During latency HSV-1 replication is blocked at the level of viral immediate early (IE) gene expression. This restriction of replication may be caused by the differentiated state of neurons, in which cellular transcription factors required for viral IE gene expression are down- regulated or transcriptional repressors are present. Reactivation signals may induce certain cellular immediate early (IE)genes which in turn up- regulate HSV IE genes, culminating in reactivation of HSV-1 from latency. The proposed project will test this hypothesis, that an altered transcriptional program in sensory neurons causes reactivation. Cellular transcription factors that bind to viral IE promoters in vitro include octamer binding proteins Oct-1 and Oct-2, SP1, GA binding protein, NF-Y, F1 and possibly NF-k3 and AP1. The role of these cellular transcription factors in sensory neurons is poorly characterized. We will evaluate the role of these cellular transcription factors in the induction of HSV-1 IE genes during viral reactivation and also will seek novel factors. HSV-1 latency will be studied in murine trigeminal ganglia (TG) using both in vivo and in vitro reactivation models. We will characterize the expression pattern of cellular transcription factors before and after stress. Electrophoretic mobility shift assays using consensus DNA binding sites will test changes in the level of these cellular transcription factors. Second, novel transcriptional regulatory proteins necessary for viral reactivation will be sought using genetic selections in the yeast S. cerevisiae. cDNA prepared from unstimulated or stimulated TG will be screened for factors that bind to viral IE gene promoter sequences or physically interact with known cellular proteins that are required for viral IE expression. Third, we will examine cell- specific patterns of expression of cellular factors relative to that of viral genes, using in situ hybridization and histochemistry. Finally, the role of these identified cellular transcription factors during reactivation will be assessed using transfection into cultured, latently- infected cells.
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