This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. 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 configuration and most viral genes are silenced. The molecular mechanisms responsible for this are unclear. We hypothesized that novel transcription factors control chromatin modifications and regulate HSV-1 gene expression during lytic infection and latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1 (RE-1) located between HSV-1 genes ICP22 and ICP4. Results from Roizman's lab suggested that ICP0 exhibited sequence similarity to CoREST and disrupted REST/CoREST/HDAC complex to regulate HSV-1 gene expression. However, they did not explain how the complex was recruited to HSV-1 genome. We hypothesize that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF) regulates expression of ICP22 and ICP4. 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 significantly 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. 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. Multiple factors/pathways have been suggested to control HSV-1 latency and reactivation. We identified thyroid hormone response elements (TREs) in the promoters of HSV-1 genes TK and LAT. TREs are the binding sites of nuclear hormone receptors such as thyroid hormone receptors (TRs). TRs are transcription factors whose activity is dependent on the ligand thyroid hormone (T3). We investigate the roles of TR and T3 on HSV-1 gene expression using a neuoroblastoma cell line constitutively expressing TR isoform (N2aTR ). In the present study, we demonstrated that TRs repressed TK promoter activity in the presence of T3 by transient transfection and viral infection. However, the liganded TRs activated LAT transcription. Using plasmids with deletion in LAT TREs, the data showed that ICP0 transcription was repressed by TR and T3 only when the LAT TREs were present. Chromatin immunoprecipitation (ChIP) illustrated that TRs were recruited to TK and LAT TREs independently of T3 and hyperacetylated H4 was associated with promoters that were transcriptionally active. In addition, our ChIP results showed that a repressive mono methylated H3 modified at lysine 9 and chromatin insulator protein CTCF was enriched in the TK and LAT TREs, respectively, in the presence of TR and T3. Besides histone modification, transcription of LAT was further regulated by chromatin remodeling factor BRG1 complex since overexpression of BRG1 enhanced the LAT transcription and dominant negative mutant of BRG1 K785R abolished the activation. T3-treated N2aTR cells were suppressive to TK expression and virus egress at low moi. However, our RT-PCR and plaque assays showed that the TK expression was enhanced and the release of infectious viruses was increased when the T3 was removed. These results suggested that T3 may regulate the HSV-1 gene expression through its receptor via histone modification and chromatin remodeling and therefore control viral latency and reactivation.
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