HSV-1 is one of the most common viral infections affecting humans from mild cold sores to severe ocular keratitis and deadly encephalitis. The virus establishes a life-long latent infection within neurons of the trigeminal ganglia (TG). Literature showed that herpesvirus reactivation is correlated to hormone imbalance. Therefore we predict that hormone alteration may regulate the HSV-1 gene expression during reactivation. In searching for hormone responsive elements in HSV-1 genome, we identified thyroid hormone receptor responsive elements (TREs) in the regulatory regions of HSV-1 thymidine kinase (TK), latency-associated transcript (LAT), and infected cell protein 0 (ICP0). TREs are the binding sites where the thyroid hormone receptor (TR) binds/acts as a transcription factor. Our central hypothesis is that thyroid hormone (TH or T3) and its receptor (TR) can control HSV-1 latency and reactivation by regulating the HSV-1 key gene expression and thus influence the transcription and replication of virus within the latently-infected neurons. This hypothesis is supported by the literature and our observations: First, HSV-1 TK and ICP0 are important for efficient viral DNA replication and transcription within the infected neurons. Second, TR is present in sensory ganglia. Third, our results have shown that TR regulates expression of TK, LAT, and ICP0 in vitro. Fourth, our publication indicated that the level of T3 controls viral replication and the release of infectious viruses in vitro. Fifth, standard procedures used to induce HSV-1 reactivation, such as hyperthermia and dexamethasone treatment, also decrease TH levels. Together our published results demonstrate that the liganded receptor (T3/TR, the hormone-receptor complex) controls the expression of HSV-1 key genes and replication. N2aTR cell has been used for pilot study because of the advantages that 1) it is convenient to control the level of T3 by adding it into media, and 2) N2aTR cells can be differentiated by T3 after 3-days of treatment, mimicking the condition of differentiated neurons. In this project, we will further study the mechanisms by which T3/TR regulates HSV-1 replication/gene expression via the following Specific Aims. Specific Aim 1. To test the hypothesis that T3/TR control the transcription of thymidine kinase (TK) by chromatin regulation using silencing mediator of retinoid and thyroid receptors (SMRT) recruited by Nuclear Orphan Receptors (NORs) to the TK T3 Response Element (TRE) via TR. Our results indicated that liganded TR was recruited to the TK TRE, and NORs were up-regulated in T3-treated N2aTR cells. NOR is shown to interact with SMRT and repress promoter activity. In Aim 1, we will make a series of TRE mutants and test them using our T3 removal N2aTR cell culture model to investigate: 1) TR binding, 2) NOR/SMRT recruitment, and 3) their functions on viral replication/release by transient transfection and viral infection in neuronal and non-neuronal cells. Specific Aim 2. To test the hypothesis that T3 /TR modulate transcription of LAT and genes by recruiting multifunctional transcription factor Early Growth Response gene (Egr-1) and chromatin insulator CTCF to the key regulatory regions of HSV-1. In different studies we showed that Egr-1 and CTCF were induced by T3 /TR in neuronal cells upon infection. They both exhibit repressive effects on HSV-1 genes. In Aim 2, we will generate a series of mutant plasmids with partial deletion of HSV-1 Repeat Element-1 (RE-1) to study the boundary effect of CTCF on LAT and genes. Egr-1-mediated regulation will be further investigated by transfection assays as well as infections using recombinant virus over-expressing Egr-1 in neuronal and non-neuronal cells. Specific Aim 3: To test the hypothesis that Regulator of Chromosome Condensation 1 (RCC1) and RAs- related Nuclear protein (Ran) complex participated in the global epigenetic control of HSV-1 transcription/ replication by maintaining repressive chromatin structure on HSV-1 genome for gene silencing via T3/TR. RCC1/Ran complex is involved in the control of RNA translocation, DNA synthesis, and cell cycle progression via modulating effects on chromatin and nucleosomes. RCC1 is partially degraded in lytic infection but induced in neuronal cells by T3/TR upon infection (C.12, Fig. 7). In Aim 3, we will study the effects of RCC1/Ran complex on HSV-1 gene expression, replication, and virus release. Our laboratory has been working on HSV-1 biology through the support from NCRR/NIH, and we have three publications supporting the hypotheses. Additional studies are proposed in the Specific Aims based on our unpublished results. Our short-term mission is to establish an active research program at the School of Pharmacy at the University of Maryland Eastern Shore (UMES), a land-grant, historically black college and university (HBCU) so the students can study the current progress of virology, molecular biology, cell biology, neuroscience, and endocrinology. The long-term goal is to identify the regulatory mechanisms to assist in the development of novel therapeutic protocols for better treatments.