Associated Regulatory Region To Drive Stable Reporter Gene Expression in the Nervous System

The ability of herpes simplex virus type 1 (HSV-1) to establish a lifelong, transcriptionally active, latent infection in neurons has led to much interest in developing HSV-based vectors for gene delivery to the nervous system. A prerequisite of such vectors is that they should be capable of directing long-term transgene expression in latently infected neurons. The continued transcription of HSV-1 latency-associated transcripts (LATs) during neuronal latency suggests that regulatory sequences which mediate expression of LATs could be utilized for long-term expression of heterologous genes in the mammalian nervous system. In addition to upstream regulatory elements which are important for LAT promoter-mediated transcription during neuronal latency, there is growing evidence that sequences downstream of the LAT transcription start site play an important role in facilitating long-term latent-phase transcription. In order to maintain the integrity of both upstream and downstream regulatory elements of the LAT promoter, we constructed viruses which contained the lacZ and lacZ-neo reporter genes linked to the encephalomyocarditis virus internal ribosomal entry site (IRES) (viruses LbA and LbB, respectively) inserted approximately 1.5 kb downstream of the LAT transcrip-tion start site. These viruses expressed low levels of b-galactosidase in lytically infected Vero cells and in cervical dorsal root ganglion neurons during the acute stage of infection in vivo. In contrast, at later times postinfection and consistent with the establishment of latency, increases both in the numbers of neuron