UTILIZATION OF THE HERPES-SIMPLEX VIRUS TYPE-1 LATENCY-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 life long, transcriptionally active, latent infection in neurons has led to much interest in developing HSV based vectors for gene delivery to th e nervous system. A prerequisite of such vectors is that they should b e capable of directing long-term transgene expression in latently infe cted neurons. The continued transcription of HSV-1 latency-associated transcripts (LATs) during neuronal latency suggests that regulatory se quences which mediate expression of LATs could be utilized for long-te rm expression of heterologous genes in the mammalian nervous system. I n addition to upstream regulatory elements which are important for LAT promoter-mediated transcription during neuronal latency, there is gro wing evidence that sequences downstream of the LAT transcription start site play an important role in facilitating long-term latent-phase tr anscription. In order to maintain the integrity of both upstream and d ownstream regulatory elements of the LAT promoter, we constructed viru ses which contained the lacZ and lacZ-neo reporter genes linked to the encephalomyocarditis virus internal ribosomal entry site (IRES) (viru ses L beta A and L beta B, respectively) inserted approximately 1.5 kb downstream of the LAT transcription start site. These viruses express ed low levels of beta-galactosidase in lytically infected Vero cells a nd in cervical dorsal root ganglion neurons during the acute stage of infection in vivo. In contrast, at later times postinfection and consi stent with the establishment of latency, increases both in the numbers of neurons expressing beta-galactosidase and in the intensity of stai ning were observed. Examination of the brain stems and spinal cords of animals latently infected with L beta A, sampled at time points from 72 to 307 days postinfection, revealed the stable expression of beta-g alactosidase within neurons located in facial and hypoglossal nerve nu clei and the upper cervical spinal cord. We conclude that the insertio n of an IRES linked to a reporter gene 1.5 kb downstream from the LAT transcription start site does not disrupt elements of the LAT promoter necessary for long-term gene expression and, in both the peripheral a nd central nervous systems, facilitates beta-galactosidase expression in a wide variety of neurons.