Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency.

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) protects cells against cold-shock-induced apoptosis by maintaining phosphorylation of protein kinase B (AKT).

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis.

The herpes simplex virus type 1 latency-associated transcript can protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis and CD8 T-cell killing.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo.

The herpes simplex virus 1 latency-associated transcript promotes functional exhaustion of virus-specific CD8+ T cells in latently infected trigeminal ganglia: a novel immune evasion mechanism.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB(498-505) tetramer(+)) CD8(+) T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT(+) virus compared to LAT(-) virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT(+) and LAT(-) viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT(+) virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT(-) TG having more functional HSV-gB(498-505) tetramer(+) CD8(+) T cells compared to LAT(+) TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8(+) T cells in latently infected TG, resulting in increased virus reactivation.

Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is expressed abundantly in latently infected sensory neurons. LAT-deletion-mutant virus strains have reduced-reactivation phenotypes in small animal models of infection, demonstrating that LAT plays an important role in the latency-reactivation cycle of HSV-1. Previous studies demonstrated that the anti-apoptosis functions of LAT are important for regulating the latency-reactivation cycle because three different anti-apoptosis genes can substitute for LAT. Although LAT inhibits caspase 3 activation, the signalling pathway by which LAT inhibits caspase 3 activation was not identified. In this study, we analysed mouse neuroblastoma cells (C1300) that express LAT stably (DC-LAT6 cells) following serum starvation. As expected, DC-LAT6 cells were resistant to apoptosis following serum withdrawal. Levels of total and phosphorylated AKT (protein kinase B), a serine/threonine protein kinase that promotes cell survival, were higher in DC-LAT6 cells after serum withdrawal than in C1300 cells or a cell line stably transfected with a LAT promoter mutant (DC-DeltaLAT311). A specific AKT inhibitor reduced the anti-apoptosis functions of LAT and phosphorylated AKT levels. After serum withdrawal, more DC-LAT6 cells sprouted neurites and exhibited a differentiated morphology. NeuN (neuronal nuclei), a neuron-specific nuclear protein, was expressed abundantly in DC-LAT6 cells, but not C1300 cells, after serum withdrawal, further supporting the concept that LAT enhanced neuronal-like morphology. Collectively, these studies suggested that LAT, directly or indirectly, maintained total and phosphorylated AKT levels, which correlated with increased cell survival and mature neuronal-like morphology.

Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5' end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5' terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection.

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell, CD4+ and CD8+ T cell epitopes.

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8(+) T cell epitope, from ovalbumin (OVA(257-264)) and an universal CD4(+) T helper (Th) epitope (PADRE). The resulting CTL-Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four alpha-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA(257-264)-specific IFN-gamma producing CD8(+) T cells; (3) PADRE-specific CD4(+) T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4(+), and CD8(+) T cell epitopes-based immunotherapeutic cancer vaccines.

Identification of herpes simplex virus type 1 proteins encoded within the first 1.5 kb of the latency-associated transcript.

Expression of the first 1.5 kb of the latency-associated transcript (LAT) that is encoded by herpes simplex virus type 1 (HSV-1) is sufficient for wild-type (wt) levels of reactivation from latency in small animal models. Peptide-specific immunoglobulin G (IgG) was generated against open reading frames (ORFs) that are located within the first 1.5 kb of LAT coding sequences. Cells stably transfected with LAT or trigeminal ganglionic neurons of mice infected with a LAT expressing virus appeared to express the L2 or L8 ORF. Only L2 ORF expression was readily detected in trigeminal ganglionic neurons of latently infected mice.

Identification of two small RNAs within the first 1.5-kb of the herpes simplex virus type 1-encoded latency-associated transcript.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice.

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT's antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus' reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT's proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand-, caspase-8-, or caspase-10-dependent pathway) or the intrinsic (mitochondria-, caspase-9-dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(-) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(-) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway.

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype.

During herpes simplex virus-1 (HSV-1) latency in sensory neurons, LAT (latency-associated transcript) is the only abundantly expressed viral gene. LAT plays an important role in the HSV-1 latency-reactivation cycle, because LAT deletion mutants have a significantly decreased reactivation phenotype. Based solely on sequence analysis, it was speculated that LAT encodes a ribozyme that plays an important role in how LAT enhances the virus' reactivation phenotype. Because LAT ribozyme activity has never been reported, we decided to test the converse hypothesis, namely, that this region of LAT does not encode a ribozyme function important for LAT's ability to enhance the reactivation phenotype. We constructed a viral mutant (LAT-Rz) in which the speculated ribozyme consensus sequence was altered such that no ribozyme was encoded. We report here that LAT-Rz had a wild-type reactivation phenotype in mice, confirming the hypothesis that the speculated LAT ribozyme is not a dominant factor in stimulating the latency-reactivation cycle in mice.

Craniosynostosis in transgenic mice overexpressing Nell-1.

Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS), one of the most common congenital craniofacial deformities. Here we describe the creation and analysis of transgenic mice overexpressing Nell-1. Nell-1 transgenic animals exhibited CS-like phenotypes that ranged from simple to compound synostoses. Histologically, the osteogenic fronts of abnormally closing/closed sutures in these animals revealed calvarial overgrowth and overlap along with increased osteoblast differentiation and reduced cell proliferation. Furthermore, anomalies were restricted to calvarial bone, despite generalized, non-tissue-specific overexpression of Nell-1. In vitro, Nell-1 overexpression accelerated calvarial osteoblast differentiation and mineralization under normal culture conditions. Moreover, Nell-1 overexpression in osteoblasts was sufficient to promote alkaline phosphatase expression and micronodule formation. Conversely, downregulation of Nell-1 inhibited osteoblast differentiation in vitro. In summary, Nell-1 overexpression induced calvarial overgrowth resulting in premature suture closure in a rodent model. Nell-1, therefore, has a novel role in CS development, perhaps as part of a complex chain of events resulting in premature suture closure. On a cellular level, Nell-1 expression may modulate and be both sufficient and required for osteoblast differentiation.

Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.

UNLABELLED: We studied the cellular function of Nell-1, a craniosynostosis-related gene, in craniofacial development. Nell-1 modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure. INTRODUCTION: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with CS. Nell-1 overexpression induced calvarial overgrowth and resulted in premature suture closure in a rodent model. On a cellular level, Nell-1 is suggested to promote osteoblast differentiation. MATERIALS AND METHODS: Different levels of Nell-1 were introduced into osteoblastic cells by viral infection and recombinant protein. Apoptosis and gene expression assays were performed. Mice overexpressing Nell-1 were examined for apoptosis. RESULTS: In this report, we further showed that overexpression of Nell-1 induced apoptosis along with modulation of apoptosis-related genes. The induction of apoptosis by Nell-1 was observed only in osteoblastic cells and not in NIH3T3 or primary fibroblasts. The CS mouse model overexpressing Nell-1 showed increased levels of apoptosis in the calvaria. CONCLUSION: We show that Nell-1 expression modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure.

The role of a glycoprotein K (gK) CD8+ T-cell epitope of herpes simplex virus on virus replication and pathogenicity.

PURPOSE: The authors recently reported that a recombinant HSV-1 expressing two extra copies of glycoprotein K (gK) exacerbated corneal scarring (CS) in mice. The authors also identified a peptide, STVVLITAYGLVLVW, within the signal sequence of gK as an immunodominant gK T-cell-stimulatory region both in vitro and in vivo and identified a highly conserved potential CD8(+) T-cell epitope (ITAYGLVL) within the peptide. In this study, the effect of giving this octamer (8mer) as an eye drop 1 hour before ocular infection with HSV-1 was investigated. METHODS: Naive mice and rabbits received the gK 8mer or control peptides as eye drops and were then ocularly infected with HSV-1. Virus replication in the eye and trigeminal ganglia (TG), survival, CS, and relative amounts of gB, gK, CD4, CD8, IFN-gamma, and granzyme A/B transcripts were determined in the cornea and TG of infected animals at various times after infection. The effect of the gK 8mer was also analyzed in immunized HLA transgenic mice. RESULTS: The gK 8mer resulted in a short-term significant increase in virus replication in the eyes of BALB/c mice, C57BL/6 mice, and NZW rabbits. gK 8mer treatment also increased viral neurovirulence and viral induced CS in ocularly infected mice. Moreover, in HSV-infected humanized HLA-A*0201 transgenic mice, the gK 8mer epitope induced strong IFN-gamma-producing cytotoxic CD8(+) T-cell responses, as assessed by CD107a/b expression and IFN-gamma ELISAs. CONCLUSIONS: gK 8mer induced CD8(+) T-cell responses were unlikely to occur soon enough to account for increased virus replication on day 1 after infection. In contrast, the data are consistent with CD8(+) T cells being involved in the appearance of CS at late times after infection. In addition, the gK peptide may affect viral replication and innate immune responses through other undefined mechanisms.

Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity.

The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene has anti-apoptosis activity that directly or indirectly enhances the virus's reactivation phenotype in small animal models. The first 1.5 kb of the primary 8.3 kb LAT is sufficient and some or all of it is necessary for LAT's anti-apoptosis in transient transfection assays and for LAT's ability to enhance the reactivation phenotype. Based on LAT's genomic sequence, the first 1.5 kb contains eight potential open reading frames (ORFs) defined as an ATG followed by an in frame termination codon. In this study, point mutations were introduced into the ATGs of ORFs present in the 1.5 kb fragment of LAT. Mutagenesis of all eight ATGs in LAT ORFs consistently reduced the anti-apoptotic activity of LAT in transiently transfected mouse neuroblastoma cells regardless of whether apoptosis was induced by caspase 8 or caspase 9. Mutation of the six ATGs located in the stable intron sequences within the 1.5 kb LAT had a dramatic effect on caspase 9, but not caspase 8, induced apoptosis. For both caspase 8 and caspase 9 induced apoptosis, mutating the two ATGs in the exon of the LAT 1.5 kb fragment reduced, but did not eliminate the anti-apoptotic activity of LAT. These studies suggest that altering the fine structure of regulatory RNA or expression of a putative LAT ORF regulates the anti-apoptosis activity of LAT. These studies also indicate that more than one function is present in the 1.5 kb LAT fragment.

Stable cell lines expressing high levels of the herpes simplex virus type 1 LAT are refractory to caspase 3 activation and DNA laddering following cold shock induced apoptosis.

The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene's anti-apoptosis activity plays a central, but not fully elucidated, role in enhancing the virus's reactivation phenotype. In transient transfection experiments, LAT increases cell survival following an apoptotic insult in the absence of other HSV-1 genes. However, the high background of untransfected cells has made it difficult to demonstrate that LAT inhibits specific apoptotic factors such as caspases. Here we report that, in mouse neuroblastoma cell lines (C1300) stably expressing high levels of LAT, cold shock induced apoptosis was blocked as judged by increased survival, protection against DNA fragmentation (by DNA ladder assay), and inhibition of caspase 3 cleavage and activation (Western blots). To our knowledge, this is the first report providing direct evidence that LAT blocks two biochemical hallmarks of apoptosis, caspase 3 cleavage and DNA laddering, in the absence of other HSV-1 gene products.

Reactivation phenotype in rabbits of a herpes simplex virus type 1 mutant containing an unrelated antiapoptosis gene in place of latency-associated transcript.

Latency-associated transcript (LAT) significantly enhances the spontaneous reactivation phenotype of herpes simplex virus type 1 (HSV-1). The mechanism by which LAT accomplishes this has been elusive. To determine if LAT's antiapoptosis activity is involved, the authors used a rabbit eye model to analyze the spontaneous reactivation phenotype of an HSV-1 mutant in which LAT was replaced by an unrelated antiapoptosis gene. This virus, dLAT-cpIAP, contains the open reading frame of the baculovirus inhibitor of apoptosis protein gene (cpIAP) in place of LAT, under control of the LAT promoter. The authors report here that in a rabbit ocular model of infection, dLAT-cpIAP had a spontaneous reactivation phenotype similar to wild-type virus and significantly higher than LAT(-) viruses. This was consistent with their previous findings using the mouse trigeminal ganglia explant-induced reactivation model. Whether LAT (and in the case of dLAT-cpIAP, cpIAP) enhances the spontaneous reactivation phenotype by functioning during establishment of latency, maintenance of latency, or reactivation from latency, or during two or more of these periods, remains to be determined. Regardless, the results presented in this study strongly support the hypothesis that LAT's antiapoptosis activity is the dominant function that enhances HSV-1's spontaneous reactivation phenotype.

Nell-1 induces acrania-like cranioskeletal deformities during mouse embryonic development.

We previously reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS). Nell-1 overexpression also results in premature suture closure/craniosynostosis in newborn transgenic mice. On a cellular level, increased levels of Nell-1 induce osteoblast differentiation and apoptosis. In this report, mice over-expressing Nell-1 were examined during embryonic development as well as shortly after birth for further analysis of craniofacial defects including neural tube defects (NTDs). The results demonstrated that overexpression of Nell-1 could induce acrania at relatively late gestation stage (E15.5) in mouse embryos, through massive apoptosis in calvarial osteoblasts and neural cells. The induced apoptosis was associated with an increase in Fas and Fas-L production. In addition, transgenic E15.5 and newborn transgenic mice with the CS phenotype displayed distortion of the chondrocranium associated with premature hypertrophy and increased apoptosis of chondrocytes. These findings were also verified in vitro with primary chondrocytes transduced with AdNell-1. In conclusion, Nell-1 overexpression can induce craniofacial anomalies associated with neural tube defects during embryonic development and may involve mechanisms of massive apoptosis associated with the Fas/Fas-L signaling pathway. NELL-1: used when describing the human gene; NELL-1: used when describing the human protein; Nell-1: used when describing the rodent gene; Nell-1: used when describing the rodent protein.

A herpes simplex virus type 1 mutant expressing a baculovirus inhibitor of apoptosis gene in place of latency-associated transcript has a wild-type reactivation phenotype in the mouse.

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT- mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT- mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT- mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse.