Corneal HSV-1: LAT Blocks Apoptosis in Rabbit TG

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DESCRIPTION (provided by applicant): Recurrent herpes simplex virus type 1 (HSV-l) infection is a major cause of viral induced blindness. Understanding the molecular mechanisms of the HSV-l latency-reactivation cycle, should lead to development of a means for reducing the incidence of HSV-I induced blindness. LAT, the only HSV-l gene abundantly transcribed during latency is essential for efficient spontaneous reactivation. However, the underlying mechanism remains unknown. We recently showed that plasmids expressing LAT block apoptosis in tissue culture. Also, in trigeminal ganglia (TG) of rabbits ocularly infected with a LAT- mutant extensive apoptosis occurs on day 7 post infection (during transition from lytic to latent infection). In contrast, little apoptosis occurs in TG of rabbits ocularly infected with LAT+ virus (wt or marker rescued). This LAT anti-apoptosis function could enhance spontaneous reactivation by promoting neuronal survival following acute infection, thereby making a larger pool of latently infected neurons available for reactivation. The main hypothesis to be tested in this proposal is: LAT enhances spontaneous reactivation by blocking apoptosis. The Specific Aims are: 1. Fine map the region of LAT responsible for blocking apoptosis in rabbit TG. Our existing library of LAT mutants will be analyzed for anti-apoptosis activity in vivo in rabbit TG. Additional mutants expressing progressively smaller LAT regions will be constructed and analyzed for anti-apoptosis activity and spontaneous reactivation. If LAT's anti-apoptosis function co-maps with LAT's spontaneous reactivation function, it would strongly support the hypothesis that LAT enhances reactivation by blocking apoptosis. 2. Determine if LAT's reactivation function can be replaced by alternative anti-apoptosis genes. Various anti-apoptosis genes under control of the LAT promoter will be individually inserted into a LAT mutant that does not block apoptosis during acute infection of rabbit TG and that has low levels of spontaneous reactivation. If blocking apoptosis and enhancing spontaneous reactivation are restored to wild type levels, it would confirm that LAT enhances reactivation by blocking apoptosis.