Regulation of T-type Ca2+ channel expression by interleukin-6 in sensory-like ND7/23 cells post-herpes simplex virus (HSV-1) infection.

Herpes simplex virus-type 1 (HSV-1) infection of sensory neurons may lead to a significant reduction in the expression of voltage-activated Na+ and Ca2+ channels, which can disrupt the transmission of pain information. Viral infection also results in the secretion of various pro-inflammatory cytokines, including interleukin (IL)-6. In this work, we tested whether IL-6 regulates the expression of Na+ and Ca2+ channels post-HSV-1 infection in ND7/23 sensory-like neurons. Our results demonstrate that HSV-1 infection causes a significant decrease in the protein expression of the Cav3.2 T-type Ca2+ channel subunit, despite increasing Cav3.2 mRNA synthesis. Neither Cav3.2 mRNA nor total protein content was affected by IL-6 treatment post-HSV-1 infection. In ND7/23 cells, HSV-1 infection caused a significant reduction in the expression of Na+ and T-type Ca2+ channels within 48 h. Exposure of ND7/23 cells to IL-6 for 24 h post-infection reverses the effect of HSV-1, resulting in a significant increase in T-type Ca2+ current density. However, Na+ currents were not restored by 24-h treatment with IL-6 post-HSV-1 infection of ND7/23 cells. The ability of IL-6 to increase the functional expression of T-type Ca2+ channels on the membrane was blocked by the inhibition of protein trafficking with brefeldin-A and ERK1/2 activation. These results indicate that IL-6 release following HSV-1 infection regulates the expression of T-type Ca2+ channels, which may alter the transmission of pain information.

Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells.

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca2+ current densities. Increased T-type Ca2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca2+ channels is mediated by several factors, including decreased expression of Cav3.2 T-type Ca2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca2+ channels, which can results in morphological and functional changes in electrical excitability.

Receipt of thyroid hormone deficiency treatment and risk of herpes zoster.

OBJECTIVE: Thyroid hormone (TH) has been suggested to control herpes virus gene expression and replication in neurons via epigenetics through its nuclear receptors. It has previously been shown that patients with hypothyroidism are predisposed to herpes zoster (HZ), suggesting that the TH deficiency may be a risk factor for varicella zoster virus (VZV) reactivation. The aim of this study was to test the hypothesis that TH treatment will ameliorate the complication of HZ. METHODS: This study investigated the hypothesis by enquiring into a comprehensive medical database at Kaiser Permanente Southern California (KPSC) to verify whether patients taking TH medication experience a reduction in HZ occurrence. RESULTS: It was shown by Kaplan-Meier analysis that hypothyroidism patients taking TH medicines had a lower risk of HZ. The fully adjusted analysis indicated that patients receiving medication for the treatment of TH deficiency exhibited a reduced risk of HZ (hazard ratio 0.60, 95% confidence interval 0.51-0.71). This lower risk of HZ was significant in all age groups except the 18-39 years cohort. In addition, female patients taking TH treatment exhibited a lower risk than their male counterparts. CONCLUSIONS: Together these findings support the hypothesis that a constant level of TH will provide a degree of protection from contracting HZ. More studies are underway to evaluate the laboratory data for an analysis of hormonal effects on individuals.

Discovery and preliminary structure-activity relationship of the marine natural product manzamines as herpes simplex virus type-1 inhibitors.

Herpes simplex virus type-1 (HSV-1) is a member of alpha-herpesviridae family and is known to cause contagious human infections. The marine habitat is a rich source of structurally unique bioactive secondary metabolites. A small library of marine natural product classes 1-10 has been screened to discover a new hit entity active against HSV-1. Manzamine A showed potent activity against HSV-1 via targeting the viral gene ICP0. Manzamine A is a ß-carboline alkaloid isolated from the Indo-Pacific sponge Acanthostrongylophora species. Currently, acyclovir is the drug of choice for HSV-1 infections. Compared with 50 µM acyclovir, manzamine A at 1 µM concentration produced potent repressive effects on viral replication and release of infectious viruses in SIRC cells in recent studies. The potent anti-HSV-1 activity of manzamine A prompted a preliminary structure-activity relationship study by testing targeted manzamines. These included 8-hydroxymanzamine A (11), to test the effect of the C-8 hydroxy substitution at the ß-carboline moiety; manzamine E (12), to assess the importance of substitution at the azacyclooctane ring; and ircinal A (13), to determine whether the ß-carboline ring is required for the activity. Manzamine A was chemically transformed to its salt forms, manzamine A monohydrochloride (14) and manzamine A monotartrate (15), to test whether improving water solubility and hydrophilicity will positively affect the activity. Compounds were tested for activity against HSV-1 using fluorescent microscopy and plaque assay. The results showed the reduced anti-HSV-1 activity of 11, suggesting that C-8 hydroxy substitution might adversely affect the activity. Similarly, manzamines 12 and 13 showed no activity against HSV-1, indicating the preference of the unsubstituted azacylcooctane and ß-carboline rings to the activity. Anti-HSV-1 activity was significantly improved for the manzamine A salts 14 and 15, suggesting that improving the overall water solubility as salt forms can significantly enhance the activity. Manzamines have significant potential for future development as anti-HSV-1 entity.

Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1.

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50 µM, equipotent to acyclovir.

Effects of thyroid hormone on HSV-1 gene regulation: implications in the control of viral latency and reactivation.

Thyroid hormone (TH) is involved in many biological functions such as animal development, cell differentiation, etc. Variation and/or disruption of plasma TH level often led to abnormalities and physiological disorders. TH exerts the effects through its nuclear receptors (TR). Literature showed that procedures resulted in TH alteration also linked to reactivation of several viruses including Herpes Simplex Virus Type -1 (HSV-1). Bioinformatic analyses revealed a number of putative TH responsive elements (TRE) located in the critical regulatory regions of HSV-1 genes such as thymidine kinase (TK), latency associated transcript (LAT), etc. Studies using neuronal cell lines have provided evidences demonstrating that liganded TR regulated viral gene expression via chromatin modification and controlled viral replication. The removal of TH reversed the inhibition and induced the viral replication previously blocked by TH. These results suggest that TH may have implication to participate in the control of reactivation during HSV-1 latency.

Lytic HSV-1 infection induces the multifunctional transcription factor Early Growth Response-1 (EGR-1) in rabbit corneal cells.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB. METHODS: SIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP). Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy. RESULTS: Western blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB) were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection. CONCLUSION: Collectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene expression, replication, inflammation, and the disease progression.

Manzamine A as a novel inhibitor of herpes simplex virus type-1 replication in cultured corneal cells.

This study investigated the putative inhibitory effect of manzamine A on HSV-1 infection. Our results indicated that manzamine A effectively inhibited viral replication and infection in the cell line SIRC, a corneal cell line, at 1 µM. The existing anti-HSV-1 drug acyclovir was analyzed and showed a comparable activity at 50 µM. Plaque assays demonstrated that manzamine A reduced the release of infectious virus by 10 (11)-fold. RTPCR assays indicated that HSV-1 virion host shutoff (vhs) activity and ICP0 transcription were decreased by manzamine A treatment. These results bode well for the development of manzamines as potential leads to reduce viral infection in corneal cells and to prevent HSV-1-induced eye infections such as keratitis.

Thyroid hormone controls the gene expression of HSV-1 LAT and ICP0 in neuronal cells.

Various factors/pathways including hormonal regulation have been suggested to control herpes simplex virus type 1 (HSV-1) latency and reactivation. Our computer analysis identified a DNA repeat containing thyroid hormone-responsive elements (TRE) in the regulatory region of HSV-1 latency-associated transcript (LAT). Thyroid hormone (triiodothyronine, T(3)) functions via its receptor TR (thyroid hormone receptor), a transcription factor. Present study investigated the roles of TR and T(3) in HSV-1 gene expression using cultured neuoroblastoma cell lines. We demonstrated that liganded TR activated LAT transcription, but repressed infected cell protein no. 0 (ICP0) transcription in the presence of LAT TRE. Chromatin immunoprecipitation (ChIP) assays showed that TRs were recruited to LAT TREs independently of T(3) and hyperacetylated H4 was associated with the LAT promoter that was transcriptionally active. In addition, ChIP results showed that the chromatin insulator protein CCCTC-binding factor was enriched at the LAT TREs in the presence of TR and T(3). In addition, the BRG1 chromatin remodeling complex is found to participate in the T(3)/TR-mediated LAT activation since overexpression of BRG1 enhanced the LAT transcription and the dominant-negative mutant K785R abolished the activation. This is the first report revealing that TR elicits epigenetic regulation on HSV-1 ICP0 expression in neuronal cells and could have a role in the complex processes of HSV-1 latency/reactivation.

Regulation of herpes simplex virus type 1 thymidine kinase gene expression by thyroid hormone receptor in cultured neuronal cells.

Herpes simplex virus type 1 (HSV-1) undergoes acute infection in epithelial cells followed by establishment of latency in the neurons of trigeminal ganglia. The latent virus maintains a dormant state and can recurs spontaneously, suggesting transcriptional silencing and reactivation occur in neurons. Computer data mining identified a nuclear hormone response element (NRE), the binding site for the thyroid hormone receptor (TR) or other nuclear hormone receptor, in the promoter of HSV-1 thymidine kinase (TK). TRs are transcription factors whose activity is dependent on their ligand thyroid hormone (T(3); triiodothyronine). We hypothesize that TR and T(3) exert regulation on HSV-1 gene expression in neurons. A neuroblastoma cell line expressing the TR isoform beta (N2aTRbeta) was utilized for in vitro investigation. Results showed that liganded TR repressed TK promoter activity but unliganded TR relieved the inhibition. The mutagenesis study demonstrated that one nucleotide mutation at the NRE abolished the T(3)/TR-mediated regulation. N2aTRbeta cells treated with T(3) were suppressive to TK expression and virus release but the removal of T(3) de-repressed TK expression and increased virus release, confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and plaque assays, respectively. Chromatin immunoprecipitation (ChIP) assays showed that TRs were enriched at TK NRE in the presence of T(3). Additional results demonstrated that hyper acetylated histone H4 and monomethylated H3 modified at lysine 9 (H3K9me1) were enriched at transcriptionally active TK promoters but were dissociated from the NRE by T(3)/TR. These results suggest that T(3) could regulate HSV-1 gene expression through its receptor via histone modification in cultured neuronal cells.

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification.

BACKGROUND: During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1) establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE) located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF) regulates expression of ICP22 and ICP4. RESULTS: Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Additionally, chromatin immuno-precipitation (ChIP) assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF. CONCLUSION: Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression.

Early growth response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression.

The molecular mechanisms mediating herpes simplex virus type 1 (HSV-1) gene silencing during latent infection are not clear. Five copies of early growth response gene 1 (Egr-1) binding elements were identified in the intron of HSV-1 ICP22 (infected cell protein No. 22) gene, leading to the hypothesis that Egr-1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 (infected cell protein No. 4) promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed. Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-1-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Sp1 to the promoters and that the co-repressor Nab2 (NGFI-A/EGR1-binding protein) was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multifunctional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment of co-repressor Nab2 and reduction of Sp1 occupancy, and thus may play a critical role in HSV-1 gene silencing during latency.

A dominant-negative thyroid hormone receptor blocks amphibian metamorphosis by retaining corepressors at target genes.

The total dependence of amphibian metamorphosis on thyroid hormone (T(3)) provides a unique vertebrate model for studying the molecular mechanism of T(3) receptor (TR) function in vivo. In vitro transcription and developmental expression studies have led to a dual function model for TR in amphibian development, i.e., TRs act as transcriptional repressors in premetamorphic tadpoles and as activators during metamorphosis. We examined molecular mechanisms of TR action in T3-induced metamorphosis by using dominant-negative receptors (dnTR) ubiquitously expressed in transgenic Xenopus laevis. We showed that T(3)-induced activation of T(3) target genes and morphological changes are blocked in dnTR transgenic animals. By using chromatin immunoprecipitation, we show that dnTR bound to target promoters, which led to retention of corepressors and continued histone deacetylation in the presence of T(3). These results thus provide direct in vivo evidence for the first time for a molecular mechanism of altering gene expression by a dnTR. The correlation between dnTR-mediated gene repression and inhibition of metamorphosis also supports a key aspect of the dual function model for TR in development: during T(3)-induced metamorphosis, TR functions as an activator via release of corepressors and promotion of histone acetylation and gene activation.

Chromatin disruption and histone acetylation in regulation of the human immunodeficiency virus type 1 long terminal repeat by thyroid hormone receptor.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) controls the expression of HIV-1 viral genes and thus viral propagation and pathology. Numerous host factors participate in the regulation of the LTR promoter, including thyroid hormone (T(3)) receptor (TR). In vitro, TR can bind to the promoter region containing the NF-kappa B and Sp1 binding sites. Using the frog oocyte as a model system for chromatin assembly mimicking that in somatic cells, we demonstrated that TR alone and TR/RXR (9-cis retinoic acid receptor) can bind to the LTR in vivo independently of T(3). Consistent with their ability to bind the LTR, both TR and TR/RXR can regulate LTR activity in vivo. In addition, our analysis of the plasmid minichromosome shows that T(3)-bound TR disrupts the normal nucleosomal array structure. Chromatin immunoprecipitation assays with anti-acetylated-histone antibodies revealed that unliganded TR and TR/RXR reduce the local histone acetylation levels at the HIV-1 LTR while T(3) treatment reverses this reduction. We further demonstrated that unliganded TR recruits corepressors and at least one histone deacetylase. These results suggest that chromatin remodeling, including histone acetylation and chromatin disruption, is important for T(3) regulation of the HIV-1 LTR in vivo.