IN-VIVO CHARACTERIZATION OF SITE-DIRECTED MUTATIONS IN THE PROMOTER OF THE HERPES-SIMPLEX VIRUS TYPE-1 LATENCY-ASSOCIATED TRANSCRIPTS

Transient expression assays in PC12 cells showed that the cAMP respons e element (CRE) and the TATA box of the herpes simplex virus type 1 la tency-associated transcripts (LATs) promoter are essential for basal e xpression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to rep licate, express LATs and reactivate from latency were compared with wi ld-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equiv alent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild -type or marker-rescued viruses. In situ hybridization of TATA box mut ant virus-infected ganglia, however, showed threefold fewer LAT-positi ve neurons than wild-type virus-infected ganglia, with consistently we aker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs C RE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hyb ridization, however, the percentage and intensity of LATs-positive neu rons were found to be comparable for the CRE mutant- and wild-type vir us-infected ganglia, suggesting that the CRE is dispensable for abunda nt LATs expression but that a reactivation function of the LATs may de pend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independen tly of the LATs.