Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript.

Latent infections with periodic reactivation are a common outcome after acute infection with many viruses. The latency-associated transcript (LAT) gene is required for wild-type reactivation of herpes simplex virus (HSV). However, the underlying mechanisms remain unclear. In rabbit trigeminal ganglia, extensive apoptosis occurred with LAT(-) virus but not with LAT(+) viruses. In addition, a plasmid expressing LAT blocked apoptosis in cultured cells. Thus, LAT promotes neuronal survival after HSV-1 infection by reducing apoptosis.

Insulin suppresses triiodothyronine-induced growth hormone secretion by GH3 rat pituitary cells.

Physiological doses of insulin were shown by us to suppress basal and hydrocortisone (HCT)-induced GH secretion and GH mRNA levels in GH3 rat pituitary cells. Because T3 stimulates GH gene expression, the effects of semisynthetic human insulin were tested on T3-induced GH secretion. Cells were first incubated for 48 h in medium containing insulin and fetal calf serum (10%) depleted of T3 and T4 by ion exchange resin. Insulin suppression of basal GH secretion was independent of T3, as insulin (0.7 nM) suppressed basal secretion of GH by 40% in T3-depleted cells. Medium glucose concentrations and cell proliferation did not differ in control or insulin-treated wells. The 4-fold increase in GH secretion induced by added T3 (0.5 nM) during 72 h was suppressed up to 40% by insulin (P less than 0.001). This suppression was maximal with 3.5 nM insulin and occurred after a lag period of 48 h. The previously described 20-fold synergistic stimulation of GH by T3 (0.5 nM) together with HCT (1 microM) was also suppressed by insulin (3.5 nM) by 80% during 72 h of incubation. The selectivity of the inhibitory effects of insulin on GH were also shown when the T3-induced suppression of PRL secretion was reversed by insulin treatment. The data show that physiological doses of insulin antagonize T3-induced stimulation of GH secretion and also partially block the synergistic stimulation of GH by T3 and HCT. As T3 and HCT probably stimulate GH gene transcription at different sites, insulin may suppress GH gene expression at a more distal site. Alternatively, the inhibitory effects of insulin on T3-induced GH secretion in these cells may be posttranscriptional.

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1.5 kilobases of the 8.3-kilobase primary transcript.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We report here that although the LAT gene is 8.3 kb in length, the first 1.5 kb of the LAT gene alone is sufficient for wild-type levels of spontaneous reactivation. We began with a LAT deletion mutant of HSV-1 strain McKrae in which the LAT promoter and the first 1.6 kb of the 5' end of the LAT gene had been deleted from both copies of LAT (one in each viral long repeat). As we previously reported, this mutant (dLAT2903) was significantly impaired for spontaneous reactivation (G. C. Perng, E. C. Dunkel, P. A. Geary, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 68:8045-8055, 1994). We then inserted the LAT promoter and the first 1.5 kb of the LAT gene into a location in the unique long region of dLAT2903 far removed from the normal location of LAT in the long repeats. This resulted in a virus (LAT15a) whose capacity for transcribing LAT RNA was limited to the first 1.5 kb of the 8.3-kb LAT primary transcript. Rabbits were ocularly infected with this mutant, and spontaneous reactivation was measured in comparison to those of the original LAT-negative mutant and its marker-rescued (wild-type) virus, dLAT2903R. LAT15a had an in vivo spontaneous reactivation rate of 12%, compared with a rate of 11% for the marker-rescued virus and 0% for the LAT-negative virus. Southern analysis confirmed that the spontaneously reactivated LAT15a virus retained the original deletions in both copies of LAT and the 1.5-kb LAT insertion in the unique long region. Thus, insertion of the first 1.5 kb of LAT (and its promoter) at a site distant from the normal LAT location appeared to completely restore in vivo spontaneous reactivation to wild-type levels, despite the remaining inability of the original LAT genes to transcribe any LAT RNA. The function of LAT involved in efficient spontaneous reactivation therefore appeared to map completely within the first 1.5 kb of the LAT gene.

A 371-nucleotide region between the herpes simplex virus type 1 (HSV-1) LAT promoter and the 2-kilobase LAT is not essential for efficient spontaneous reactivation of latent HSV-1.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. However, neither the mechanism by which LAT carries out this function nor the region of LAT responsible for this function in known. LAT is transcribed as an unstable 8.3-kb RNA that gives rise to a very stable 2-kb LAT RNA that is readily detected in latently infected sensory neurons. We show here that 371 of the 662 nucleotides located between the start of LAT transcription and the 5' end of the 2-kb LAT RNA do not appear to be essential for wild-type levels of spontaneous reactivation in the rabbit ocular model of HSV-1 latency. We deleted LAT nucleotides 76 to 447 from both copies of the LAT gene (one in each viral long repeat) to produce the mutant dLAT371. Rabbits were ocularly infected with dLAT371, and spontaneous reactivation was measured in comparison with the marker-rescued virus dLAT371R. Both dLAT371 and dLAT371R had spontaneous reactivation rates of approximately 13 to 14%. This was consistent with the parental McKrae wild-type virus (11.7%; P = 0.49) and significantly higher than the LAT transcription-negative mutant dLAT2903 (2.4%; P < 0.0001). Southern analysis confirmed that the spontaneously reactivated dLAT371 virus retained the deletion in both copies of LAT. Therefore, it appeared that the function of LAT involved in efficient spontaneous reactivation mapped outside the 371-nucleotide region deleted from the LAT gene of dLAT371.

High-dose ocular infection with a herpes simplex virus type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation.

We report here that in the rabbit ocular model of herpes simplex virus type 1 (HSV-1) latency, spontaneous reactivation of the HSV-1 ICP34.5 deletion mutant d34.5 increased significantly in response to increasing infectious doses. At the highest infectious dose of d34.5, the spontaneous reactivation rate was indistinguishable from that of wild-type virus (average spontaneous reactivation rates for d34.5, 0.3 to 1.4% at 2 x 10(5) PFU per eye, 3.4% at 2 x 10(6) PFU per eye, and 6.3 to 11.5% at 1 x 10(8) PFU per eye; average spontaneous reactivation rates for marker-rescued virus, 7.7 to 19.6% at 2 x 10(5) PFU per eye). The percentage of latency-associated transcript (LAT) RNA-positive neurons in sections from trigeminal ganglia (TG) of rabbits latently infected with d34.5 demonstrated a similar dose-response effect as estimated by in situ hybridization (0.05% LAT RNA-positive neurons at 2 x 10(5) PFU per eye and 0.1% LAT RNA-positive neurons at 1 x 10(8) PFU per eye; P = 0.002). In contrast, even at the highest infectious dose (1 x 10(8) PFU per eye), d34.5 was less virulent (23 of 23 survivors) than the normal infectious dose (2 x 10(5) PFU per eye) of marker-rescued virus (14 of 27 survivors; P < 0.0001). In addition, at 1 x 10(8) PFU per eye, d34.5 produced virtually no corneal disease, compared with the production of severe corneal disease by 2 x 10(5) PFU of marker-rescued virus per eye (P < 0.0001). Thus, at increasing infectious doses of d34.5, both spontaneous reactivation and the percentage of neurons expressing LAT appeared to increase, without a corresponding increase in virulence. These results strongly suggest that (i) the phenotypes of neurovirulence and spontaneous reactivation are separable, (ii) the phenotypes of corneal disease and spontaneous reactivation are separable, and (iii) the decreased rate of spontaneous reactivation previously reported for d34.5 (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995) is at least partially due to a reduced rate of establishing latency.

Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames.

The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICP0 and is transcribed in the opposite direction from ICP0, producing LR-RNAs that are complementary (antisense) to ICP0 mRNA. The LR gene is thought to be involved in HSV-1 latency. We report here the fine mapping and partial sequence analysis of this HSV-1 LR gene. 32P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LR-RNA 2, respectively. Their 5' ends started approximately 1,210 nucleotides downstream from the 3' end of the ICP0 mRNA. Their 3' ends overlapped ICP0 by nearly 1,000 nucleotides. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.

Therapeutic periocular vaccination with a subunit vaccine induces higher levels of herpes simplex virus-specific tear secretory immunoglobulin A than systemic vaccination and provides protection against recurrent spontaneous ocular shedding of virus in latently infected rabbits.

Rabbits latently infected with herpes simplex virus type 1 (HSV-1) were vaccinated either periocularly or systemically with a subunit vaccine (gB2 + gD2) plus adjuvant or adjuvant alone. Tear films were collected daily to measure recurrent infectious HSV-1 shedding. After systemic vaccination, the latently infected rabbits were not protected against recurrent ocular viral shedding (HSV-1-positive tear film cultures/total cultures) compared with either the systemic or periocular adjuvant controls (systemic vaccination = 49 of 972, 5.0%; systemic control = 46 of 972, 4.7%; periocular control = 43 of 930, 4.6%; P > 0.8). In contrast, latently infected rabbits vaccinated periocularly with the same vaccine had significantly reduced recurrent shedding (20 of 1026, 2.0%) compared with controls (P < 0.001) or systemic vaccination (P = 0.0002). Thus, recurrent HSV-1 shedding was significantly reduced by therapeutic local periocular subunit vaccination but not by therapeutic systemic subunit vaccination. Neutralizing antibody titers in the serum of systemically and ocularly vaccinated rabbits was similar. In contrast, HSV-specific tear secretory immunoglobulin A was significantly higher in the ocularly vaccinated group (P < 0.01). These results strongly suggest that in the rabbit, and presumably in humans, the local ocular (mucosal) immune response is much more important than the systemic immune response for therapeutic protection against recurrent ocular HSV-1. Thus development of a therapeutic vaccine against recurrent ocular HSV-1 should be directed at enhancing the local ocular (mucosal) immune response.

The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits.

The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LAT-negative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (DeltaLAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than DeltaLAT-EGFP-infected neurons (4.9% versus 2%, P < 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for DeltaLAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (P = 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two- to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.

Herpes simplex virus type 1 serum neutralizing antibody titers increase during latency in rabbits latently infected with latency-associated transcript (LAT)-positive but not LAT-negative viruses.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation in the rabbit ocular model of HSV-1 latency and reactivation. LAT is also the only viral gene abundantly expressed during latency. Rabbits were ocularly infected with the wild-type HSV-1 strain McKrae or the McKrae-derived LAT null mutant dLAT2903. Serum neutralizing antibody titers were determined at various times during acute and latent infection. The neutralizing antibody titers induced by both viruses increased and were similar throughout the first 45 days after infection (P > 0.05). However, by day 59 postinfection (approximately 31 to 45 days after latency had been established), the neutralizing antibody titers induced by wild-type virus and dLAT2903 diverged significantly (P = 0.0005). The dLAT2903-induced neutralizing antibody titers decreased, while the wild-type virus-induced neutralizing antibody titers continued to increase. A rescuant of dLAT2903, in which spontaneous reactivation was fully restored, induced wild-type neutralizing antibody levels on day 59 postinfection. A second LAT mutant with impaired spontaneous reactivation had neutralizing antibody levels comparable to those of dLAT2903. In contrast to the results obtained in rabbits, in mice, neutralizing antibody titers did not increase over time during latency with any of the viruses. Since LAT is expressed in both rabbits and mice during latency, the difference in neutralizing antibody titers between these animals is unlikely to be due to expression of a LAT protein during latency. In contrast, LAT-positive (LAT(+)), but not LAT-negative (LAT(-)), viruses undergo efficient spontaneous reactivation in rabbits, while neither LAT(+) nor LAT(-) viruses undergo efficient spontaneous reactivation in mice. Thus, the increase in neutralizing antibody titers in rabbits latently infected with LAT(+) viruses may have been due to continued restimulation of the immune system by spontaneously reactivating virus.

Expression of the first 811 nucleotides of the herpes simplex virus type 1 latency-associated transcript (LAT) partially restores wild-type spontaneous reactivation to a LAT-null mutant.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is required for efficient spontaneous reactivation in the rabbit ocular model. We recently showed that insertion of 1.8 kb of the LAT promoter and the first 1.5 kb of the 8.3-kb primary LAT transcript into a novel, ectopic location in the virus unique long (UL) region restored wild-type spontaneous reactivation to a LAT-null mutant. To further map the LAT spontaneous reactivation function within the first 1.5 kb of LAT, we rescued the same LAT-null mutant by inserting 1.8 kb of the LAT promoter and just the first 811 nucleotides of LAT into the same location in the UL. In a series of three experiments, the resulting virus, designated LAT2.6A, had a spontaneous reactivation rate that was midway between the original LAT-null mutant and wild-type virus. Thus expression of the first 811 LAT nucleotides produced a spontaneous reactivation rate that was significantly higher than that of the LAT-null mutant but significantly less than that of wild type. This suggests that part, but not all, of the LAT function involved in efficient spontaneous reactivation is located within the first 811 nucleotides of the primary 8.3-kb LAT.

The role of interleukin (IL)-2 and IL-4 in herpes simplex virus type 1 ocular replication and eye disease.

To assess the relative effect of interleukin (IL)-2- and IL-4-dependent immune responses on herpes simplex virus (HSV)-1 infection, naive, vaccinated, and mock-vaccinated IL-20/0 and IL-40/0 knockout mice were challenged ocularly with HSV-1. Naive IL-20/0 mice were significantly more susceptible to lethal infection than IL-40/0 or parental BALB/c mice. Vaccinated, IL-20/0, IL-40/0, and BALB/c mice induced similar neutralizing antibody titers and were completely protected against HSV-1-induced death and corneal scarring. Vaccinated and mock-vaccinated IL-20/0 mice had significantly higher HSV-1 titers in their eyes than BALB/c mice, while vaccinated and mock-vaccinated IL-40/0 mice had significantly lower HSV-1 titers in their eyes than BALB/c mice. Recombinant (r) IL-2 treatment of the IL-20/0 mice significantly reduced ocular HSV-1 replications, but rIL-4 treatment of IL-40/0 mice significantly increased ocular HSV-1 replications. Th1 (IL-2) cytokine responses may help protect mice against ocular HSV-1 challenge and reduce ocular HSV-1 replication.

An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.

The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation.

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.

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.

During herpes simplex virus type 1 (HSV-1) neuronal latency, the only viral RNA detected is from the latency-associated transcript (LAT) gene. We have made a LAT deletion mutant of McKrae, an HSV-1 strain with a very high in vivo spontaneous reactivation rate. This mutant (dLAT2903) lacks the LAT promoter and the first 1.6 kb of the 5' end of LAT. dLAT2903 was compared with its parental virus and with a rescued virus containing a restored LAT gene (dLAT2903R). Replication of the LAT mutant in tissue culture, rabbit eyes, and rabbit trigeminal ganglia was similar to that of the rescued and parental viruses. On the basis of semiquantitative PCR analysis of the amount of HSV-1 DNA in trigeminal ganglia, the LAT mutant was unimpaired in its ability to establish latency. In contrast, spontaneous reactivation of dLAT2903 in the rabbit ocular model of HSV-1 latency and reactivation was decreased to approximately 33% of normal. This decrease was highly significant (P < 0.0001) and demonstrates that in an HSV-1 strain with a high spontaneous reactivation rate, deletion of LAT can dramatically decrease in vivo spontaneous reactivation. We also report here that deletion of LAT appeared to eliminate rather than just reduce in vivo induced reactivation.

The region of the herpes simplex virus type 1 LAT gene involved in spontaneous reactivation does not encode a functional protein.

We previously showed that the LAT function required for efficient spontaneous reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency in the rabbit maps within the first 1.5 kb of the 8.3-kb primary LAT transcript. This demonstrated that LAT does not function via an antisense mechanism, since the first 1.5 kb of LAT does not overlap any other known HSV-1 gene. Furthermore, if LAT encodes a protein essential for efficient spontaneous reactivation, it must map within the functional first 1.5 kb of LAT. Thus, the absence of a well-conserved LAT open reading frame in this region among all HSV-1 LAT genes capable of supporting high levels of spontaneous reactivation would demonstrate that LAT does not encode a protein essential for efficient spontaneous reactivation. In this report, we sequenced the first 1.5 kb of LAT from HSV-1 McKrae, a strain with a very high spontaneous reactivation rate. Of the HSV-1 LAT sequences available for comparison (17syn+, KOS, and F), only strain 17syn+ has a high spontaneous reactivation rate. However, as shown in this report, a chimeric virus containing the KOS LAT gene on an HSV-1 McKrae genetic background had a spontaneous reactivation rate indistinguishable from McKrae (15 versus 13.6%; P > 0.05). Thus, the spontaneous reactivation competency of the LAT gene from HSV-1 KOS was similar to that of the McKrae LAT gene. Comparative sequence analysis of the LAT genes from McKrae, 17syn+, and KOS revealed that none of the eight potential McKrae LAT ORFs were well conserved. Additional types of sequence analyses further confirmed that none of the potential ORFs were likely to encode a functional LAT protein. These results strongly support the notion that the LAT function involved in spontaneous reactivation is mediated by a direct DNA or RNA mechanism rather than a protein.

A herpes simplex virus type 1 latency-associated transcript mutant with increased virulence and reduced spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We previously reported that insertion of the LAT promoter and just the first 1.5 kb of the 8. 3-kb LAT gene into an ectopic location in the virus restored wild-type spontaneous reactivation to a LAT null mutant. This mutant, LAT3.3A (previously designated LAT1.5a), thus showed that the expression of just the first 1.5 kb of LAT is sufficient for wild-type spontaneous reactivation. We also showed that in the context of the entire LAT gene, deletion of LAT nucleotides 76 to 447 (LAT mutant dLAT371) had no effect on spontaneous reactivation or virulence. We report here on a LAT mutant designated LAT2.9A. This mutant is similar to LAT3.3A, except that the ectopic LAT insert contains the same 371-nucleotide deletion found in dLAT371. We found that LAT2.9A had a significantly reduced rate of spontaneous reactivation compared to marker-rescued and wild-type viruses. This was unexpected, since the combined results of dLAT371 and LAT3.3A predicted that spontaneous reactivation of LAT2.9A would be wild type. We also found that LAT2.9A was more virulent than wild-type or marker-rescued viruses after ocular infection of rabbits. This was unexpected, since LAT null mutants and LAT3.3A have wild-type virulence. These results suggest for the first time (i) that regions past the first 1.5 kb of LAT can compensate for deletions in the first 1.5kb of LAT and may therefore play a role in LAT dependent spontaneous reactivation and (ii) that regions of LAT affect viral virulence.

Three herpes simplex virus type 1 latency-associated transcript mutants with distinct and asymmetric effects on virulence in mice compared with rabbits.

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920-929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014-2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5' end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex.

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.

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 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, 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 of the more stable 2- and 1.3-kb LATs.