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 virus latency and reactivation at the molecular level. In the present application, we propose to continue to examine the latency associated transcripts (LATs) to determine what role they play in viral latency and reactivation. Their regulation through the cellular early growth response (EGR) proteins will also be studied. These goals will be achieved using the techniques of molecular virology. We will study expression of a newly identified latency associated transcription unit, which appears to map upstream of the well recognized LAT. This transcription unit is encoded in a region of the viral genome to which the slow or inefficient reactivation phenotype of some HSV mutants has been mapped, and we are presently defining this genetic locus. A second phenotype has recently been associated with the 2 kb LAT RNA. Mutants that are deleted in part of this transcript appear to form latency in a reduced number of neuronal cells. Key to the understanding of this phenotype will be knowledge of the mechanism underlying the cellular localization of the 2 kb LAT RNA. Our funding that the 2 kb LAT RNA is present in the cytoplasm during lytic infection and appears associated with ribosomal subunits suggests a role in translation for this RNA. In order to understand why the LAT RNA is present in the nucleus during latency and in the cytoplasm during reactivation and the lytic cycle of infection, we will examine the structure of the 2 kb LAT through comparisons of, the 5' end position, the presence of a cap, splicing, and promoter studies, between nuclear and cytoplasmic LAT species. In order to further study the 2 kb LAT and the role that its cellular distribution plays in the apparent reduction of latently infected cells, we will develop cell lines expressing 2 kb LAT. This will be done in fibroblasts, CV-1 cells, cells of neuronal origin, Pc12 cells and ES cells. Finally, from cell lines expressing 2 kb LAT in ES cells, transgenic animals will be made to study LAT expression and cellular localization after reactivation stimuli are applied. We will study the role of EGR proteins in modulating the LAT gene expression as once we understand what the LAT gene does, it will be important to understand how the LAT gene function is regulated.
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