Identification of a second ATF/CREB-like element in the herpes simplex virus type 1 (HSV-1) latency-associated...

The herpes simplex virus type-1 (HSV-1) latency-associated transcript (LAT) promoter (LP) has been shown to function in a cell type-specific manner. We have constructed an extensive series of PCR deletion mutations of the LP from nucleotides +1 to -348 to delineate the specific sequences involved in the cell type-specific activity of the HSV-1 LP. This series of 5' LP deletion constructs has been transiently transfected into both C1300 (neuronal) and L929 (nonneuronal) cells. When nucleotides -75 to -83 were added to nucleotides +1 to -74, a three- to fourfold C1300-specific increase in promoter activity was observed. In addition, when sequences upstream of nucleotide -211 were added to nucleotides +1 to -211, a second threefold increase in promoter activity was observed in C1300 cells. To begin to understand the biochemical basis for these observations, we have examined the interaction of a segment of the HSV-1 LP (nucleotides -54 to -134) with factors present in neuronal and nonneuronal nuclear extracts. This region of the LP contains the sequence most proximal to the transcriptional start site demonstrated to be involved in cell type-specificity (nucleotides -75 to -83). By coupling the functional studies with electrophoretic mobility shift (EMS), oligonucleotide competition EMS, and antibody supershift EMS analyses, we have demonstrated that members of the activating transcription factor (ATF)/cyclic-AMP response element binding protein (CREB) transcription factor family interact with nucleotides -75 to -83 of the HSV-1 LP. The identification of a novel ATF/CREB-like element in the HSV-1 LP may facilitate the understanding of neuronal factors which regulate LAT expression during HSV-1 infection. These studies may ultimately provide additional insight concerning the role of HSV-1 LAT in the regulation of viral latency and reactivation.