Repeated herpes simplex virus type I (HSV-l) occular recurrences as a result of HSV-l reactivation from latency, is a major cause of corneal scarring and blindness. LAT is the only viral gene known to be active during HSV-L latency, and is therefore a potential handle for studying the molecular mechanisms of HSV-1 latency and reactivation. In CAT assays, our new data show that the LAT promoter has specificity for primary cultures of corneal stromal keratocytes. This is particulary interesting in light of recent interest in the cornea as an alternate site of HSV-L latency and the recent demonstration of LAT RNA in stromal keratocytes of latently infected mice. We therefore plan to fine map the LAT promoter and its regulatory regions and characterize LAT regulation in stromal keratocytes from man, rabbit, and mouse (compared to neuronal and non-neuronal cells). These studies will test the theory that 'regulation of LAT is identical in corneal cells (stromal keratocytes) and sensory neurons' ' . If correct, the notion of 'true' corneal latency would be greatly supported. If incorrect, the likelihood of 'true' corneal latency will be decreased. A better understanding of the molecular mechanisms of HSV-L latency and reactivation should allow the development of procedures to effectively intervene in HSV-L infections and block latency and/or reactivation, thereby reducing the incidence of HSV-L induced blindness.
The specific aims to test the above hypothesis and to understand and compare the molecular mechanisms of HSV-1 latency in stromal keratocytes and neurons include: 1. Fine mapping of the LAT promoter in corneal stromal keratocytes and neuronal derived cells by means of CAT assays. 2. Identification and characterization of cis-acting regulatory regions in stromal keratocytes and neuronal cells using CAT assays. 3. Sequence determination of LAT regulatory sequences by DNA binding studies and preliminary characterization of stromal keratocyte cell factors that bind to these sequences (and thereby control LAT expression).
| Perng, G C; Zwaagstra, J C; Ghiasi, H et al. (1994) Similarities in regulation of the HSV-1 LAT promoter in corneal and neuronal cells. Invest Ophthalmol Vis Sci 35:2981-9 |
| Perng, G C; Ghiasi, H; Kaiwar, R et al. (1994) An improved method for cloning portions of the repeat regions of herpes simplex virus type 1. J Virol Methods 46:111-6 |
| Ghiasi, H; Nesburn, A B; Kaiwar, R et al. (1991) Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D. Arch Virol 121:163-78 |
| Zwaagstra, J C; Ghiasi, H; Nesburn, A B et al. (1991) Identification of a major regulatory sequence in the latency associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1). Virology 182:287-97 |
