College of Pharmacy at ULM, Dean's Message

Infection with HSV-1 typically causes diseases of the mouth, face, skin, esophagus, or brain. The virus initially infects mucosal epithelium and undergoes latency in ganglion neurons. The mechanism of latency is not well understood due to the lack of cell culture model. Our long-term goal is to elucidate the regulatory mechanisms controlling viral gene expression during latency as a necessary prerequisite to the development of therapeutic protocols. We hypothesize that neuronal factors and the nucleosomal structure formed on the latent viral genome repressed the viral DNA replication and the immediate-early (IE) gene transcription in sensory neurons. This hypothesis is based on the observations that: 1) the latent viral genome remained in a circular form and nucleosomes are associated with the DNA. 2) The latent virus is incapable of DNA replication in neuron. 3) Most viral transcriptions including the essential genes are silenced during latency. and 4) the major drawback of using HSV-1 as gene transfer vehicle for treating neurological conditions is the occurrence of transgene silencing. The experimental focus of this proposal is to understand the effect of chromatin on viral promoters in the chromatin context. The specific aims are to: 1. Define the relationship between IE gene transcription and the nucleosomal structure during lytic infection. In a prerequisite experiment, we were surprised to learn that the nucleosomal structure was formed on the viral genome efficiently during lytic infection. We will confirm the results using viral inhibitors to analyze the changes of histone modification and putative nucleosomal phasing and remodeling. 2. Characterize the regulation of IE genes by chromatin and a neuronal repressor in a novel episomal neuronal cell culture model. Given the fact that latent viral genome acquires the characteristics of circular form in differentiated neurons, it is of importance to study the latent viral gene regulation in neuronal cell culture system where cells can differentiate and plasmids harboring viral promoters remain as episome. We will construct such a system and study the effect of differentiation and role of neuronal repressor on gene regulation and chromatin modification. 3. Investigate the role of nuclear receptor in viral replication, transcription, and latency. Several observations suggested that viral DNA replication may have a critical role in transcription in peripheral neurons. We speculated that HSV-1 tk gene, essential for DNA synthesis in neurons, may be regulated by thyroid hormone receptor (TR), thus contributing to latency. We will test the regulatory effect of TR in our episomal systems and perform the viral infection in neuronal cell lines to evaluate the consequence of replication and transcription by TR.

Function of Thyroid Hormone Receptor in Gene Regulation and Development

2.1: Role of proteasome mediated degradation in the thyroid hormone induced transcription: Recently using yeast two hybrid screening against TR, I found an interacting fragment exhibiting strong homology to sug-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Encouraged by several literatures showing that proteasome mediated degradation of nuclear hormone receptor during the transactivation event, I hypothesize that sug-1 could be recruited to liganded TR and selectively specify the degradation through the ubiquitin-proteasome pathway and thus completed the transactivation. I will construct the appropriate cell lines and the plasmids and hope to obtain all the inhibitors and the required antibodies for the study. I would like to first confirm the requirement of degradation in the process of transcription activation. If this holds true, I will then analyze the domain of TR that interacts with sug-1. I can also study the ubiquitylation status during the process of degradation and transactivation. Finally, I will define the phosphorylation of liganded TR and the correlation to transactivation since phosphorylation has been shown to play a role in proteasome mediated degradation.

2.2: Functional study of proteasome mediated degradation during organ development: To study the profile of TR regulation during intestinal development, colleague in the laboratory Tosi Amano reported that several ubiquitin activating enzymes and proteasome regulatory subuits were up-regulated at the later stages of metamorphosis. It is feasible to postulate that this specified degradation contribute to many developmental events such as apotosis, cell proliferation, transription regulation, etc. To investigate its role in thyroid hormone induced development, I will utilize our tadpole transgenic system by making some dominant negative mutants of sug-1 and TR to block the proposed pathway and observe the phenotype.