus (43).

It is possible that the patterns of HSV-1 gene expression detected during latent infection in TG neurons reflects the fact that these neurons are regularly exposed to HSV-1 reactivation triggers. Under these circumstances there may be a requirement for multiple reactivation control mechanisms. The trigeminal nerve endings, located in the face and the cornea, often undergo microtraumas, such as ultraviolet irradiation. The cellular transcription factors such as oct-1, c-jun, and c-fos-1, which rapidly increase in TG neurons following an experimental nerve injury (74, 81), could promote both LATs and ICP0 gene expression, since they have binding sites in the LAT and ICP0 promoters (33, 50, 57, 72). Owing to the established role of ICP0 in the viral productive cycle and reactivation process (7, 18, 56), tight regulation of ICP0 gene expression would be advantageous to the survival of HSV-1 by regulating reactivation events. We hypothesize that the LATs could either inhibit splicing of ICP0 transcripts and/or increase the stability of intron-containing ICP0 transcripts, resulting in their retention within the nuclei of latently infected neurons and thus the blockade of viral replication.

This hypothesis is consistent with several previous results. An alternative splicing of ICP0 transcripts has already been shown to regulate viral gene expression in acutely infected cells in culture (69, 83), and such a regulation could persist during the latent infection. Furthermore, overexpression of LATs reduces IE gene expression and ICP0 RNA levels in neuronal cells (47) but not in nonneuronal cells (5), whereas LAT-null mutants result in increased expression of ICP4 and TK mRNA in latently infected mouse ganglia (9), increased productive-cycle gene expression in acutely infected ganglia (22), and decreased amounts of intron-containing ICP0 transcripts in latently infected TG (10).

Other mechanisms for the maintenance of HSV-1 latency involving LATs and/or ICP0 have been previously suggested. An antisense interaction between these transcripts has been invalidated (5). A role for the putative protein ORFP, encoded by either L/ST transcripts or by minor LAT species (4), has not been confirmed (10). Finally, recent experiments suggest a posttranscriptional constraint on the expression of ICP0 protein mediated by LATs (79), which is consistent with our findings.

The putative regulation of ICP0 expression involving LATs is not exclusive of the other properties previously assigned to these transcripts, especially antiapoptotic effects, leading to an increased reservoir of latently infected neurons (32, 54, 55), inhibition of viral superinfection (48), and anti-interferon effects (52).

Finally, our results showed that ICP0 transcripts and 2-kb LATS are detected in all HSV-1 latently infected tissues in the mouse. Since the splicing efficiency of ICP0 transcripts seems related to the amount of LATs, it will be of interest to determine the importance of the expression and interaction of these two viral products in the regulation of HSV-1 latency.


arrow
ACKNOWLEDGMENTS
 
We are grateful to Jane Arthur (Cambridge) for providing plasmid pSLAT5 and to Roger Everett (Glasgow) for gently providing anti-ICP0 antibodies. Many warm thanks to Anne Flamand for helpful discussions and continuous encouragement.

This work was supported in part by grants from the Fédération des Aveugles et Handicapés Visuels de France, the Fondation pour la Recherche Médicale, the Association des Amblyopes Unilatéraux, and the Agence Nationale pour la Recherche (grant 05 MIIM 008 02). S. Efstathiou acknowledges support from the Medical Research Council and Wellcome Trust. There are no conflicts of interest.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Virologie Moléculaire et Structurale, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France. Phone: 33 1 69 82 38 40. Fax: 33 1 69 82 43 08. E-mail: marc.labetoulle{at}bct.aphp.fr. Back


arrow
REFERENCES
 
    1
  1. Alvira, M. R., W. F. Goins, J. B. Cohen, and J. C. Glorioso. 1999. Genetic studies exposing the splicing events involved in herpes simplex virus type 1 latency-associated transcript production during lytic and latent infection. J. Virol. 73:3866-3876.[Abstract/Free Full Text]
    2. 2
  2. Arthur, J., S. Efstathiou, and A. Simmons. 1993. Intranuclear foci containing low abundance herpes simplex virus latency-associated transcripts visualized by non-isotopic in situ hybridization. J. Gen. Virol. 74:1363-1370.[Abstract/Free Full Text]
    3. 3
  3. Block, T. M., and J. M. Hill. 1997. The latency associated transcripts (LAT) of herpes simplex virus: still no end in sight. J. Neurovirol. 3:313-321.[Medline]
    4. 4
  4. Bruni, R., and B. Roizman. 1996. Open reading frame P—a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA. Proc. Natl. Acad. Sci. USA 93:10423-10427.[Abstract/Free Full Text]
    5. 5
  5. Burton, E. A., C. S. Hong, and J. C. Glorioso. 2003. The stable 2.0-kilobase intron of the herpes simplex virus type 1 latency-associated transcript does not function as an antisense repressor of ICP0 in nonneuronal cells. J. Virol. 77:3516-3530.[Abstract/Free Full Text]
    6. 6
  6. Cai, W., T. L. Astor, L. M. Liptak, C. Cho, D. M. Coen, and P. A. Schaffer. 1993. The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency. J. Virol. 67:7501-7512.[Abstract/Free Full Text]
    7. 7
  7. Cai, W., and P. A. Schaffer. 1992. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J. Virol. 66:2904-2915.[Abstract/Free Full Text]
    8. 8
  8. Cai, W. Z., and P. A. Schaffer. 1989. Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J. Virol. 63:4579-4589.[Abstract/Free Full Text]
    9. 9
  9. Chen, S. H., M. F. Kramer, P. A. Schaffer, and D. M. Coen. 1997. A viral function represses accumulation of transcripts from productive- cycle genes in mouse ganglia latently infected with herpes simplex virus. J. Virol. 71:5878-5884.[Abstract]
    10. 10
  10. Chen, S. H., L. Y. Lee, D. A. Garber, P. A. Schaffer, D. M. Knipe, and D. M. Coen. 2002. Neither LAT nor open reading frame P mutations increase expression of spliced or intron-containing ICP0 transcripts in mouse ganglia latently infected with herpes simplex virus. J. Virol. 76:4764-4772.[Abstract/Free Full Text]
    11. 11
  11. Cohrs, R. J., J. Randall, J. Smith, D. H. Gilden, C. Dabrowski, K. H. van Der, and R. Tal-Singer. 2000. Analysis of individual human trigeminal ganglia for latent herpes simplex virus type 1 and varicella-zoster virus nucleic acids using real-time PCR. J. Virol. 74:11464-11471.[Abstract/Free Full Text]
    12. 12
  12. Deshmane, S. L., and N. W. Fraser. 1989. During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. J. Virol. 63:943-947.[Abstract/Free Full Text]
    13. 13
  13. Devi-Rao, G. B., S. A. Goodart, L. M. Hecht, R. Rochford, M. K. Rice, and E. K. Wagner. 1991. Relationship between polyadenylated and nonpolyadenylated herpes simplex virus type 1 latency-associated transcripts. J. Virol. 65:2179-2190.[Abstract/Free Full Text]
    14. 14
  14. Dyson, H., C. Shimeld, T. J. Hill, W. A. Blyth, and D. L. Easty. 1987. Spread of herpes simplex virus within ocular nerves of the mouse: demonstration of viral antigen in whole mounts of eye tissue. J. Gen. Virol. 68:2989-2995.[Abstract/Free Full Text]
    15. 15
  15. Efstathiou, S., and C. M. Preston. 2005. Towards an understanding of the molecular basis of herpes simplex virus latency. Virus Res. 111:108-119.[CrossRef][Medline]
    16. 16
  16. Ellison, K. S., S. A. Rice, R. Verity, and J. R. Smiley. 2000. Processing of {alpha}-globin and ICP0 mRNA in cells infected with herpes simplex virus type 1 ICP27 mutants. J. Virol. 74:7307-7319.[Abstract/Free Full Text]
    17. 17
  17. Everett, R. D. 1989. Construction and characterization of herpes simplex virus type 1 mutants with defined lesions in immediate early gene 1. J. Gen. Virol. 70:1185-1202.[Abstract/Free Full Text]
    18. 18
  18. Everett, R. D. 2000. ICP0, a regulator of herpes simplex virus during lytic and latent infection. Bioessays 22:761-770.[CrossRef][Medline]
    19. 19
  19. Farrell, M., A. Dobson, and L. Feldman. 1991. Herpes simplex latency-associated transcript is a stable intron. Proc. Natl. Acad. Sci. USA 88:790-794.[Abstract/Free Full Text]
    20. 20
  20. Feldman, L. T., A. R. Ellison, C. C. Voytek, L. Yang, P. Krause, and T. P. Margolis. 2002. Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. Proc. Natl. Acad. Sci. USA 99:978-983.[Abstract/Free Full Text]
    21. 21
  21. Fraser, N. W., W. C. Lawrence, Z. Wroblewska, D. H. Gilden, and H. Koprowski. 1981. Herpes simplex type 1 DNA in human brain tissue. Proc. Natl. Acad. Sci. USA 78:6461-6465.[Abstract/Free Full Text]
    22. 22
  22. Garber, D. A., P. A. Schaffer, and D. Knipe. 1997. A LAT-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type 1. J. Virol. 71:5885-5893.[Abstract]
    23. 23
  23. Gerdes, J. C., D. S. Smith, and S. L. Forstot. 1981. Restriction endonuclease cleavage of DNA obtained from herpes simplex isolates of two patients with bilateral disease. Curr. Eye Res. 1:357-360.[Medline]
    24. 24
  24. Gupta, A., J. J. Gartner, P. Sethupathy, A. G. Hatzigeorgiou, and N. W. Fraser. 2006. Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 442:82-85.[Medline]
    25. 25
  25. Hagglund, R., and B. Roizman. 2004. Role of ICP0 in the strategy of conquest of the host cell by herpes simplex virus 1. J. Virol. 78:2169-2178.[Free Full Text]
    26. 26
  26. Halford, W. P., C. D. Kemp, J. A. Isler, D. J. Davido, and P. A. Schaffer. 2001. ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells. J. Virol. 75:6143-6153.[Abstract/Free Full Text]
    27. 27
  27. Halford, W. P., and P. A. Schaffer. 2001. ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency. J. Virol. 75:3240-3249.[Abstract/Free Full Text]
    28. 28
  28. Harris, R. A., and C. M. Preston. 1991. Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814. J. Gen. Virol. 72:907-913.[Abstract/Free Full Text]
    29. 29
  29. Hill, J. M., F. Sedarati, R. T. Javier, E. K. Wagner, and J. G. Stevens. 1990. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 174:117-125.[CrossRef][Medline]
    30. 30
  30. Hill, T. J., H. J. Field, and W. A. Blyth. 1975. Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. J. Gen. Virol. 28:341-353.[Abstract/Free Full Text]
    31. 31
  31. 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.[Abstract/Free Full Text]
    32. 32
  32. Inman, M., G. C. Perng, G. Henderson, H. Ghiasi, A. B. Nesburn, S. L. Wechsler, and C. Jones. 2001. Region of herpes simplex virus type 1 latency-associated transcript sufficient for wild-type spontaneous reactivation promotes cell survival in tissue culture. J. Virol. 75:3636-3646.[Abstract/Free Full Text]
    33. 33
  33. Jones, C. 1998. Alphaherpesvirus latency: its role in disease and survival of the virus in nature. Adv. Virus. Res. 51:237-347.[Medline]
    34. 34
  34. Kramer, M. F., and D. M. Coen. 1995. Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus. J. Virol. 69:1389-1399.[Abstract]
    35. 35
  35. Kubat, N. J., A. L. Amelio, N. V. Giordani, and D. C. Bloom. 2004. The herpes simplex virus type 1 latency-associated transcript (LAT) enhancer/rcr is hyperacetylated during latency independently of LAT transcription. J. Virol. 78:12508-12518.[Abstract/Free Full Text]
    36. 36
  36. Kubat, N. J., R. K. Tran, P. McAnany, and D. C. Bloom. 2004. Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression. J. Virol. 78:1139-1149.[Abstract/Free Full Text]
    37. 37
  37. Labetoulle, M., P. Kucera, G. Ugolini, F. Lafay, E. Frau, H. Offret, and A. Flamand. 2000. Neuronal propagation of HSV-1 from the oral mucosa to the eye. Investig. Ophthalmol. Vis. Sci. 41:2600-2606.[Abstract/Free Full Text]
    38. 38
  38. Labetoulle, M., P. Kucera, G. Ugolini, F. Lafay, E. Frau, H. Offret, and A. Flamand. 2000. Neuronal pathways for the propagation of HSV-1 from one retina to the other in a murine model. J. Gen. Virol. 81:1201-1210.[Abstract/Free Full Text]
    39. 39
  39. Labetoulle, M., S. Maillet, S. Efstathiou, S. Dezelee, E. Frau, and F. Lafay. 2003. HSV-1 latency sites after inoculation in the lip: assessment of their localization and connections to the eye. Investig. Ophthalmol. Vis. Sci. 44:217-225.[Abstract/Free Full Text]
    40. 40
  40. Lachmann, R. H., and S. Efstathiou. 1997. Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system. J. Virol. 71:3197-3207.[Abstract]
    41. 41
  41. Lachmann, R. H., M. Sadarangani, H. R. Atkinson, and S. Efstathiou. 1999. An analysis of herpes simplex virus gene expression during latency establishment and reactivation. J. Gen. Virol. 80:1271-1282.[Abstract]
    42. 42
  42. Leib, D. A., D. M. Coen, C. L. Bogard, K. A. Hicks, D. R. Yager, D. M. Knipe, K. L. Tyler, and P. A. Schaffer. 1989. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J. Virol. 63:759-768.[Abstract/Free Full Text]
    43. 43
  43. Liesegang, T. J. 2001. Herpes simplex virus epidemiology and ocular importance. Cornea 20:1-13.[CrossRef][Medline]
    44. 44
  44. Loiacono, C. M., R. Myers, and W. J. Mitchell. 2002. Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice. J. Virol. 76:2449-2459.[Abstract/Free Full Text]
    45. 45
  45. Lonsdale, D. M., S. M. Brown, J. H. Subak-Sharpe, K. G. Warren, and H. Koprowski. 1979. The polypeptide and the DNA restriction enzyme profiles of spontaneous isolates of herpes simplex virus type 1 from explants of human trigeminal, superior cervical and vagus ganglia. J. Gen. Virol. 43:151-171.[Abstract/Free Full Text]
    46. 46
  46. Mador, N., E. Braun, H. Haim, I. Ariel, A. Panet, and I. Steiner. 2003. Transgenic mouse with the herpes simplex virus type 1 latency-associated gene: expression and function of the transgene. J. Virol. 77:12421-12429.[Abstract/Free Full Text]
    47. 47
  47. Mador, N., D. Goldenberg, O. Cohen, A. Panet, and I. Steiner. 1998. Herpes simplex virus type 1 latency-associated transcripts suppress viral replication and reduce immediate-early gene mRNA levels in a neuronal cell line. J. Virol. 72:5067-5075.[Abstract/Free Full Text]
    48. 48
  48. Mador, N., A. Panet, and I. Steiner. 2002. The latency-associated gene of herpes simplex virus type 1 (HSV-1) interferes with superinfection by HSV-1. J. Neurovirol. 8(Suppl. 2):97-102.[CrossRef][Medline]
    49. 49
  49. Martin, R. G., C. R. Dawson, P. Jones, B. Togni, C. Lyons, and J. O. Oh. 1977. Herpesvirus in sensory and autonomic ganglia after eye infection. Arch. Ophthalmol. 95:2053-2056.[Abstract/Free Full Text]
    50. 50
  50. O'Hare, P., and C. R. Goding. 1988. Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell 52:435-445.[CrossRef][Medline]
    51. 51
  51. Paxinos, G. 1995. The rat nervous system, 2 ed. Academic Press, San Diego, Calif.
    52. 52
  52. Peng, W., G. Henderson, M. Inman, L. BenMohamed, G. C. Perng, S. L. Wechsler, and C. Jones. 2005. The locus encompassing the latency-associated transcript of herpes simplex virus type 1 interferes with and delays interferon expression in productively infected neuroblastoma cells and trigeminal ganglia of acutely infected mice. J. Virol. 79:6162-6171.[Abstract/Free Full Text]
    53. 53
  53. Perng, G. C., E. C. Dunkel, P. A. Geary, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1994. The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency. J. Virol. 68:8045-8055.[Abstract/Free Full Text]
    54. 54
  54. Perng, G. C., C. Jones, J. Ciacci-Zanella, M. Stone, G. Henderson, A. Yukht, S. M. Slanina, F. M. Hofman, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler. 2000. Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science 287:1500-1503.[Abstract/Free Full Text]
    55. 55
  55. Perng, G. C., B. Maguen, L. Jin, K. R. Mott, N. Osorio, S. M. Slanina, A. Yukht, H. Ghiasi, A. B. Nesburn, M. Inman, G. Henderson, C. Jones, and S. L. Wechsler. 2002. A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels. J. Virol. 76:1224-1235.[Abstract/Free Full Text]
    56. 56
  56. Preston, C. M. 2000. Repression of viral transcription during herpes simplex virus latency. J. Gen. Virol. 81:1-19.[Free Full Text]
    57. 57
  57. Preston, C. M., M. C. Frame, and M. E. Campbell. 1988. A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence. Cell 52:425-434.[CrossRef][Medline]
    58. 58
  58. Preston, C. M., and M. J. Nicholl. 1997. Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis. J. Virol. 71:7807-7813.[Abstract]
    59. 59
  59. Rezuchova, I., M. Kudelova, V. Durmanova, A. Vojvodova, J. Kosovsky, and J. Rajcani. 2003. Transcription at early stages of herpes simplex virus 1 infection and during reactivation. Intervirology 46:25-34.[CrossRef][Medline]
    60. 60
  60. Rock, D. L., and N. W. Fraser. 1983. Detection of HSV-1 genome in central nervous system of latently infected mice. Nature 302:523-525.[CrossRef][Medline]
    61. 61
  61. Rodahl, E., and J. G. Stevens. 1992. Differential accumulation of herpes simplex virus type 1 latency-associated transcripts in sensory and autonomic ganglia. Virology 189:385-388.[CrossRef][Medline]
    62. 62
  62. Roizman, B., and D. M. Knipe. 2001. Herpes simplex viruses and their replication, p. 2399. In B. N. Fields and D. M. Knipe (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
    63. 63
  63. Sacks, W. R., and P. A. Schaffer. 1987. Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICP0 exhibit impaired growth in cell culture. J. Virol. 61:829-839.[Abstract/Free Full Text]
    64. 64
  64. Sawtell, N. M., and R. L. Thompson. 1992. Herpes simplex virus type 1 latency-associated transcription unit promotes anatomical site-dependent establishment and reactivation from latency. J. Virol. 66:2157-2169.[Abstract/Free Full Text]
    65. 65
  65. Shimeld, C., S. Efstathiou, and T. Hill. 2001. Tracking the spread of a lacZ-tagged herpes simplex virus type 1 between the eye and the nervous system of the mouse: comparison of primary and recurrent infection. J. Virol. 75:5252-5262.[Abstract/Free Full Text]
    66. 66
  66. Shimeld, C., A. B. Tullo, T. J. Hill, W. A. Blyth, and D. L. Easty. 1985. Spread of herpes simplex virus and distribution of latent infection after intraocular infection of the mouse. Arch. Virol. 85:175-187.[CrossRef][Medline]
    67. 67
  67. Shimeld, C., J. L. Whiteland, N. A. Williams, D. L. Easty, and T. J. Hill. 1997. Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1. J. Gen. Virol. 78:3317-3325.[Abstract]
    68. 68
  68. Shimomura, Y., J. B. Dudley, L. P. Gangarosa, Sr., and J. M. Hill. 1985. HSV-1 quantitation from rabbit neural tissues after epinephrine-induced reactivation. Investig. Ophthalmol. Vis. Sci. 26:121-125.[Abstract/Free Full Text]
    69. 69
  69. Spatz, S. J., E. C. Nordby, and P. C. Weber. 1996. Mutational analysis of ICP0R, a transrepressor protein created by alternative splicing of the ICP0 gene of herpes simplex virus type 1. J. Virol. 70:7360-7370.[Abstract]
    70. 70
  70. Spivack, J. G., G. Woods, and N. W. Fraser. 1991. Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT. J. Virol. 65:6800-6810.[Abstract/Free Full Text]
    71. 71
  71. Steiner, I., J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser. 1989. Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection. EMBO J. 8:505-511.[Medline]
    72. 72
  72. Stern, S., M. Tanaka, and W. Herr. 1989. The Oct-1 homoeodomain directs formation of a multiprotein-DNA complex with the HSV transactivator VP16. Nature 341:624-630.[CrossRef][Medline]
    73. 73
  73. Stevens, J. G., E. K. Wagner, G. B. Devi-Rao, M. L. Cook, and L. T. Feldman. 1987. RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons. Science 235:1056-1059.[Abstract/Free Full Text]
    74. 74
  74. Tal-Singer, R., T. M. Lasner, W. Podrzucki, A. Skokotas, J. J. Leary, S. L. Berger, and N. W. Fraser. 1997. Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures. J. Virol. 71:5268-5276.[Abstract]
    75. 75
  75. Thackray, A. M., and H. J. Field. 2000. The effects of antiviral therapy on the distribution of herpes simplex virus type 1 to ganglionic neurons and its consequences during, immediately following and several months after treatment. J. Gen. Virol. 81:2385-2396.[Abstract/Free Full Text]
    76. 76
  76. Thompson, R. L., M. L. Cook, G. B. Devi-Rao, E. K. Wagner, and J. G. Stevens. 1986. Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS. J. Virol. 58:203-211.[Abstract/Free Full Text]
    77. 77
  77. Thompson, R. L., and N. M. Sawtell. 1997. The herpes simplex virus type 1 latency-associated transcript gene regulates the establishment of latency. J. Virol. 71:5432-5440.[Abstract]
    78. 78
  78. Thompson, R. L., and N. M. Sawtell. 2001. Herpes simplex virus type 1 latency-associated transcript gene promotes neuronal survival. J. Virol. 75:6660-6675.[Abstract/Free Full Text]
    79. 79
  79. Thompson, R. L., M. T. Shieh, and N. M. Sawtell. 2003. Analysis of herpes simplex virus ICP0 promoter function in sensory neurons during acute infection, establishment of latency, and reactivation in vivo. J. Virol. 77:12319-12330.[Abstract/Free Full Text]
    80. 80
  80. Tullo, A. B., D. L. Easty, W. A. Blyth, and T. J. Hill. 1982. Ocular herpes simplex and the establishment of latent infection. Trans. Ophthalmol. Soc. UK 102:15-18.[Medline]
    81. 81
  81. Valyi-Nagy, T., S. L. Deshmane, A. Dillner, N. W. Fraser, and S. Deshmane. 1991. Induction of cellular transcription factors in trigeminal ganglia of mice by corneal scarification, herpes simplex type 1 infection, and explantation of trigeminal ganglia. J. Virol. 65:4142-4152.[Abstract/Free Full Text]
    82. 82
  82. Warren, K. G., S. M. Brown, Z. Wroblewska, D. Gilden, H. Koprowski, and J. Subak-Sharpe. 1978. Isolation of latent herpes simplex virus from the superior cervical and vagus ganglions of human beings. N. Engl. J. Med. 298:1068-1069.[Medline]
    83. 83
  83. Weber, P. C., S. J. Spatz, and E. C. Nordby. 1999. Stable ubiquitination of the ICP0R protein of herpes simplex virus type 1 during productive infection. Virology 253:288-298.[CrossRef][Medline]
    84. 84
  84. Wu, T. T., Y. H. Su, T. M. Block, and J. M. Taylor. 1998. Atypical splicing of the latency-associated transcripts of herpes simplex type 1. Virology 243:140-149.[CrossRef][Medline]
    85. 85
  85. Zwaagstra, J. C., H. Ghiasi, S. M. Slanina, A. B. Nesburn, S. C. Wheatley, K. Lillycrop, J. Wood, D. S. Latchman, K. Patel, and S. L. Wechsler. 1990. 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. J. Virol. 64:5019-5028.[Abstract/Free Full Text]

Journal of Virology, September 2006, p. 9310-9321, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.02615-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Girlich, D., Poirel, L., Nordmann, P. (2009). CTX-M Expression and Selection of Ertapenem Resistance in Klebsiella pneumoniae and Escherichia coli. Antimicrob. Agents Chemother. 53: 832-834 [Abstract] [Full Text]  
  • Terry-Allison, T., Smith, C. A., DeLuca, N. A. (2007). Relaxed Repression of Herpes Simplex Virus Type 1 Genomes in Murine Trigeminal Neurons. J. Virol. 81: 12394-12405 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maillet, S.
Right arrow Articles by Labetoulle, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maillet, S.
Right arrow Articles by Labetoulle, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»