Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript

Abstract

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells, promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern (RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor of the more stable 2- and 1.3-kb LATs.