Enhancer and long-term expression functions of herpes simplex virus type 1latency-associated promoter are both located in the same region

During herpes simplex virus type I (HSV-1) latent infection in vivo, the la tency-associated promoter (LAP) is the only promoter to remain highly activ e long term. In a previous attempt to characterize LAP activity in vitro an d in a mouse model, we showed that a 1.5-kb fragment called the long-term e xpression element (LTE), located immediately downstream from the transcript ional start site of LAP, was able to (i) increase gene expression in an ori entation-independent manner, regardless of the cell type or the promoter us ed in vitro (enhancer activity) and (ii) keep LAP active during latency in vivo (long-term expression activity) (H. Berthomme, J, Lokensgard, L, Yang, T, Margolis, and L, T, Feldman, J, Virol, 74:3613-3622, 2000), To determin e if these two functions could be separated genetically, we conducted a mut ational analysis on the LTE and analyzed the effect on the LAP-LTE properti es in both transient expression in fell culture and mouse dorsal root gangl ia lytic and latent infection. In this report, we show that the first half of the LTE sequence, corresponding to the region previously described as LA P2 or exon1, encodes the enhancer function. This same region is also requir ed to keep the LAP active during latency. These results exclude the intron region as containing any significant enhancer activity or any ability to ke ep the LAP active during latency, The results also show that these two func tions have not been separated, leaving open the possibility that there is n o long-term expression function per se but that the enhancer itself may fun ction to keep the LAP active during latency by raising the level of express ion to a detectable one. Further mutational analysis will be required to de termine if these two potential functions continue to cosegregate.