Identification of cis-acting sequences in the promoter of the herpes simplex virus type 1 latency-associated transcripts required for activation by nerve growth factor and sodium butyrate in PC12 cells

Abstract

In the absence of detectable viral proteins, expression of the latency-associated transcripts (LATs) is likely regulated by cellular factors during latent infection of neurons with herpes simplex virus type 1. The amounts and activation states of these factors may in turn be regulated by extracellular regulatory factors. Consistent with this hypothesis, we have recently demonstrated that LAT expression is significantly enhanced by nerve growth factor (NGF) and sodium butyrate (NaB) in neurally derived PC12 cells. With the ultimate goal of identifying trans-acting cellular factors involved in regulating LAT expression during latency, we have attempted to identify the cis-acting elements to which these putative cellular factors bind by characterizing the LAT promoter and a series of 5' promoter deletion mutants in PC12 cells following treatment with the LAT-enhancing agents NGF and NaB. Transient expression assays demonstrated that distinct cis-acting sequences mediate basal and induced LAT promoter expression. Basal activity in PC12 cells is mediated by two elements: a negative regulatory element between -435 and -270 and a positive element between -240 and -204. The positive element contains binding sites for the transactivator Sp-1, whereas the negative element bears some resemblance to known neuron-specific silencer elements. In contrast to basal expression, maximum induction of the LAT promoter by NGF and NaB requires sequences between -159 and -81. Using gel mobility shift assays, we have identified three sets of protein-DNA complexes that bind to this 78-bp region and shown by competition analysis that binding is specific. The abundance and mobility of these complexes were altered by treatment with NGF or NaB. The nucleotide sequences to which these complexes bind were fine mapped by competition analysis with oligonucleotide probes containing substitution mutations. The target sequences identified exhibit no homology to binding sites of known transcription factors. These regions were critical for complex formation in vitro and for maximum induction of the LAT promoter by NGF and NaB in transient expression assays. The protein complexes that form with target sequences likely participate in the regulation of LAT expression in response to physiological stimuli in neurons in vivo.