Although the ability of herpes simplex virus (HSV) to establish long term latency is sensory ganglion neurons is well documented, the virus-host interactions involved in transcriptional control of viral genes during latency remain largely unknown. Two latency active promoters have been discovered in the LAT region of the genome. LAP1 contains a TATA box while LAP2 is TATA-less. Reports indicate that LAP1 expresses a large unstable polyA+ 8.7kb transcript from which a small stable 2.0kb polyA- LAT RNA is derived by splicing. Moreover, numerous less abundant LATs of unknown origin have been described. Recent evidence, however, suggests that the 2kb LAT is a primary transcript and associated with polyribosomes. Our laboratory has evidence that LAP2 functions to express reporter genes in both the LAT and an ectopic locus. Experiments are described in the following three interrelated aims to carefully (i) determine whether one or both LAPs express LATs and under what circumstances, (ii) define the cis and trans elements which determine LAP activity and (iii) examine the potential role of chromatin structure in governing latency gene expression. (1) The question of whether LAP1 or LAP2 drive expression of either the major or minor LATs during lytic infection in vitro and during latent infection of the mouse trigeminal ganglion will be addressed through careful examination of transcript termini and through the use of mutants altered in LAP regulatory elements and putative LAT splice donor/acceptor sites. (2) Depending on the outcome of experiments in Aim 1, genetic studies involving recombinant viruses carrying linker-scanner and site-directed mutations in one of both LAPs will be conducted to determine the cis-acting signals that positively and/or negatively regulate LAP function in vitro and in vivo. Biochemical assays are also planned to study the binding of nuclear proteins to specific promoter sequences which contribute to the ability of LAP to function during latency, thereby establishing a connection between LAP functional elements and transcriptional activator or repressor proteins. (3) Studies re outlined to evaluate the role of chromatin structure in governing latency gene expression. The distribution of nucleosomes on latent HSV DNA will be determined by nuclease digestion and correlated with the pattern of gene expression during latency. Additional assays using cell lines carrying chromosomally integrated lytic and latency genes will be employed to evaluate differences between transcriptionally active and silent promoters.
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