The 2-kilobase intron of the herpes simplex virus type 1 latency-associated transcript has a half-life of approximately 24 hours in SY5Y and COS-1 cells

The herpes simplex virus type I (HSV-1) 2-kb latency-associated transcript (LAT) is a stable intron, which accumulates in cells both lytically and latently infected with HSV-1. We have used a tetracycline-repressible expression system to determine the half-life of the 2-kb LAT RNA intron in the human neuroblastoma cell line SY5Y. Using Northern hybridization analyses of RNA isolated from transiently transfected SY5Y cells over time after repression of LAT expression, we measured the half-life of the 2-kb LAT to be approximately 24 h. Thus, unlike typical introns that are rapidly degraded in a matter of seconds following excision, the 2-kb LAT intron has a half-life similar to those of some of the more stable cellular mRNAs. Furthermore, a similar half-life was measured for the 2-kb LAT in transiently transfected nonneuronal monkey COS-1 cells, suggesting that the stability of the 2-kb LAT is neither cell type nor species specific. Previously, we found that the determinant responsible for the unusual stability of the 2-kb LAT maps to the 3' terminus of the intron. At this site is a nonconsensus intron branch point located adjacent to a predicted stem-loop structure that is hypothesized to prevent debranching by cellular enzymes. Here we show that mutations which alter the predicted stem-loop structure, such that branching is redirected, either reduce or abolish the stability of the 2-kb LAT intron.