• Anti-apoptotic activity of the herpes simplex virus type 2 gene ICP10 PK: Implications for therapy of neurological disorders that involve apoptosis

      Perkins, Dana Stela; Aurelian, Laure (2002)
      Apoptosis is an etiologic component of neurodegenerative disorders [e.g. Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS)] and acute brain injury (ischemia/hypoxia and trauma). Our laboratory has previously shown that a Herpes Simplex Virus Type 2 (HSV-2) gene, ICP10 PK, activates the Ras/MEK/ERK pathway in non-neuronal cells. Because this pathway was implicated in neuronal cell survival, the present studies tested whether ICP 10 PK blocks apoptosis in various experimental paradigms of neuronal apoptosis: (1) virus infection of primary hippocampal cultures, (2) trophic factor deprivation of NGF-dependent PC12 cells and primary hippocampal cultures, (3) naturally occurring genetic disorders such as the mouse trisomy 16 (Ts16), and (4) oxidative stress of N2a neuronal cells that express a mutant superoxide dismutase-1 (G85R). In the virus infection paradigm, HSV-1 and an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) induced apoptosis, but apoptosis was not seen for HSV-2 and an HSV-2 mutant that retains the ICP10 PK (ICP10DeltaRR), suggesting that ICP10 PK has anti-apoptotic activity. This activity was dependent on activation of the Raf/MEK/ERK survival pathway and inhibition of the pro-apoptotic JNK/c-Jun pathway. It involved inhibition of caspase-3 activation and PARP cleavage, likely resulting from induction of the anti-apoptotic protein Bag-1 and inhibition of the pro-apoptotic protein Bad. Ectopically expressed ICP10 PK inhibited apoptosis in the three other tested paradigms. The broad anti-apoptotic activity of ICP10 PK suggests that it may be used in gene therapy of neurological disorders that involve apoptosis. A replication-deficient HSV-2 mutant (ICP10DeltaRR) engineered in our laboratory, was used for non-invasive delivery of ICP10 PK to the CNS. Expression of the therapeutic ICP10 PK gene following intranasal administration in the mouse, was consistent with a central spread of the vector through the central olfactory pathways to the hippocampus and related limbic structures. Collectively, the data suggest that ICP10 PK has broad anti-apoptotic activity and can be delivered to the CNS by peripheral administration of a replication-deficient HSV-2 vector.
    • Broad spectrum neuroprotection by the HSV-2 gene ICP10PK includes multiple targets and crosstalk between neurons and glial cells

      Laing, Jennifer; Aurelian, Laure (2009)
      Neurodegenerative diseases are characteristically multi-target and multi-cell conditions that are resistant to presently available therapeutics options. It is widely believed that gene therapy strategies could be a promising alternative to current therapies, but a clinical challenge is the identification of the target gene(s) and the cell to cell interactions that regulate pathogenesis. ICP10PK is an anti-apoptotic herpes simplex virus type 2 (HSV-2) gene. Here we describe the ability of ICP10PK to provide neuroprotection against a wide range of toxic stimuli including caspase and calpain-dependent programmed cell death (PCD), oxidative stress and death caused by genetic alterations, such as SOD1. The mechanism of neuroprotection, including modulation of cell-cell interactions is emphasized. Our data indicate that ICP10PK, delivered with the growth compromised HSV-2 vector deltaRR or by transfection, inhibits PCD induced through the activation of the Ras-dependent MEK/ERK and PI3K/Akt survival pathways. Neuroprotection was seen both in vitro and in vivo, including models of acute excitotoxicity (kainic acid and NMDA), an in vitro model of Parkinson's disease (MPP+), and in vitro and in vivo models of Amyotrophic lateral sclerosis (G93A and G85R SOD1). ICP10PK-mediated neuroprotection was associated with MEK/ERK and PI3K/Akt-dependent release of soluble factors that protected uninfected (ICP10PK-) neurons. They include VEGF, which has paracrine protective activity on adjacent (ICP10PK-) neurons and the chemokine fractalkine (FKN) which functions as a bi-directional mediator of crosstalk between neurons and microglia. FKN released by the ICP10PK+ neurons stimulated microglia to release increased levels of the neuroprotective cytokine (IL-10) while inhibiting the release of the inflammatory cytokine TNF-alpha. Factors released by the ICP10PK+ neurons also modulated astrocytes to release nerve growth factor. ICP10PK has the distinct advantage over other neuroprotective strategies that, in addition to protecting the infected neurons, it modulates them to release neuroprotective soluble factors in a balanced proportion such as to create a self-propagating cycle of neuronal inputs and release of chemical mediators that inhibit the progression of acute and chronic neurodegeneration through protection of uninfected neurons.
    • Characterization of the intrinsic protein kinase activity and the kinase catalytic motifs of the large subunit of the herpes simplex virus type 2 ribonucleotide reductase

      Luo, Jianhua; Aurelian, Laure (1991)
      The large subunit of Herpes Simplex Virus type 2 (HSV-2) ribonucleotide reductase (ICP10) is a chimera protein consisting of a serine/threonine protein kinase (PK) domain at the amino-terminus and a ribonucleotide reductase (RR) domain at the carboxyl-terminus. Like growth factor receptor PKs, ICP10 is myristylated, it has features of a signal peptide and putative transmembrane (TM) segment, and its PK activity is modulated by basic proteins and by antibodies to amino acid residues upstream of the TM. To further characterize this PK domain, we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29 KDa protein (designated pp29{dollar}\sp{lcub}\rm la1{rcub}){dollar} that represents a truncated portion of the ICP10 PK domain as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10 positive eukaryotic cells, and peptide mapping. pp29{dollar}\sp{lcub}\rm la1{rcub}{dollar} has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn{dollar}\sp{lcub}2+{rcub}{dollar} but not by Mg{dollar}\sp{lcub}\rm 2+{rcub}{dollar} ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine. To determine the significance of ICP10 PK catalytic motifs, site-directed and deletion mutants in PK motifs I and II, the putative signal peptide and the TM segment were used to determine the role of these elements in ICP10-PK activity. PK activity was lost by deletion of the putative TM segment (amino acid residues 85-106). However, mutation of the central Gly in PK catalytic motif I (Gly{dollar}\sp{lcub}106{rcub}{dollar}) or of the invariant Lys in PK catalytic motif II (Lys{dollar}\sp{lcub}176{rcub}{dollar}) or deletion of both of these catalytic motifs (amino acid residues 106-178) did not abolish the kinase activity as determined both in auto- and transphosphorylation assays. PK activity of the mutant deleted in domains I and II was 4-fold lower than that of the wild type ICP10 and it was insensitive to Mn{dollar}\sp{lcub}2+{rcub}{dollar}, suggesting that these motifs are involved in Mn{dollar}\sp{lcub}2+{rcub}{dollar} activation of kinase activity. The result of immunoblotting demonstrated that ICP10 complexes with GTPase activating protein (GAP). Ras GTPase activity is significantly inhibited in ICP10 transformed (JHLa1) cells. These results suggested that ICP10 may constitutively activate ras activity by blocking its down-regulation process, implying a potential signal transduction mechanism for ICP10 induced transformation.
    • Construction and characterization of herpes simplex virus type 2 mutants deleted in the protein kinase or ribonucleotide reductase domains of the chimeric ICP10 protein

      Peng, Tao; Aurelian, Laure (1996)
      The large subunit of ribonucleotide reductase (RR1) encoded by Herpes Simplex Virus Type 1 and 2 (ICP6 and ICP10, respectively) is a chimeric protein consisting of a Ser/Threonine protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase (RR) domain at the carboxy terminus. The findings that the PK domain is present only in HSV RR1 proteins, it is dispensable for ribonucleotide reduction and it functions as immediate-early (IE) protein during HSV infection, suggest that the PK activity plays a significant role in virus pathogenesis. The present work was initiated to confirm the intrinsic nature of ICP10 PK activity and to elucidate its role in HSV-2 infection. In stably transfected eukaryotic cells, ICP10 PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the transmembrane (TM) segment and it was significantly impaired by mutation of the invariant Lys (Lys{dollar}\sp{lcub}176{rcub}{dollar}). Loss of PK activity by Lys{dollar}\sp{lcub}176{rcub}{dollar} mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29{dollar}\rm\sp{lcub}la1{rcub}{dollar}) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. In cells infected with laboratory and patient isolates of HSV, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and Immunoglobulin G (IgG). Two HSV-2 (G) mutants deleted in the protein kinase or ribonucleotide reductase domains of ICP10 (ICP10{dollar}\Delta{dollar}PK and ICP10{dollar}\Delta{dollar}RR, respectively) were constructed by marker transfer. ICP10{dollar}\Delta{dollar}PK virus lost its intrinsic PK activity but retained its RR activity; ICP10{dollar}\Delta{dollar}RR virus retained its PK activity but lost its RR activity. ICP10{dollar}\Delta{dollar}PK virus does not replicate during the first 10 hrs postinfection (p.i.). However, its titers catch up with those of the wild type virus by 24 hrs p.i.; ICP10{dollar}\Delta{dollar}RR virus replicates as well as the wild type virus in exponentially growing cells but it is significantly impaired for growth in growth-restricted cells. HSV-2 and ICP10{dollar}\Delta{dollar}RR virus produce similar clear plaques but ICP10{dollar}\Delta{dollar}PK virus produces hazy plaques, which under magnification consist of a mixture of lysed and unlysed cells. The studies suggest that (i) ICP10 has intrinsic auto- and transphosphorylating PK activity, (ii) ICP10 PK and RR are functionally dissociable in virus infected cells, (iii) ICP10 PK is required for virus replication during the first 10 hrs p.i. and (iv) ICP10 PK may be involved in cell death in virus infected cells.
    • Identification and characterization of the protein kinase activity associated with the large subunit of the herpes simplex virus type 2 ribonucleotide reductase and domain mapping of the multi-functional protein

      Chung, Theodore Dookjong K.; Aurelian, Laure (1991)
      The transforming region of the herpes simplex virus type 2 (HSV-2) genome encodes a 140 kDa protein, designated ICP10, which was previously demonstrated to be the large subunit of the viral ribonucleotide reductase (RR1) and to share identity with the cervical cancer associated antigen AG-4. The present work was initiated to further characterize the biochemical and functional nature of ICP10 and to elucidate its role, if any, in the induction of neoplastic transformation. Combining computer assisted sequence analyses with conventional biochemical and molecular techniques, ICP10 is demonstrated to possess multiple functionally distinct domains. Most significantly, the amino terminal one-third of ICP10, previously shown to be unique to HSV, possesses protein kinase (PK) activity, a transmembrane helical segment and an extracellular modulatory domain analogous to growth factor receptor kinases. Analysis of the RR activity reveals that leucine-rich stretch of amino acids representing the junction between the PK and RR domains is critical for association of the two virally encoded RR subunits and a previously uncharacterized cellular 180 kDa protein that functions to stabilize the RR activity. It is proposed that the ICP10 protein represents a chimera between a cellular receptor kinase-related oncoprotein and the HSV-2 RR1. Stabilization of the RR active complex may be one factor that has favored conservation of this union.
    • Molecular mechanisms of neuroprotection by the herpes simplex virus type 2 gene ICP10PK

      Wales, Samantha Q.; Aurelian, Laure (2008)
      Recent progress in molecular biology has focused interest on gene therapy as a strategy for the control of chronic and acute neurodegenerative disorders. However, the selection of the appropriate gene and delivery vector is a clinical challenge. Herpes simplex virus type 2 (HSV-2) is a promising gene delivery vector, as it is neurotropic, has a large genome that is amenable to genetic manipulation, and unlike HSV-1, it does not cause encephalitis in adult humans. HSV-2 contains an anti-apoptotic serine/threonine protein kinase (known as ICP10PK), that acts as a constitutively activated growth factor receptor. It activates Ras and its downstream MEK/ERK survival pathway and inhibits apoptosis caused by virus infection of primary hippocampal cultures (Perkins et al. 2003b, Perkins et al. 2002a). The studies described in this report were designed to examine the molecular mechanisms of ICP10PK-mediated neuroprotection, and ensure that it can act independently of other viral proteins. Rat pheochromocytoma (PC12) cells stably transfected with ICP10PK (PC47 and PC70 cells) or its kinase-negative mutant p139(TM) (PC139 cells), were neuronally differentiated by culture with nerve growth factor (NGF) and examined for cell survival after NGF withdrawal. Apoptosis was seen in PC12 and PC139, but not PC47 and PC70 cells. In PC47 cells, neuroprotection was MEK- and PKA-dependent, associated with stabilization/activation of the transcription factor cAMP-responsive element binding protein (CREB), inhibition (phosphorylation) of the pro-apoptotic protein Bad and stabilization of the anti-apoptotic proteins Bcl-2 and Bag-1. In PC70 cells, neuroprotection occurred downstream of caspase activation, and involved MEK-dependent up-regulation of the anti-apoptotic protein XIAP and down-regulation of the XIAP inhibitor Smac/DIABLO. To examine whether ICP10PK is also neuroprotective in other paradigms, we examined its effect in an in vitro model of Parkinson's disease, using the neurotoxin MPP+. ICP10PK, but not p139(TM), inhibited MPP +-induced programmed cell death through inhibition of calpain-dependent Bax translocation to the mitochondria, AIF nuclear translocation, and caspase activation, indicating that the actions of ICP10PK are kinase-dependent. Collectively, the data indicate that ICP10PK has broad-spectrum neuroprotective activity that extends beyond apoptotic cellular programs. Further study of its use as a gene therapy strategy is warranted.
    • Molecular mechanisms of oncolysis by the herpes simplex virus type 2 mutant ΔPK

      Colunga, Aric Gabriel; Aurelian, Laure (2012)
      Cancer is a largely incurable and fatal disease that is resistant to standard therapeutics. This resistance is mediated by enhanced proliferative and prosurvival signals caused by activating mutations in the Ras/Raf/MEK/ERK and PI3K/AKT pathways, and the contribution of highly resilient cancer stem cells (CSC). Oncolytic virotherapy is based upon cancer specific virus replication and cell lysis. The specificity of engineered oncolytic viruses (OV) is accomplished through the deletion of one or more genes that are critical to virus replication. The mutations responsible for uncontrolled proliferation of cancer cells compensate for these deletions, thereby allowing OV to target cancer cells. HSV-2 encodes a unique serine/threonine protein kinase (PK)--known as ICP10PK--that activates the Ras and PI3K survival pathways. The mutant virus ΔPK, which lacks this PK domain, exhibits severely reduced levels of virus replication and latency reactivation while simultaneously inducing programmed cell death (PCD) in neurons. This dissertation work investigated the ability of ΔPK to selectively replicate and induce PCD in cancer cells and to define the molecular mechanisms involved therein. The data demonstrate that ΔPK eradicates cancer cultures and melanoma xenografts while sparing normal cells, and is well tolerated in treated mice. ΔPK-induced lysis of melanoma monolayer cultures is dominated by caspase- and calpain-mediated PCD, but also involves autophagy induction and JNK/cJun activation. Importantly, ΔPK eradication of breast and melanoma CSC requires autophagy and/or calpain but not caspase activation, and the contribution of calpain-mediated facilitation of autophagic flux represents a novel node of cross-talk between these two death pathways. In addition, ΔPK infection of melanoma cultures concomitantly inhibits anti-inflammatory IL-10 secretion while inducing the expression and secretion of numerous pro-inflammatory cytokines. Melanoma xenografts treated with ΔPK also exhibit a marked innate immune response. Significantly, ΔPK induces its own cancer specific replication through the activation of JNK, whereas the induction of PCD has no effect on virus growth. Collectively, the data indicate that ΔPK differentially induces a spectrum of PCD and pro-inflammatory programs that preclude the selection of resistant subpopulations in both quiescent CSC and proliferative tumor cells. These mechanisms uniquely potentiate its oncolytic activity and highlight its clinical promise.
    • Neoplastic transformation, cellular localization and protein kinase activity of the RR1 subunit of herpes simplex virus type 2 (ICP10)

      Hunter, James Colin Ramsay; Aurelian, Laure (1996)
      The large subunit of the herpes simplex virus (HSV) Type 1 and Type 2 ribonucleotide reductase (ICP6 and ICP10, respectively) is a chimaera consisting of a protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase domain at the carboxy terminus. The present work was performed in order to further characterize this PK activity and determine its biological significance. Computer-assisted analyses of ICP10 and ICP6 in comparison to other, known protein kinases suggest that they are members of a previously unidentified sub-family of serine/threonine (Ser/Thr) growth factor receptors. Using cells infected with laboratory and patient isolates of HSV, ICP10 was demonstrated to possess a Ser/Thr specific auto- and transphosphorylating kinase activity. ICP6 exhibited a similar PK activity, but was considerably weaker. Cells that constitutively express ICP10 or a library of ICP10 mutants illustrated a direct relationship between ICP10 PK activity and its ability to transform cells as determined by anchorage-independent growth. All mutations which abolish PK activity resulted in a concomitant abrogation in its transforming potential. Cells transfected to constitutively express ICP10 demonstrated its internalization from the membrane and degradation in lysosomes in a manner characteristic of a typical activated growth factor receptor. ICP10 was also found bound to cytoskeletal actin, similar to epidermal growth factor receptor. A PK-negative mutant of ICP10, deleted in the transmembrane domain was recoevered from the cytosol, but did not localize to the membrane, nor was it found in endosomes or lysosomes, nor in association with actin. In ICP10 constitutively expressing cells, Raf-1 and Erk evidenced decreased electrophoretic mobility consistent with an activated state, suggesting that ICP10 PK may cause transformation through modulation of the MAP kinase pathway. It is suggested that the PK domains of ICP6 and ICP10 were cellular genes integrated into the genome of an ancestral herpes simplex virus and subsequently conserved to promote viral replication and/or reactivation from latency.
    • Oncolytic Mechanisms of the HSV-2 Mutant deltaPK and Enhancement by Valproic Acid

      Bollino, Dominique; Aurelian, Laure (2015)
      Carcinogenesis is the process by which normal cells become malignant. It typically occurs through the accumulation of mutations that dysregulate intracellular signaling pathways and lead to unchecked growth and proliferation. Oncolytic viruses (OV) are replication conditional virus mutants that take advantage of these overactivated growth pathways to selectively replicate in and lyse tumor cells. In addition to the direct infection and lysis of tumor cells, OVs also kill tumor cells through disruption of tumor vasculature and the induction of potent anti-tumor immune responses. Clinical efficacy of OVs remains relatively poor, attributed to therapeutic barriers such as poor tumor penetration, premature viral clearance, and the presence of highly resistant cancer stem cell (CSC) subpopulations. Efforts to enhance OV efficacy include the addition of transgenes to enhance anti-tumor immunity, as well as combination therapy with cytotoxic and/or immunosuppressive drugs to increase tumor cell death and reduce innate antiviral responses. The growth compromised HSV-2 mutant ΔPK, has robust oncolytic activity in both melanoma cultures and xenografts associated with the induction of multiple pathways of programmed cell death. However, the impact of ΔPK on putative CSC populations, as well as its ability to harness immune responses that contribute to tumor cell death is still poorly understood. This thesis work sought to answer these questions as well as investigate the potential benefits of combining ΔPK with valproic acid (VPA), a histone deacetylase inhibitor with demonstrated cytotoxic and immunosuppressive properties. We report that: (i) ΔPK prevents anchorage-independent growth and lyses 3D cultures through calpain-dependent clearance of the autophagy protein p62/SQSTM1, (ii) ΔPK oncolysis includes several features of immunogenic cell death, such as the inhibition of Th2-based immunosuppressive conditions, promotion of a Th1-biased microenvironment, and the induction of anti-tumor immune surveillance mechanisms, (iii) VPA induces a novel calpain-dependent necroptotic form of cell death in neuronal cells, and (iv) the combination of VPA and ΔPK treatment in melanoma increases ΔPK -induced cell death through enhanced caspase activation. These findings suggest that ΔPK is a multi-mechanistic OV with particularly promising cancer therapeutic potential, and warrant further in vivo investigation into the oncolytic potential of the ΔPK and VPA combination.
    • Transcriptional regulation and neoplastic transforming potential of the large subunit of ribonucleotide reductase from herpes simplex virus type 2

      Wymer, James Paul; Aurelian, Laure (1991)
      Herpes simplex virus (HSV) genes are regulated in a cascade of immediate early (IE), delayed early (DE), late (L). The large subunit of ribonucleotide reductase (RR) from HSV-2, designated ICP10, has been grouped with DE proteins. The amino-terminal domain of ICP10 has protein kinase (PK) activity and properties similar to growth factor receptor kinases that can be activated to transforming potential. The studies described in this dissertation sought to develop a better understanding of regulatory aspects of ICP10 regulation as well as the role of ICP10 expression in neoplastic transformation. Regulation of expression of the ICP10 gene was studied by immunofluorescence with the intact ICP10 gene or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 promoter constructions containing the wild type ICP10 promoter or site-directed mutants deficit in specific cis-response motifs. Co-transfection of these constructions with DNA encoding an HSV nonspecific transactivator (IE110) or an IE gene-specific transactivator (Vmw65), enhanced expression at least 10-fold, regardless of the assay system. In contrast, expression was minimally enhanced by DNA encoding a DE gene transactivator (IE175) at low doses and slightly reduced at high doses. Sequence analysis of the ICP10 promoter revealed the presence of both herpesvirus IE gene-specific (TAATGARAT, GA-rich, and {dollar}\alpha{dollar}H2-{dollar}\alpha{dollar}H3 motifs), as well as cellular cis-response motifs (potential SP-1, consensus AP-1, and octamer transcription factor-1 (OTF-1) binding elements). Factors that bind to the ICP10 promoter were identified by gel retardation analysis with mixtures of uninfected cell nuclear extracts and virion lysates or in vitro synthesized OTF-1 and Vmw65. The Oct-1 motif (ATGCAAAT) was necessary for optimal Vmw65 binding to, but not for transactivation of the ICP10 promoter as evidenced by competition experiments with oligonucleotides overlapping the consensus IE110 promoter virion response element and by site-directed mutagenesis of the motif. The 3{dollar}\sp\prime{dollar} portion of the TAATGARAT motif (GARAT) was dispensible for binding but necessary for activation. These data suggest that ICP10 behaves as an IE gene and could therefore affect host gene regulation independent of lytic infection. ICP10-PK has neoplastic transforming potential in vitro. Anchorage independent growth was observed in cells transfected with the vectors that express the entire ICP10 protein or just the PK domain, but not a frameshift (not expressing ICP10) mutant or a carboxy-terminus (RR domain) expression vector.
    • Vaccinia recombinants as probes of relevant HSV-2-specific immunity

      Wachsman, Matthew; Aurelian, Laure (1991)
      Vaccinia recombinants expressing the glycoprotein D (gD) of HSV-1 (VP176) or HSV-2 (VP221) under control of an early vaccinia virus promoter or of HSV-1 under the control of a late promoter (VP254) were studied as probes for relevant immunity. The recombinants expressed comparable amounts of gD and grew equally well in vitro and in vivo VP176 immunization protected guinea pigs against primary (p {dollar}<{dollar} 0.005) and recurrent (p {dollar}<{dollar} 0.005) cutaneous HSV-2 disease, VP221 protected against recurrent disease and VP254 immunization afforded no protection. All vaccines protected mice at 10 days post infection, but VP176 gave superior protection on day 50 post immunization (p {dollar}<{dollar} 0.0001). Although neutralizing anti-HSV antibody and immunity to vaccinia antigens induced were identical, immunization with VP176 as compared to VP254 induced significantly higher levels of T-cell mediated immunity to HSV antigen as defined by lymphocyte transformation or delayed type hypersensitivity (P {dollar}<{dollar} 0.01). L3T4+ LNC from VP176 immunized animals mediated these effects and could transfer protection but required a second non-HSV immune, radiosensitive cell. Immunity induced by prior HSV-2 immunization was less effective than that induced by VP176 in terms of preventing primary disease (p {dollar}<{dollar} 0.01) and local viral replication (100 fold higher virus titers). This was associated with lower HSV-specific blastogenic and DTH response in HSV but not VP176 immune animals following HSV reexposure. VP254 but not VP176 infected antigen presenting cells failed to produce fully glycosylated gD. This defect correlated with the failure of VP254 infected epidermal or spleen cells to express gD on the cell surface or to present antigen.