Latent Infection In Neurons Research Articles

Tegument Assembly and Secondary Envelopment of Alphaherpesviruses.

Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Herpes simplex virus type 2 infection induces AD-like neurodegeneration markers in human neuroblastoma cells

Herpes simplex virus (HSV) types 1 and 2 are neurotropic viruses that establish lifelong latent infections in neurons. Mounting evidence suggests that HSV-1 infection is involved in the pathogenesis of Alzheimer's disease (AD). The relationships between other herpesvirus infections and events associated with neurodegeneration have not, however, been extensively studied. The present work reports that HSV-2 infection leads to the strong accumulation of hyperphosphorylated tau and the amyloid-β peptides Aβ40 and Aβ42 (all major pathological hallmarks of AD) in human SK-N-MC neuroblastoma cells. Infection is also associated with a marked reduction in the amount of Aβ40 secreted and in the proteolytic fragments of the amyloid-β precursor protein (APP) (secreted APPα and the α-C-terminal fragment). These results indicate that HSV-2 infection inhibits the nonamyloidogenic pathway of APP processing and impairs Aβ secretion in these cells. In addition, HSV-2 induces the accumulation of intracellular autophagic compartments containing Aβ due to a failure in the late stages of autophagy. To our knowledge, this is the first report to show that HSV-2 infection strongly alters the tau phosphorylation state, APP processing, and autophagic process in human neuroblastoma cells, leading to the appearance of AD-like neurodegeneration markers.

Nutraceutical activators of AMPK/Sirt1 axis inhibit viral production and protect neurons from neurodegenerative events triggered during HSV-1 infection

Herpes simplex virus type-1 (HSV-1) is ubiquitous and is able to establish a lifelong persistent latent infection in neurons of infected individuals. It has been estimated that in approximately 70% of the population over 50 years old, the virus enters the brain and infects neurons, and possibly undergoes recurrent reactivation episodes during lifetime, especially in immunodepressed individuals. We previously showed that the sensors AMP-dependent kinase (AMPK) and Sirtuin 1 (Sirt1), involved in survival pathways and neuroprotection, were affected during the course of HSV-1 infection. To evaluate if natural activators of the AMPK/Sirt1 axis, such as Resveratrol and Quercetin could reduce viral propagation and/or counteract the effects of neuronal infection, we analyzed progeny virion production, neuronal viability and neurodegenerative events during HSV-1 infection. We found that the activators of AMPK/Sirt1 axis, increased the viability of infected neurons, significantly reduced the viral titer in the supernatant and the expression of viral genes. More importantly, pretreatment of neurons with Resveratrol or Quercetin significantly reduced the levels of caspase-3 cleaved- and hyperphosphorylated tau associated with HSV-1 infection. These results suggest that activators of the AMPK/Sirt1 axis could be potentially useful in reducing the risk of HSV-1 productive infection in neurons and the cellular damage associated with reactivation episodes.

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na + /H + Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

Intrinsic innate immunity fails to control herpes simplex virus and vesicular stomatitis virus replication in sensory neurons and fibroblasts.

Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β, and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state. Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores, blindness, and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study, we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice, we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence or absence of innate immunity. In contrast, neurons were able to mount a robust antiviral response when provided with beta interferon, a molecule that strongly stimulates innate immunity, and that HSV-1 can combat this response through the γ34.5 viral gene. Our results have important implications for understanding how the nervous system defends itself against virus infections.

Dynamic 1H NMR-based extracellular metabonomic analysis of oligodendroglia cells infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a large, neurotropic, double-stranded DNA virus that establishes a lifelong latent infection in neurons and glial cells. Previous studies reveal that several metabolic perturbations are associated with HSV-1 infection. However, the extracellular metabolic alterations associated with HSV-1 infection have not been systematically profiled in human cells. Here, a proton nuclear magnetic resonance-based metabonomic approach was applied to differentiate the extracellular metabonomic profiles of HSV-1 infected human oligodendroglia cells (n = 18) and matched control cells (n = 18) at three time points (12, 24, and 36 h post-infection). Resulting spectra were analyzed by chemometric and statistical methods. Metabonomic profiling revealed perturbations in 21 extracellular metabolites. Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between HSV-1 infected human oligodendroglia cells and control cells. Eight extracellular metabolites, seven of which were amino acids, were primarily responsible for score plot discrimination between HSV-1 infected human oligodendroglia cells and control cells at 36 h post-infection: alanine, glycine, isoleucine, leucine, glutamate, glutamine, histidine, and lactate. HSV-1 infection alters amino acid metabolism in human oligodendroglia cells cultured in vitro. HSV-1 infection may disturb these host cellular pathways to support viral replication. Through elucidating the extracellular metabolic changes incident to HSV-1 infection, this study also provides future directions for investigation into the pathogenic mechanism of HSV-1.

Kinetics of Facultative Heterochromatin and Polycomb Group Protein Association with the Herpes Simplex Viral Genome during Establishment of Latent Infection

ABSTRACTThe herpes simplex virus (HSV) genome is associated with heterochromatic histone modifications, including trimethylation of the lysine 27 residue of histone H3 (H3K27me3), during latent infection of neurons. Here we have examined the kinetics of general chromatin and H3K27me3 association with the viral genome during establishment of latent infection. Using both wild-type virus and a mutant virus that is unable to undergo replication in neurons, we found that histone H3 associates with viral gene promoters by 7 days postinfection (dpi). Levels of H3K27me3 were low at 7 dpi but increased dramatically by 14 dpi. Hence, general chromatin association and/or other factors may play a key role(s) in the initial silencing of lytic genes, and H3K27me3 may play a role in further suppression of the genome and/or the maintenance of latency. A component of Polycomb repressive complex 2 (PRC2), which mediates the addition of K27me3 to histone H3 (Suz12), was also recruited by 14 dpi. We have shown previously that the levels of H3K27me3 during latent infection are increased in the presence of the latency-associated transcript (LAT). However, the initial targeting of PRC2 was not found to be dependent on the LAT. We found that a component of the PRC1 complex (Bmi1), which binds to H3K27me3, was not enriched at promoters found previously to be enriched for H3K27me3. Our results are consistent with (i) chromatinization of viral DNA or other mechanisms causing the initial silencing of HSV lytic genes and (ii) facultative heterochromatin maintaining that silencing during latent infection of neurons.

Chromatin Assembly on Herpes Simplex Virus 1 DNA Early during a Lytic Infection Is Asf1a Dependent

Herpes simplex virus (HSV) is a large DNA virus which is characterized by its ability to form latent infections in neurons of the peripheral nervous system. Although histones are found in the capsids of small DNA viruses (papovaviruses), none are found in the capsids of large HSV. However, after entry into the infected cell nucleus, the HSV genome begins to associate with nucleosomes during the earliest stages of infection. In contrast, late during infection, newly replicated viral DNA does not appear to associate with nucleosomes, suggesting that histones are deposited specifically on input viral DNA. The mechanisms of deposition and removing histones from the viral genome are unclear. Recently, histone chaperones, involved in the assembly and disassembly of nucleosomes, have been identified. Human antisilencing factor 1 (Asf1) is one such factor which is involved in both the assembly and disassembly of nucleosomes in cellular systems. In this study, we have examined the effect of small interfering RNA (siRNA) knockdown of Asf1a on HSV infections in HeLa cells. Both viral replication and growth were found to be decreased. Also, viral DNA was significantly less protected from micrococcal nuclease (MNase) digestion up to 6 h postinfection (hpi). However, transcription of the immediate early (IE) genes ICP0 and ICP4 was significantly upregulated at 3 h postinfection. Also, these genes were found to be less protected from MNase digestion and, therefore, less associated with nucleosomes. These results suggest that Asf1a plays a role in regulating IE genes by assembling chromatin onto histone-free viral DNA by 3 h postinfection.

TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects double-stranded RNA and induces type I IFN expression. Humans with defects in the TLR3/IFN pathway have an elevated susceptibility to HSV infections of the CNS. However, it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN responses in astrocytes. Tlr3-/- mice were hypersusceptible to HSV-2 infection in the CNS after vaginal inoculation. HSV-2 exhibited broader neurotropism in Tlr3-/- mice than it did in WT mice, with astrocytes being most abundantly infected. Tlr3-/- mice did not exhibit a global defect in innate immune responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.

Herpes simplex virus infections among rural residents in eastern China

BackgroundHerpes simplex virus (HSV) has two types: HSV-1 and HSV-2. Both infect epithelial cells and establish latent infections in neurons causing an infection that persists for life. Information on age- and gender-specific seroprevalence of HSV-1 and HSV-2 is valuable for understanding HSV transmission dynamics and designing population-based prevention and intervention programs for HSV. However, such information is not available for China.MethodsCryopreserved serum samples of all subjects aged 5 to 60 years from two randomly selected rural villages in Zhejiang province in Eastern China who had participated in the China national seroepidemiological survey of hepatitis B virus (HBV) infection conducted in 2006 were tested. Seroprevalence of HSV-1 and HSV-2 infections were determined by type-specific IgG antibody tests using an ELISA technique. Their 95% confidence intervals adjusted for the sampling fraction were calculated according to the Clopper-Pearson method.ResultsA total of 2,141 residents participated in the survey, with a response rate of 82.3%. HSV-1 seroprevalence was 92.0% overall, 89.1% for males and 94.2% for females. HSV-1 seroprevalence was 61.6% among children aged 5-9 years, 90.3% among 25-29 years, and nearly 100% among those aged > = 40 years. HSV-2 seroprevalence was 13.2% overall, 10.5% for males and 15.3% for females. No children aged 5-14 years were HSV-2 positive, and HSV-2 seroprevalence was 7.1% among 15-19 years and peaked at 24.3% among those aged 45-49 years. Neither HSV-1 nor HSV-2 infections were significantly different by gender. About 11.8% of study subjects were co-infected with both types of HSV. Among 549 participating couples, 8.6% were HSV-1 serodiscordant and 11.8% were HSV-2 serodiscordant. No one tested positive for HIV. The overall prevalence of HBsAg was 16.2%, 16.9% for males and 15.4% for females.ConclusionsHSV-1 was highly prevalent among all rural residents aged between 5-60 years in Eastern China, whereas HSV-2 was prevalent among sexually active people. HSV-1 and HSV-2 have different transmission modes and dynamics. Future HSV prevention and control programs in China should be type specific.

QSAR Studies on HSV-1 Inhibitors by CoMFA, CoMSIA and HQSAR Approaches

HSV infection has high incidence rate and high prevalence. HSV-1 may cause cold sores, fever blisters, and encephalitis. Moreover, HSV-1 has the ability to cause the latent infection of neurons. Therefore, the QSAR studies of HSV-1 inhibitors and drug design based on QSAR have great importance. This paper focused on the pyrazolo[1,5- a]pyridines, which are not acyclic nucleoside analog. The reported pyrazolopyridines showed potent and selective inhibition activity. In order to find novel compounds with higher activity and to develop better predictive models, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and hologram quantitative structure activity relationship (HQSAR) methods were performed on a series of HSV-1 inhibitors. The models built by CoMFA, CoMSIA, and HQSAR were found to be suitable for the compounds investigated with good predictive power (CoMFA q2=0.669, R2=0.953; CoMSIA q2=0.693, R2=0.919; HQSAR q2=0.619, R2=0.921). 3D contour maps obtained from PLS models of CoMFA and CoMSIA, and the atom contribution map from PLS model of HQSAR indicated that small steric volumes and electron-withdrawing groups in the R2, R3 regions would be useful to improve the activity. Results showed that the established models of the present work would be helpful in studying the relationship between the bioactivity and the molecular structures and discovering more potent HSV-1inhibitors.

Ultrastructural Analysis of Virion Formation and Anterograde Intraaxonal Transport of the Alphaherpesvirus Pseudorabies Virus in Primary Neurons

A hallmark of alphaherpesviruses is their capacity to be neuroinvasive and establish latent infections in neurons. After primary replication in epithelial cells at the periphery, entry into nerve endings occurs, followed by retrograde transport of nucleocapsids to the nucleus where viral transcription, genome replication, and nucleocapsid formation take place. Translocation of nucleocapsids to the cytoplasm is followed by axonal transport to infect synaptically linked neurons. Two modes of intraaxonal anterograde herpesvirus transport have been proposed: transport of complete, enveloped virions within vesicles ("married model"), and separate transport of capsids and envelopes ("subassembly model"). To assess this in detail for the alphaherpesvirus pseudorabies virus (PrV), we used high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia after infection with wild-type and gB-deficient PrV. Our data show that intranuclear capsid maturation, nuclear egress and cytoplasmic secondary envelopment occur as in cultured nonpolarized cells (H. Granzow, F. Weiland, A. Jöns, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:2072-2082, 1997). PrV virions were present in axons as enveloped particles within vesicles associated with microtubules and apparently leave the neuron by exocytosis primarily at the growth cone. Only a few nonenveloped nucleocapsids were found in the axon. The same picture was observed after infection by phenotypically complemented gB-deficient PrV, which is able to complete only a single round of replication. Our data thus support intraaxonal anterograde transport of enveloped PrV virions within vesicles following the "married model."

Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript

MicroRNAs (miRNAs) are a class of small RNA molecules that regulate the stability or the translational efficiency of target messenger RNAs (mRNAs). The latency-associated transcript (LAT) of herpes simplex virus-1 (HSV-1) is the only viral gene expressed during latent infection in neurons. LAT inhibits apoptosis and maintains latency by promoting the survival of infected neurons. No protein product has been attributed to the LAT gene and the mechanism by which LAT protects cells from apoptosis is not yet known. Here we show that a miRNA encoded by the HSV-1 LAT gene confers resistance to apoptosis. Neuroblastoma cells transfected with a fragment of the LAT gene show reduced susceptibility to cell death. The anti-apoptotic function of LAT has been mapped to a region within the first exon. We have identified and characterized a microRNA (miR-LAT) generated from the exon 1 region of the HSV-1 LAT gene. The LAT miRNA was found to accumulate in cells transiently transfected with the LAT gene fragment or infected with a wild-type strain of HSV-1. A mutant virus in which a 372-nucleotide fragment encompassing the mature miRNA was deleted neither protected the infected cells from apoptosis nor generated an miRNA. miR-LAT exerts its anti-apoptotic effect by downregulation of transforming growth factor (TGF)-beta 1 and SMAD3 expression, both of which are functionally linked in the TGF-beta pathway. Our results suggest that the miRNA encoded by the HSV-1 LAT gene regulates the induction of apoptosis in infected cells by modulation of TGF-beta signalling and thus contributes to the persistence of HSV in a latent form in sensory neurons.

HSV-1-Derived Recombinant and Amplicon Vectors for Gene Transfer and Gene Therapy

Herpes simplex virus type 1 (HSV-1) is a major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections. Although in most cell types infection with HSV-1 will induce toxic effects ending in the death of the infected cells, the very deep knowledge we possess on the genetics and molecular biology of HSV-1 has permitted the deletion of most toxic genes and the development of non-pathogenic HSV-1-based vectors for gene transfer. Several unique features of HSV-1 make vectors derived from this virus very appealing for preventive or therapeutic gene transfer. These include (i) the very high transgenic capacity of the virus particle, authorizing to convey very large pieces of foreign DNA to the nucleus of mammalian cells, (ii) the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and (iii) the ability of HSV-1 vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia and probably in other neurons as well, from where transgenes can be strongly and long-term expressed. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors, and defective helper-dependent vectors known as amplicons. Each of these different vectors attempts to exploit one or more of the above-mentioned features of HSV-1. In this review we will update the current know-how concerning design, construction, and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

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.

A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels.

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500-1503, 2000; M. Inman et al., J. Virol. 75:3636-3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (P < 0.0004 and P = 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.

The transgenic ICP4 promoter is activated in Schwann cells in trigeminal ganglia of mice latently infected with herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the beta-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for beta-galactosidase-positive cells. The numbers of beta-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.

Human neuron-committed teratocarcinoma NT2 cell line has abnormal ND10 structures and is poorly infected by herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.

Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1.