Latent Herpes Simplex Virus Type 1 Research Articles

P4-029: Herpes simplex virus type 1 (HSV1) in brain: A cause of the neuropathological features of Alzheimer's disease?

The causes of AD remain unknown, despite extensive studies on amyloid plaques and neurofibrillary tangles. We have implicated the virus, HSV1, as a major factor. We discovered that latent HSV1 is present in a high proportion of elderly brains (Jamieson et al., 1991), and in carriers of the type 4 allele of the apolipoprotein E gene (APOE-e4) it confers a strong risk of AD (Itzhaki et al., 1997); also, it reactivates from latency in brain, causing an acute infection – possibly recurrently (Wozniak et al., 2005). Immunocytochemistry; ELISA; Western blotting; in situ PCR. We have linked HSV1 directly to AD brain abnormalities: (1) HSV1 infection of cultured cells causes intracellular deposition of Aβ (Wozniak et al., 2007); (2) HSV1 infection of mice results in Aβ deposition in brain (Wozniak et al., 2007); (3) HSV1 infection of cultured cells causes AD-like phosphorylation of tau (Wozniak et al., to be submitted). These changes result from increases in the relevant secretases and kinases, and are likely due to HSV1 subverting cell processes to aid its replication, taking over the protein-synthesising machinery, and causing entry into cycle, although as in AD, only up to G2/M. As for the role of APOE-e4 with HSV1 in AD, we propose that it determines extent of HSV1 damage. This is strongly supported by our discovery that APOE determines outcome of infection in several diseases of known microbial cause (Wozniak & Itzhaki, 2006). Further, on examining HSV1 location in relation to amyloid plaques in AD brain, we found 90% of plaques associated with viral DNA and most of the viral DNA is located in plaques (Wozniak et al., submitted). This co-localisation, together with our discovery that HSV1 induces amyloid deposition, strongly implicates HSV1 in plaque formation and AD development (we can refute explanations that co-localisation results from plaque formation). Our data strongly implicate HSV1 as a major factor in AD. They support antiviral usage to prevent deterioration of patients and possibly in future, vaccination against HSV1 (which protects against establishment of viral latency in mouse brain – Lin et al., 2001) to prevent AD occurrence.

Presence of HSV‐1 Immediate Early Genes and Clonally Expanded T‐cells with a Memory Effector Phenotype in Human Trigeminal Ganglia

The latent persistence of herpes simplex virus type 1 (HSV-1) in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltrate. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to characterize the immune cell infiltrates by this feature. We combined in situ hybridization, laser cutting microscopy, and single cell RT-PCR to demonstrate the expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T-cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8+ T-cells had features of the memory effector phenotype. The voltage-gated potassium channel Kv1.3, a marker of chronic activated memory effector cells, and the chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells. The corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all CD8 T-cells. In addition, T-cells expressed granzymes and perforin. In contrast to animal models of HSV-1 latency, hardly any FoxP3-positive regulatory T-cells were detected in human TG. Thus, HSV-1 IE genes are expressed in human TG and the infiltrating T-cells bear several characteristics that suggest viral antigenic stimulation.

CD8 T cells and latent herpes simplex virus type 1: keeping the peace in sensory ganglia

Herpes simplex virus type 1 (HSV-1) infections represent a significant worldwide heath problem. The lack of an effective therapy to curtail reactivation of HSV-1 from a state of neuronal latency has lead to significant morbidity and mortality. Effective therapies to prevent reactivation must likely elicit a protective CD8 T-cell response that could act to prevent reactivation from sensory neurons prior to release of infectious virus at the periphery. This review focuses on the present understanding of how CD8 T cells maintain HSV-1 latency and how this knowledge could facilitate the generation of more effective therapeutic modalities.

The latency-associated transcript of herpes simplex virus type 1 promotes survival and stimulates axonal regeneration in sympathetic and trigeminal neurons

Herpes simplex virus type 1 (HSV-1) primarily infects mucoepithelial tissues of the eye, the orofacial region, and to a lesser extent the genitalia. The virus is retrogradely transported through the axons of sensory and sympathetic neurons to their cell bodies to establishe a life-long latent infection. Throughout this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). The function of LAT is still largely unknown. To understand how HSV-1 latency might affect neurons, the authors transfected primary cultures of sympathetic neurons and trigeminal sensory neurons obtained from rat embryos with LAT-expressing plasmids. LAT increased the survival of both sympathetic and trigeminal neurons after induction of cell death by nerve growth factor (NGF) deprivation. Because HSV-1 is transported through axons both after initial infection and during reactivation, the authors considered the possibility that LAT may affect axonal growth. They found that LAT expression increased axonal regeneration by twofold in both types of neurons. Inhibition of the mitogen-activated protein kinase (MAPK) pathway reverses stimulation of both neuronal survival and axonal regeneration, which indicates that these effects are mediated through the MAPK pathway. These data provide evidence that HSV-1 LAT promotes survival of sympathetic as well as trigeminal neurons. The authors show for the first time that LAT stimulates axonal regeneration in both sympathetic and trigeminal neurons.

Modulation of HLA-G and HLA-E Expression in Human Neuronal Cells After Rabies Virus or Herpes Virus Simplex Type 1 Infections

Human Leukocyte Antigen (HLA)-G and E are nonclassical human MHC class I molecules. They may promote tolerance leading to virus and tumor immune escape. We recently described that the herpes simplex virus type 1 (HSV-1), a neurotropic virus inducing chronic infection and neuron latency, and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection, up-regulate HLA-G expression in human neurons (NT2-N). Surface expression was only detected after RABV infection. We investigated here whether RABV and HSV-1 up-regulate HLA-E expression in human neuronal precursors (Ntera-2D/1). We found that RABV, not HSV-1, up-regulates HLA-E expression, nevertheless HLA-E could not be detected on the surface of RABV-infected Ntera-2D/1. Altogether these data suggest that HLA-G and not HLA-E could contribute to the immune escape of RABV. In contrast, there was no evidence that these molecules are used by latent HSV-1 infection. Thus, neurotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially.

Two alphaherpesvirus latency-associated gene products influence calcitonin gene-related peptide levels in rat trigeminal neurons

Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons. HSV-1 establishes a life-long latent infection in these neurons, during which the transcription of the viral genome is silent, except for the sequences encoding the latency-associated transcript (LAT). To determine if HSV-1 latency might affect calcitonin gene-related peptide (CGRP) expression in trigeminal sensory neurons, we transfected primary neuronal cultures of trigeminal ganglia from rat embryos with plasmids expressing LAT. In the presence of Bone Morphogenetic Protein-7 (BMP7), CGRP was expressed in 49% of sensory neurons. However, this percentage was reduced to 19% in neurons transfected with LAT expressing plasmids. We also found that transfection of the IE63 gene of varicella-zoster virus (VZV) reduced the percentage of trigeminal neurons containing CGRP. However, the observed effect of IE63 in contrast to that of LAT was completely reversed by treatment of cultures with MgCl2, which indicates that the effect of IE63 was due to increased release of CGRP from trigeminal neurons. We provide here the first evidence that HSV-1 LAT decreases the level of CGRP in trigeminal neurons. These effects may be important for reducing the neuroinflammatory response, thus protecting host neuronal cells during HSV-1 latency in trigeminal neurons. In contrast, increased release of CGRP in the presence of IE63 protein may contribute to the neuralgias associated with VZV infection.

Herpes simplex virus type-1 latency-associated transcript-induced immunoreactivity of substance P in trigeminal neurons is reversed by bone morphogenetic protein-7

Herpes simplex virus type-1 (HSV-1) primarily infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). To understand how HSV-1 latency might affect the expression of substance P in sensory neurons, we transfected primary cultures of trigeminal neurons obtained from rat embryos, with LAT expressing plasmids. The expression of LAT increased the percentage of substance P-immunoreactive neurons by two thirds. To examine the effect of bone morphogenetic protein-7 (BMP7) on the LAT-induced increase in substance P expression in trigeminal neurons, cultures transfected with LAT were treated with BMP7. Treatment with BMP7 reversed the effects of LAT on substance P expression in trigeminal neurons. Our data show for the first time that LAT increases substance P expression in trigeminal neurons and BMP7 can reverse these effects of LAT.

Herpes simplex virus type-1 latency inhibits dendritic growth in sympathetic neurons

Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the orofacial region, the eye and to a lesser extent the genitalia. Subsequently, the virus is retrogradely transported through the axons of the sensory and sympathetic neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). LAT has been shown to affect apoptosis, but little else is known regarding its effects on neurons. To understand how HSV-1 latency might affect dendrites in sympathetic neurons, we transfected primary cultures of sympathetic neurons obtained from rat embryos, with LAT expressing plasmids. LAT inhibited initial dendritic growth and induced dendritic retraction in sympathetic neurons. Latent HSV-1 infection of cultured sympathetic neurons inhibited dendritic growth indicating that this is likely also a consequence of natural infection.

Herpes simplex virus type 1 latently infected neurons differentially express latency-associated and ICP0 transcripts.

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

A Chromatin Insulator-Like Element in the Herpes Simplex Virus Type 1 Latency-Associated Transcript Region Binds CCCTC-Binding Factor and Displays Enhancer-Blocking and Silencing Activities

A previous study demonstrated that the latency-associated transcript (LAT) promoter and the LAT enhancer/reactivation critical region (rcr) are enriched in acetyl histone H3 (K9, K14) during herpes simplex virus type 1 (HSV-1) latency, whereas all lytic genes analyzed (ICP0, UL54, ICP4, and DNA polymerase) are not (N. J. Kubat, R. K. Tran, P. McAnany, and D. C. Bloom, J. Virol. 78:1139-1149, 2004). This suggests that the HSV-1 latent genome is organized into histone H3 (K9, K14) hyperacetylated and hypoacetylated regions corresponding to transcriptionally permissive and transcriptionally repressed chromatin domains, respectively. Such an organization implies that chromatin insulators, similar to those of cellular chromosomes, may separate distinct transcriptional domains of the HSV-1 latent genome. In the present study, we sought to identify cis elements that could partition the HSV-1 genome into distinct chromatin domains. Sequence analysis coupled with chromatin immunoprecipitation and luciferase reporter assays revealed that (i) the long and short repeats and the unique-short region of the HSV-1 genome contain clustered CTCF (CCCTC-binding factor) motifs, (ii) CTCF motif clusters similar to those in HSV-1 are conserved in other alphaherpesviruses, (iii) CTCF binds to these motifs on latent HSV-1 genomes in vivo, and (iv) a 1.5-kb region containing the CTCF motif cluster in the LAT region possesses insulator activities, specifically, enhancer blocking and silencing. The finding that CTCF, a cellular protein associated with chromatin insulators, binds to motifs on the latent genome and insulates the LAT enhancer suggests that CTCF may facilitate the formation of distinct chromatin boundaries during herpesvirus latency.

ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

BackgroundThe herpes simplex virus type 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase, which is encoded within the HSV-1 latency-associated locus. When ICP0 is not synthesized, the HSV-1 genome is acutely susceptible to cellular repression. Reciprocally, when ICP0 is synthesized, viral replication is efficiently initiated from virions or latent HSV-1 genomes. The current study was initiated to determine if ICP0's putative role as a viral interferon (IFN) antagonist may be relevant to the process by which ICP0 influences the balance between productive replication versus cellular repression of HSV-1.ResultsWild-type (ICP0+) strains of HSV-1 produced lethal infections in scid or rag2-/- mice. The replication of ICP0- null viruses was rapidly repressed by the innate host response of scid or rag2-/- mice, and the infected animals remained healthy for months. In contrast, rag2-/- mice that lacked the IFN-α/β receptor (rag2-/- ifnar-/-) or Stat 1 (rag2-/- stat1-/-) failed to repress ICP0- viral replication, resulting in uncontrolled viral spread and death. Thus, the replication of ICP0- viruses is potently repressed in vivo by an innate immune response that is dependent on the IFN-α/β receptor and the downstream transcription factor, Stat 1.ConclusionICP0's function as a viral IFN antagonist is necessary in vivo to prevent an innate, Stat 1-dependent host response from rapidly repressing productive HSV-1 replication. This antagonistic relationship between ICP0 and the host IFN response may be relevant in regulating whether the HSV-1 genome is expressed, or silenced, in virus-infected cells in vivo. These results may also be clinically relevant. IFN-sensitive ICP0- viruses are avirulent, establish long-term latent infections, and induce an adaptive immune response that is highly protective against lethal challenge with HSV-1. Therefore, ICP0- viruses appear to possess the desired safety and efficacy profile of a live vaccine against herpetic disease.

A Triple Entente: Virus, Neurons, and CD8<sup>+</sup> T Cells Maintain HSV-1 Latency

Herpes simplex virus type 1 (HSV-1) travels by retrograde transport to sensory ganglia where latency is established. Recurrent disease results from virus reactivation and anterograde transport to nerve termini. Prevention of reactivation requires a complex interplay among virus, neuron, and immune response. Study of this tripartite relationship suggests possible interaction, and even communication among these components, that direct an immune response that allows for control of virus while preserving the viability of host tissue. Exciting new evidence supports the view that CD8+ effector T cells employ both lytic granule-dependent and interferon gamma-dependent effector mechanisms in maintaining HSV-1 latency.

Genotypic analysis of sequential genital herpes simplex virus type 1 (HSV-1) isolates of patients with recurrent HSV-1 associated genital herpes.

Clinical recurrences of Herpes simplex virus type 1 (HSV-1)-associated genital herpes are thought to be caused by reactivation of latent endogenous HSV-1. However, the possibility of reinfection with exogenous HSV-1 cannot be excluded. This study aimed to determine the incidence of genital HSV-1 superinfection in patients by investigating the genotype of sequential HSV-1 isolates obtained from the same anatomical site of patients with clinical recurrences of genital HSV-1 recurrent genital herpes. Sequential genital HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1 and US12. Whereas the sequential HSV-1 isolates in 11 of the 13 patients studied had the same genotypes, the sequential isolates of 2 patients showed a different genotype. The data suggest that HSV-1-induced recurrent genital herpes can be associated with genital reinfection with an exogenous HSV-1 strain.

Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6–7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT’s anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT’s anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(−) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT’s anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.

Immunity to latent viral infection: many skirmishes but few fatalities

During early childhood, herpes simplex virus type 1 (HSV-1) can establish a latent infection in sensory neurons, which then serves as a reservoir of the virus during recurrent disease. Accordingly, recurrent herpetic disease appears to result primarily from the reactivation of latent virus and its transport down nerve axons to the periphery, rather than from exogenous re-infection. Eradicating latent HSV-1 from sensory neurons is not currently feasible but recent findings implicating host immunity in maintaining HSV-1 in a latent state provide promise for immunological intervention in recurrent herpetic disease. We propose, and provide supporting evidence, for the concept that CD8 + T cells regulate HSV-1 gene expression during latency.

Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6–7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT’s anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT’s anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(−) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT’s anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.

Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression.

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

Transgenic mouse with the herpes simplex virus type 1 latency-associated gene: expression and function of the transgene.

During herpes simplex virus type 1 (HSV-1) latent infection in human peripheral sensory ganglia, the major viral gene transcribed is the latency-associated transcript (LAT) gene. In order to facilitate the study of this gene, we generated a transgenic mouse that contains the DNA fragment that transcribes the LAT RNAs (2.0 kb and its 1.5-kb spliced transcript) under control of the cytomegalovirus promoter. The tissue distribution of these transcripts and their effects upon HSV-1 replication, latency, and reactivation in the transgenic-mouse model were examined. Different steady-state amounts of both transcripts were found in various tissues. While the highest levels of the 2.0-kb RNA were detected in heart and skeletal muscle, the 1.5-kb transcript was found at elevated levels in the brain and at much higher levels in the trigeminal ganglia (TG). Replication of both the wild-type and a LAT-negative mutant virus was suppressed in primary embryonic fibroblasts obtained from LAT-expressing transgenic mice compared to that in cells obtained from normal mice. HSV-1 DNA amounts in latently infected TG of transgenic mice were similar to those in normal mice. Reactivation of latent HSV-1 LAT-negative mutants by explant cocultivation of TG from transgenic mice was more efficient than reactivation from normal-mouse TG. Considering our present and previous results, we propose that the significantly higher steady-state level of the 1.5-kb RNA in the TG may link this transcript to latency functions and that by inhibition of virus replication, the LAT gene may protect ganglion cells and thereby increase the probability of reactivation.

Herpes Simplex Virus Latency-Associated Transcript Gene Function

A major area of interest in the study of herpes simplex virus type 1 (HSV-1) involves the persistence of the virus within a latent state in neuronal cells of infected humans. The latency-associated transcripts (LATs) are believed to play a key role during HSV-1 latency. This review will discuss the most recent findings on the involvement of the LAT region with apoptotic pathways and how this relates to other potential functions of the LATs.

Capsaicin-induced reactivation of latent herpes simplex virus type 1 in sensory neurons in culture.

Herpes simplex virus type 1 (HSV-1) produces a life-long latent infection in neurons of the peripheral nervous system, primarily in the trigeminal and dorsal root ganglia. Neurons of these ganglia express high levels of the capsaicin receptor, also known as the vanilloid receptor-1 (VR-1). VR-1 is a non-selective ion channel, found on sensory neurons, that primarily fluxes Ca(2+) ions in response to various stimuli, including physiologically acidic conditions, heat greater than 45 degrees C and noxious compounds such as capsaicin. Using an in vitro neuronal model to study HSV-1 latency and reactivation, we found that agonists of the VR-1 channel - capsaicin and heat - resulted in reactivation of latent HSV-1. Capsaicin-induced reactivation of HSV-1 latently infected neurons was dose-dependent. Additionally, activation of VR-1 at its optimal temperature of 46 degrees C caused a significant increase in virus titres, which could be attenuated with the VR-1 antagonist, capsazepine. VR-1 activation that resulted in HSV-1 reactivation was calcium-dependent, since the calcium chelator BAPTA significantly reduced reactivation following treatment with caspsaicin and forskolin. Taken together, these results suggest that activation of the VR-1 channel, often associated with increases in intracellular calcium, results in HSV-1 reactivation in sensory neurons.