Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.