Mapping herpes simplex virus type 1 latency-associated transcript sequences that protect from apoptosis mediated by a plasmid expressing caspase-8.

LAT (latency-associated transcript) is the only herpes simplex virus type 1 (HSV-1) transcript abundantly expressed during neuronal latency. LAT expression is required for the high reactivation phenotype of HSV-1 and this phenotype correlates with LAT's anti-apoptosis properties. LAT nucleotides 1 to 1499 inhibit caspase-8 (death receptor apoptotic pathway), but not caspase-9 (mitochondrial apoptotic pathway), -induced apoptosis as efficiently as larger LAT fragments. LAT sequences important for inhibiting caspase-8-induced apoptosis were also localized. The ability of LAT nucleotides 1 to 1499 to efficiently inhibit caspase-8-induced apoptosis correlates with the high reactivation phenotype of a mutant virus expressing just the first 1.5 kb of LAT (nucleotides 1 to 1499).

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.

Recombinant herpes simplex virus type 1 expressing murine interleukin-4 is less virulent than wild-type virus in mice.

The effect of interleukin-4 (IL-4) on herpes simplex virus type 1 (HSV-1) infection in mice was evaluated by construction of a recombinant HSV-1 expressing the gene for murine IL-4 in place of the latency-associated transcript (LAT). The mutant virus (HSV-IL-4) expressed high levels of IL-4 in cultured cells. The replication of HSV-IL-4 in tissue culture and in trigeminal ganglia was similar to that of wild-type virus. In contrast, HSV-IL-4 appeared to replicate less well in mouse eyes and brains. Although BALB/c mice are highly susceptible to HSV-1 infection, ocular infection with HSV-IL-4 resulted in 100% survival. Furthermore, 57% of the mice survived coinfection with a mixture of HSV-IL-4 and a lethal dose of wild-type McKrae, compared with only 10% survival following infection with McKrae alone. Similar to wild-type BALB/c mice, 100% of IL-4(-/-) mice also survived HSV-IL-4 infection. T-cell depletion studies suggested that protection against HSV-IL-4 infection was mediated by a CD4(+)-T-cell response.

A herpes simplex virus type 1 mutant deleted for gamma34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation.

To create an oncolytic herpes simplex virus type 1 (HSV-1) that is inhibited for reactivation, we constructed a novel herpes recombinant virus with deletions in the gamma34.5 and LAT genes. The LAT gene was replaced by the gene for green fluorescent protein, thereby allowing viral infection to be followed. This virus, designated DM33, is effective in killing primary and established human glioma cell lines in culture. DM33 is considerably less virulent following intracerebral inoculation of HSV-susceptible BALB/c mice than the wild-type HSV-1 strain McKrae. The safety of this virus is further supported by the retention of its sensitivity to ganciclovir and its relatively limited toxicity against cultured human neuronal cells, astrocytes, and endothelial cells. The ability of DM33 to spontaneously reactivate was tested in a rabbit ocular infection model that accurately depicts human herpes infection and reactivation. Following ocular infection of rabbits, spontaneous reactivation was detected in 83% (15/18) of the eyes infected with wild-type McKrae. In contrast, none of the eyes infected with DM33 had detectable reactivation. The efficacy of this virus in cultured human glioma cell lines, its safety, confirmed by its inability to reactivate, and its attenuated neurovirulence make DM33 a promising oncolytic agent for tumor therapy.

Region of herpes simplex virus type 1 latency-associated transcript sufficient for wild-type spontaneous reactivation promotes cell survival in tissue culture.

The latency-associated transcript (LAT) is the only abundant herpes simplex virus type 1 (HSV-1) transcript expressed during latency. In the rabbit eye model, LAT null mutants do not reactivate efficiently from latency. We recently demonstrated that the LAT null mutant dLAT2903 induces increased levels of apoptosis in trigeminal ganglia of infected rabbits compared to LAT+ strains (G.-C. Perng, C. Jones, J. Ciacci-Zarella, M. Stone, G. Henderson, A. Yokht, S. M. Slanina, F. M. Hoffman, H. Ghiasi, A. B. Nesburn, and C. S. Wechsler, Science 287:1500-1503, 2000). The same study also demonstrated that a plasmid expressing LAT nucleotides 301 to 2659 enhanced cell survival of transfected cells after induction of apoptosis. Consequently, we hypothesized that LAT enhances spontaneous reactivation in part, because it promotes survival of infected neurons. Here we report on the ability of plasmids expressing different portions of the 5' end of LAT to promote cell survival after induction of apoptosis. A plasmid expressing the first 1.5 kb of LAT (LAT nucleotides 1 to 1499) promoted cell survival in neuro-2A (mouse neuronal) and CV-1 (monkey fibroblast) cells. A plasmid expressing just the first 811 nucleotides of LAT promoted cell survival less efficiently. Plasmids expressing the first 661 nucleotides or less of LAT did not promote cell survival. We previously showed that a mutant expressing just the first 1.5 kb of LAT has wild-type spontaneous reactivation in rabbits, and a mutant expressing just the first 811 nucleotides of LAT has a reactivation frequency higher than that of dLAT2903 but lower than that of wild-type virus. In addition, mutants reported here for the first time, expressing just the first 661 or 76 nucleotides of LAT, had spontaneous reactivation indistinguishable from that of the LAT null mutant dLAT2903. In summary, these studies provide evidence that there is a functional relationship between the ability of LAT to promote cell survival and its ability to enhance spontaneous reactivation.

Antibody-dependent enhancement of HSV-1 infection by anti-gK sera.

We previously reported that vaccination of BALB/c mice with the baculovirus expressed HSV-1 glycoprotein K (gK) or passive transfer of gK purified IgG to naive BALB/c mice causes severe exacerbation of HSV-1 induced corneal scarring following ocular challenge. In addition, a productive chronic infection, rather than a latent infection, is found in most trigeminal ganglia. These phenomena are accompanied by a very high T(H)1+T(H)2 response in the eye (Ghiasi, H., Cai, S., Nesburn, A.B., Wechsler, S.L., 1996. Vaccination with herpes simplex virus type 1 glycoprotein K impairs clearance of virus from the trigeminal ganglia resulting in chronic infection. Virology 224, 330-333; Ghiasi, H., Cai, S., Slanina, S., Nesburn, A. B., Wechsler, S.L., 1997. Nonneutralizing antibody against the glycoprotein K of herpes simplex virus type-1 exacerbates herpes simplex virus type-1-induced corneal scarring in various virus-mouse strain combinations. Invest. Ophthalmol. Vis. Sci. 38, 1213-1221; Ghiasi, H., Hofman, F.M., Cai, S., Perng, G.C., Nesburn, A.B., Wechsler, S.L., 1999. Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice. Vaccine 17, 2576-2582). In the studies reported here, we investigated the hypothesis that anti-gK serum produces antibody-dependent enhancement (ADE) of ocular HSV-1 infection. We found that gK vaccinated mice had significantly higher HSV-1 titers in their eyes than gD or mock-vaccinated mice and that anti-gK sera enhanced HSV-1 infection in the macrophage cell line U937. In addition, passive transfer of anti-gK sera to naive mice 24 h prior to ocular HSV-1 challenge also increased viral replication. These results were consistent with ADE of HSV-1 by sera to gK. This suggests that the severely exacerbated corneal disease seen following HSV-1 ocular challenge of gK vaccinated mice is a result of ADE. The ability of gK sera to cause harmful ADE may impact HSV-1 vaccine development.

The role of natural killer cells in protection of mice against death and corneal scarring following ocular HSV-1 infection.

C57BL/6 mice depleted of NK (natural killer) cells with anti-asialo-GM1 antibody were more susceptible to lethal HSV-1 ocular challenge (12% survival) than control C57BL/6 mice (100% survival), CD4+ depleted mice (100% survival), CD8+ depleted mice (80% survival), or macrophage depleted mice (85% survival). NK depletion also resulted in significantly higher levels of HSV-1 induced corneal scarring than was seen with any of the other groups. C57BL/6 mice depleted of NK cells with PK136 (anti-NK1.1 antibody which is more specific for NK cells than is anti-asialo-GM1 antibody) were also more susceptible to HSV-1 ocular challenge than T cell or macrophage depleted mice. Vaccination completely protected NK depleted mice against death and corneal scarring. In contrast to C57BL/6 mice, in BALB/c mice, NK depletion had no effect on survival or corneal scarring following ocular HSV-1 challenge. Experiments with IFN-gamma knockout mice (IFN-gamma(o/o) mice) suggested that IFN-gamma played a minor role in protection of naïve mice against death following HSV-1 challenge. However, IFN-gamma did not appear to be an important factor in protection against HSV-1 induced eye disease. Thus, protection against HSV-1 induced corneal scarring in naive mice appeared to be due to a non-INF-gamma NK function. Our results therefore suggest that NK cells were very important in protecting naive C57BL/6 mice but not vaccinated C57BL/6 mice against corneal scarring and death following ocular HSV-1 challenge.

Both CD4+ and CD8+ T cells are involved in protection against HSV-1 induced corneal scarring.

AIM: To determine the relative impact of CD4+ T cells and CD8+ T cells in protecting mice against ocular HSV-1 challenge. METHODS: CD4+ T cell knockout mice (CD4-/- mice), CD8+ T cell knockout mice (CD8-/- mice), and mice depleted for CD4+ or CD8+ T cells by antibody (CD4+ depleted and CD8+ depleted mice), were examined for their ability to withstand HSV-1 ocular challenge. The parental mice for both knockout mice were C57BL/6J. RESULTS: These results suggest that: (1) both CD4+ deficient mice (CD4-/- and CD4+ depleted mice) and CD8+ deficient mice (CD8-/-, and CD8+ depleted mice) developed significantly more corneal scarring than their C57BL/6J parental strain; (2) the duration of virus clearance from the eyes of the CD4+ deficient mice was 4 days longer than that of the CD8+ deficient mice; and (3) the severity of corneal scarring in the CD4+ deficient mice was approximately twice that of the CD8+ deficient mice. CONCLUSIONS: It was reported here that: (1) CD4+ and CD8+ T cells were both involved in protection against lethal ocular HSV-1 infection; and (2) CD4+ and CD8+ T cells were both involved in protection against HSV-1 induced corneal scarring.

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.

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.

Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice.

We previously reported that vaccination of BALB/c mice with different baculovirus expressed HSV-1 glycoproteins induced varying degrees of protection against HSV-1 ocular challenge, ranging from complete protection to no protection, to exacerbation of eye disease. To correlate specific local immune responses with protection and exacerbation of corneal scarring, we examined immune cell infiltrates in the cornea after ocular HSV-1 challenge of vaccinated mice. Mice were vaccinated with gD, which completely protects against corneal scarring, gG, which produces no protection against corneal scarring, or gK, which exacerbates corneal scarring. Cryostat sections of cornea were taken at different times after challenge and examined for infiltrating cells containing IL-2, IL-4, IFN-gamma, IL-6, or TNF-alpha. No corneal infiltrates were seen before challenge or 1 day after ocular challenge in any groups. By days 3-7, many cells containing IL-4 and IFN-gamma, but few cells containing IL-2, had infiltrated into the corneas of gG or mock vaccinated mice. At the same times, many cells containing IL-2, but few cells containing IL-4 or IFN-gamma, were seen in the corneas of gD vaccinated mice. In contrast, the corneas of mice vaccinated with gK contained large amounts of IL-2, IFN-gamma, and IL-4. Our results suggest that: (1) corneas from gD vaccinated mice had no corneal disease and developed a response highly biased toward IL-2 responses; (2) corneas from gG or mock vaccinated eyes had significant corneal disease and developed a mostly IL-4 and IFN-gamma cytokine response; and (3) corneas from gK vaccinated mice had exacerbated corneal disease and developed strong IL-2, IL-4 and IFN-gamma cytokine responses.

Specific and nonspecific immune stimulation of MHC-II-deficient mice results in chronic HSV-1 infection of the trigeminal ganglia following ocular challenge.

Ocular herpes simplex virus type 1 (HSV-1) infection of MHC-II-deficient mice (AO/Obeta mice) or their parental C57BL/6J wild-type mice resulted in the establishment of typical HSV-1 latent infections in the trigeminal ganglia (TG) of the surviving mice by day 28 postinfection. Latency was characterized by the complete absence of infectious virus in TG extracts, the ability to recover latent virus only following prolonged tissue culture cultivation of explanted TG, and the presence of HSV-1 DNA in TG extracts. When mice were vaccinated prior to ocular HSV-1 challenge, latency appeared unaltered in the C57BL/6J wild-type mice. However, in AO/Obeta mice, clearance of virus from the TG appeared to be seriously impaired, resulting in a chronic productive infection, rather than a latent infection. Infectious virus was readily detected in TG extracts of vaccinated AO/Obeta mice until at least 63 days postinfection. Glycoprotein B mRNA was also readily detected, confirming continued viral transcription. These chronic infections occurred regardless of whether the AO/Obeta mice were vaccinated with HSV-1-specific antigens (i.e., live HSV-1 strain KOS, recombinantly expressed HSV-1 glycoprotein D plus Freund's adjuvant, or a mixture of seven recombinantly expressed HSV-1 glycoproteins plus adjuvant) or non-HSV-1-specific antigens (i.e., tissue culture medium plus 5% fetal bovine serum, the expression vector plus adjuvant, or adjuvant alone). Passive transfer of HSV-1 neutralizing antibody to vaccinated AO/Obeta mice between days 0 and 28 post-ocular challenge did not clear infectious virus from the TG. Passive transfer of anti-HSV-1 antibody or purified naive mouse serum to unvaccinated AO/Obeta mice on days 3 or 6 post-HSV-1 ocular challenge also resulted in chronic, rather than latent, infection of the TG. Passive transfer of naive sera from B-cell-deficient mice or injection of keyhole limpet hemocyanin or purified IgG, but not PBS or dextran, 3 days after HSV-1 challenge also resulted in chronic infection of the TG.

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.

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.

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.

Identical 371-base-pair deletion mutations in the LAT genes of herpes simplex virus type 1 McKrae and 17syn+ result in different in vivo reactivation phenotypes.

The herpes simplex virus type 1 (HSV-1) LAT gene is the only viral gene abundantly transcribed during latency. LAT null mutants created with strains McKrae and 17syn+ are impaired for both in vivo spontaneous and in vivo-induced reactivation. Thus, LAT is essential for efficient in vivo-induced and spontaneous reactivation. Different investigators have studied two LAT mutants containing a StyI-StyI region deletion corresponding to LAT nucleotides 76 to 447. One mutant, dLAT371 (parent strain, McKrae), had parental high frequencies of spontaneous reactivation. In vivo-induced reactivation was not examined. The other mutant, 17DeltaSty (parent strain, 17syn+), had parental frequencies of in vitro reactivation following cocultivation of explanted ganglia but reduced frequencies of in vivo-induced reactivation. Spontaneous reactivation frequency was not reported for 17DeltaSty. These combined results suggested the possibility that in vivo spontaneous reactivation and in vivo-induced reactivation may map to different regions within the LAT domain. We now report that dLAT371 has in vivo-induced reactivation frequencies of the parent and that 17DeltaSty has reduced frequencies of in vivo spontaneous reactivation. Thus, dLAT371 demonstrated the parental phenotype for both in vivo spontaneous and -induced reactivation while the apparently identical 17DeltaSty was impaired for both in vivo spontaneous and -induced reactivation. These results suggest that one or more differences between the genetic backgrounds of McKrae and 17syn+ result in different in vivo reactivation phenotypes of otherwise identical deletion mutations and that McKrae may have compensating sequences sufficient to overcome the loss of the StyI-StyI region of the LAT transcript.

The persistent elevated cytokine mRNA levels in trigeminal ganglia of mice latently infected with HSV-1 are not due to the presence of latency associated transcript (LAT) RNAs.

Trigeminal ganglia (TG) from mice latently infected with wild type HSV-1 contain detectable levels of cytokine transcripts that are not present in TG from uninfected mice. This suggests that during HSV-1 neuronal latency, the immune system is stimulated by the production of one or more viral proteins. Since the LAT (latency associated transcript) gene is essential for wild type levels of spontaneous reactivation and is the only highly active viral gene during latency, the stimulation of cytokines may indicate the presence of a LAT encoded latency protein. We therefore compared the cytokine transcript profiles in the TG of mice latently infected with wild type and LAT negative viruses. Mice were latently infected with either: (1) the LAT null mutant dLAT2903; (2) its marker rescued virus dLAT2903R; or (3) the parental wild type HSV-1 strain McKrae. As expected, reactivation following explant cultivation of TG from latently infected mice was significantly decreased with dLAT2903 (P < 0.05)(40 +/- 8%, n = 24) compared with dLAT2903R (85 +/- 7.6%, n = 36) or the parental virus (70 +/- 10.0%, n = 36). The relative levels of various cytokines was determined by RT-PCR analysis of TG extracts. None of the cytokine transcripts detected in mice latently infected with the wild type or marker rescued viruses were missing or decreased in mice latently infected with the LAT null mutant 30 or 60 days post infection. There were also no differences in the HSV-1 antibody titers induced by the LAT negative virus compared to the LAT positive viruses. Thus, although LAT facilitated reactivation of HSV-1 from explanted mouse TG, expression of LAT during latency did not appear to be involved in persistent cytokine expression in TG. This suggests that during latency, HSV-1 does not produce a highly antigenic abundant LAT encoded protein.

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.

The UL3 open reading frame of herpes simplex virus type 1 codes for a phosphoprotein.

Based on sequence analysis, the protein encoded by the UL3 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encoding the complete sequence of the UL3 ORF in a baculovirus expression system. Western blotting was done using polyclonal antibody raised against synthetic UL3 peptides. Two major baculovirus-UL3 expressed protein bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 kDa were detected. None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated. Cell fractionation studies indicated that the UL3 protein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylation. In contrast, the baculovirus expressed UL3 protein was phosphorylated as judged by 32Pi-labeling. Immunoprecipitation followed by SDS-PAGE demonstrated a single 32Pi-labeled UL3 related band with an apparent molecular weight of 33 kDa, indicating that the UL3 protein was a phosphoprotein. Antibodies produced in mice vaccinated with baculovirus-UL3 protein reacted with two UL3 related HSV-1 bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3.