Construction and Characterization of Recombinant HSV-1 Expressing Early Growth Response-1.

Early Growth response-1 (Egr-1) is a transcription factor that possesses a variety of biological functions. It has been shown to regulate HSV-1 gene expression and replication in different cellular environments through the recruitment of distinct cofactor complexes. Previous studies demonstrated that Egr-1 can be induced by HSV-1 infection in corneal cells but the level was lower compared to other cell types. The primary goal of this report is to generate a recombinant HSV-1 constitutively expressing Egr-1 and to investigate the regulation of viral replication in different cell types or in animals with Egr-1 overexpression. The approach utilized was to introduce Egr-1 into the BAC system containing complete HSV-1 (F) genome. To assist in the insertion of Egr-1, a gene cassette was constructed that contains the Egr-1 gene flanked byloxP sites. In this clone Egr-1 is expressed under control of CMV immediate-early promoter followed by another gene cassette expressing the enhanced green fluorescent protein (EGFP) under the control of the elongation factor 1α (EF-1 α) promoter. The constructed recombinant viruses were completed containing the Egr-1 gene within the viral genome and the expression was characterized by qRT-PCR and Western blot analyses. Our results showed that Egr-1 transcript and protein can be generated and accumulated upon infection of recombinant virus in Vero and rabbit corneal cells SIRC. This unique virus therefore is useful for studying the effects of Egr-1 during HSV-1 replication and gene regulation in epithelial cells and neurons.

A Novel Thyroid Hormone Mediated Regulation of HSV-1 Gene Expression and Replication is Specific to Neuronal Cells and Associated with Disruption of Chromatin Condensation.

Previously we showed that thyroid hormone (T3) regulated the Herpes Simplex Virus Type -1 (HSV-1) gene expression and replication through its nuclear receptor TR via histone modification and chromatin remodeling in a neuroblastoma cell line neuro-2a cells (N2a). This observation suggested that T3 regulation may be neuron-specific and have implication in HSV-1 latency and reactivation. In this study, our in vitro latency/reactivation model demonstrated that removal of T3 can de-repress the HSV-1 replication and favor reactivation. Transfection studies and infection assays indicated that HSV-1 thymidine kinase (TK), a key viral gene during reactivation, was repressed by TR/T3 in cells with neuronal origin but not in non-neuronal cells. Additional studies showed that RCC1 (Regulator of Chromosome Condensation 1) was sequestered but efficiently detected upon viral infection in N2a cells. Western blot analyses indicated that addition of T3 repressed the RCC1 expression upon infection. It is likely that diminution of RCC1 upon infection in neuronal cells under the influence of TR/T3 may lead to repression of viral replication/gene expression thus promote latency. Together these results demonstrated that TR/T3 mediated regulation is specific to neuronal cells and differential chromosome condensation may play a critical role in this process.

Thyroid hormone-dependent epigenetic suppression of herpes simplex virus-1 gene expression and viral replication in differentiated neuroendocrine cells.

A global HSV-1 gene repression occurs during latency in sensory neurons where most viral gene transcriptions are suppressed. The molecular mechanisms of gene silencing and how stress factors trigger the reactivation are not well understood. Thyroid hormones are known to be altered due to stress, and with its nuclear receptor impart transcriptional repression or activation depending upon the hormone level. Therefore we hypothesized that triiodothyronine (T3) treatment of infected differentiated neuron like cells would reduce the ability of HSV-1 to produce viral progeny compared to untreated infected cells. Previously we identified putative thyroid hormone receptor elements (TREs) within the promoter regions of HSV-1 thymidine kinase (TK) and other key genes. Searching for a human cell line that can model neuronal HSV-1 infection, we performed HSV-1 infection experiments on differentiated human neuroendocrine cells, LNCaP. Upon androgen deprivation these cells undergo complete differentiation and exhibit neuronal-like morphology and physiology. These cells were readily infected by our HSV-1 recombinant virus, expressing GFP and maintaining many processes iconic of dendritic morphology. Our results demonstrated that differentiated LNCaP cells produced suppressive effects on HSV-1 gene expression and replication compared to its undifferentiated counterpart and T3 treatment has further decreased the viral plaque counts compared to untreated cells. Upon washout of the T3 viral plaque counts were restored, indicating an increase of viral replication. The qRT-PCR experiments using primers for TK showed reduced expression under T3 treatment. ChIP assays using a panel of antibodies for H3 lysine 9 epigenetic marks showed increased repressive marks on the promoter regions of TK. In conclusion we have demonstrated a T3 mediated quiescent infection in differentiated LNCaP cells that has potential to mimic latent infection. In this HSV-1 infection model thyroid hormone treatment caused decreased viral replication, repressed TK expression and increased repressive histone tail marks on the TK promoter.

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.