The mechanisms that regulate reactivation of latent herpes simplex virus type 1 (HSV-1) after exposure to external stimuli remain an enigma despite years of research. The current state of knowledge recognizes that approximately 0.1% of latently infected neurons of sensory ganglia contain HSV-1 DNA, the latent viral genome is maintained in neuronal nuclei in low copy number, and the expression of the HSV-1 genome is extremely restricted during latency. The only region of the latent viral genome transcriptionally active is a gene found within the long repeat regions. A family of RNAs termed latency associated transcripts (LATs) are transcribed by this diploid gene. While the function of LATs is not yet clear, recent evidence has suggested that LATs restrict HSV-1 reactivation, and various stimuli induce reactivation via signal transduction that release its repressive effects. This hypothesis is supported by our findings that i) proteins derived from neuronal cells bind specifically to a region (-280 to -238) of the LAT promoter after exposure to ultraviolet (Uv) light and heat shock (HS), ii) an UV response element (AP1) is present within this region, and iii) the expression of LATs is down-regulated in neuronal cells exposed to UV irradiation as determined by transient chloramphenicol acetyltransferase (CAT) assay. This project will further explore the role of DNA binding proteins induced by UV and HS in the expression of LATs, and the role of signal transduction in oral HSV-1 reactivation. The promoter sequences of LATs (HSV-1) that bind proteins induced by UV and HS will be identified using electrophoretic mobility shift assay and DNase I footprinting. To determine if these proteins are transcription factors, transient CAT assays will be performed using reporter plasmids carrying 5' deletions of LATs. A lambdagt11 cDNA expression library made from mouse neuroblastoma cells will be screened to identify the gen encoding the LATs binding protein. The sequence of the cDNA will be analyzed to determine if it encodes a known or a novel protein. The cDNA will be used in co-transfection experiments to determine if the expressed protein regulates the expression of LATS. Probes generated from the cDNA clone will be used to study reactivation in mice that have been latently infected with HSV-1 wild-type or HSV-1 deleted of the HS binding site(s), after exposure to hyperthermia. Characterization of HSV-1 reactivation will be performed by explant cultivation of latently infected ganglia and by in situ hybridization. These studies should provide insight into the mechanism by which external stimuli induce HSV-1 reactivation.
