THE SPONTANEOUS REACTIVATION FUNCTION OF THE HERPES-SIMPLEX VIRUS TYPE-1 LAT GENE RESIDES COMPLETELY WITHIN THE FIRST 1.5 KILOBASES OF THE 8.3-KILOBASE PRIMARY TRANSCRIPT

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV- 1 from latency, We report here that although the WT gene is 8.3 kb in length, the first 1.5 kb of the LAT gene alone is sufficient for wild- type levels of spontaneous reactivation, We began with a LAT deletion mutant of HSV-1 strain McKrae in which the LAT promoter and the first 1.6 kb of the 5' end of the LAT gene had been deleted from both copies of LAT (one in each viral long repeat), As we previously reported, th is mutant (dLAT2903) was significantly impaired for spontaneous reacti vation (G. C. Perng, E. C. Dunkel, P. A. Geary, S. M. Slanina, H. Ghia si, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 68:8045-80 55, 1991). We then inserted the LAT promoter and the first 1.5 kb of t he LAT gene into a location in the unique long region of dLAT2903 far removed from the normal location of LAT in the long repeats, This resu lted in a virus (LAT15a) whose capacity for transcribing LAT RNA was l imited to the first 1.5 kb of the 8.3-kb LAT primary transcript, Rabbi ts were ocularly infected with this mutant, and spontaneous reactivati on was measured in comparison to those of the original LAT-negative mu tant and its marker-rescued (wild-type) virus, dLAT2903R. LAT15a had a n in vivo spontaneous reactivation rate of 12%, compared with a rate o f 11% for the marker-rescued virus and 0% for the LAT-negative virus, Southern analysis confirmed that the spontaneously reactivated LAT15a virus retained the original deletions in both copies of LAT and the 1. 5-kb LAT insertion in the unique long region, Thus, insertion of the f irst 1.5 kb of LAT (and its promoter) at a site distant from the norma l LAT location appeared to completely restore in vivo spontaneous reac tivation to wild-type levels, despite the remaining inability of the o riginal LAT genes to transcribe any LAT RNA, The function of LAT invol ved in efficient spontaneous reactivation therefore appeared to map co mpletely within the first 1.5 kb of the LAT gene.