Herpes Simplex virus (HSV) can cause a wide range of diseases including skin lesions which are common, encephalitis which is rare, an HSV infection of the eye which is a leading cause of blindness in the USA (400,000 cases). The seroprevalence of HSV in the US adult population is very high (~70%). Transmission of virus from a mother bearing a vaginal infection to her baby during birth can have life threatening consequences. Herpes virus infections are characterized by the ability of the virus to form latent infections in the nervous system. It is this ability, which leads to recurrent episodes of the disease causing much human suffering, which is the focus of our application. The overall goal of this proposal is to understand the mechanism of HSV latency using both a mouse model system and tissue culture studies. We have previously used mouse model systems of HSV latency to study physical state of the latent viral genome, and to initiate studies on viral gene expression during latency. From our data, we have formulated models for the mechanism of HSV-1 latency. We now wish to continue to refine these models using the techniques of molecular virology in order to identify drug targets to intervene in the latency process. The program consists of four scientific projects arid two supporting cores. The scientific projects are titled: 1. Gene Expression during HSV-1 Latency and Reactivation;2. The role of Chromatin boundaries in HSV gene expression;3. The Role of Cellular Transcription Factors in the Regulation of HSV-1 Latency and Reactivation;4. Herpes Simplex Virus and Neuronal Cell Interactions. Successful completion of these studies will permit the mechanisms of HSV latency to be described in more detail allowing formulation of new strategies for the prevention of latency and recurrence. In addition, it is anticipated that the knowledge gained will continue to be of use to the fields of gene therapy and cancer therapy in the nervous system, and continue to provide patentable findings and potential drug targets. Project 1 Title: Gene Expression During HSV-1 Latency and Reactivation Principal Investigator: Nigel Fraser, Ph.D. DESCRIPTION (provided by applicant): Human neurotropic herpes viruses such as HSV cause much disease and suffering. The long-term objective of this project is to understand the mechanism of herpes simplex virus latency and reactivation at the molecular level. In this application, we propose to expand our study of the nucleosomal state of the viral DNA, as we believe that these structures play an important role in regulating viral gene expression. We will continue to examine the function of stable intron of the latency associated transcript (LAT) genes. Furthermore we will expand on our finding of differences in cell gene expression in latently infected and normal trigeminal ganglia. These goals will be achieved using the techniques of molecular virology and a mouse model of HSV infection. In the first specific aim, we will determine the distribution and role of the nucleosome in acute infection of mice. Further studies will relate these results to the situation during viral reactivation. In the second aim, we will continue our study of the functionality of the LAT gene. We will study the 2kb LAT intron, which is found in the nucleus of latently infected neurons yet in the cytoplasm of lytically infected cells. Our recent studies suggest that the intron plays a role in cellular stress response. We will determine jLAT intron function through studying its structure and the proteins associated with it. Our studies on the mechanism of the LAT antiapoptotic effect will be expanded in light of our recent progress. In the third specific aim, we will continue our study of the cell genes that are changed in expression in latently infected trigeminal ganglia. We will perform in situ hybridization to confirm the cell types that are altered in transcript levels, and immunohistochemistry to identify if the protein products are altered in amount for distribution. An understanding of these changes will help- us recognize ways in which the latent virus alters the infected sensory neuron, presumably in order to maintain its latency. These studies will continue our investigation of herpes simplex virus latency and reactivation and further our understanding of the mechanism of latency in the peripheral nervous system.
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