Recurrent herpes simplex virus type 1 (HSV-1) infection is a major cause of viral induced blindness. HSV-1 establishes a life long latent infection with intermittent periods of reactivation. Reactivations can cause recurrent corneal disease leading to blindness. LAT, the only transcriptionally active viral gene during latency, is essential for efficient spontaneous reactivation. Understanding how LAT functions, along with elucidation of other molecular mechanisms behind the HSV-1 latency-reactivation cycle, should lead to the development of a means for reducing the incidence of HSV-1 induced blindness.
The specific aims i nclude: 1. Fine map the region within the first 1.5 kb of LAT that is required for spontaneous reactivation. The PI has recently shown that spontaneous reactivation can be completely rescued in a LAT negative virus by inserting the LAT promoter and the first 1.5 kb of the 8.3 LAT into a novel location in the unique long region. Since this virus can only transcribe LAT from the insert, this mapped LAT's function to within the first 1.5 kb of the primary LAT transcript. He will use this novel system to insert and test smaller and smaller regions of LAT to fine map the minimal region required for efficient spontaneous reactivation. 2. Determine if LAT enhances spontaneous reactivation by enhancing establishment of latency. The amount of latency established by LAT negative mutants versus marker rescued viruses will be estimated by in situ PCR for HSV-1 DNA, in situ hybridization for LAT RNA in mutants that transcribe only nonessential LAT RNA, and in situ hybridization for HSV-1 mRNAs to determine if LAT mutants are defective in terminating acute infection. 3. Determine if there is a LAT protein involved in LAT's function. This will be comparative sequence analysis of the first 1.5 kb of LAT in HSV-1 strains with high spontaneous reactivation rates to find conserved potential open reading frames, followed by site-directed mutagenesis of such potential open reading frames to determine if they are essential for LAT's function.
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