Recurrent herpes simplex virus type 1 (HSV-1) infection is a major cause of viral induced blindness. Elucidation of the underlying molecular mechanisms behind the HSV-1 latency-reactivation cycle, should lead to development of a means for reducing the incidence of HSV-1 induced blindness. The HSV-1 LAT gene is essential for the high wild type reactivation phenotype and this appears to be related to LAT's antiapoptosis activity. We plan to confirm that LAT functions as an RNA, that LAT's ability to block multiple apoptosis pathways is critical for its ability to enhance reactivation, and that LAT plays an important role at the time of reactivation.
Our specific aims are: 1. Confirm the hypothesis that LAT blocks apoptosis via its RNA rather than via a LAT protein. We will: a) Knock out all 4 potential LAT ORFs within the functional region of LAT and confirm that these plasmids still efficiently block apoptosis and that introducing these knock outs into the virus do not reduce the reactivation phenotype; b) Confirm that expression of the LAT ORFs (each in a separate plasmid) do not block apoptosis, either individually or together; c) Show that LAT can block apoptosis even when protein synthesis is blocked. 2. Confirm the hypothesis that LAT enhances the reactivation phenotype by blocking apoptosis at multiple steps and/or pathways. We will replace LAT in the virus with alternative anti-apoptosis genes that block specific apoptotic pathways. We predict that only alternative anti-apoptosis genes that block multiple apoptosis steps will be able to substitute for LAT and restore the high reactivation phenotype. We will also investigate the mechanism by which LAT RNA blocks apoptosis. 3. Test the hypothesis that LAT's anti-apoptotic activity plays an important role at the time of reactivation. HSV-1 mutants will be engineered such that LAT expression will be turned on or off in specific transgenic mice following treatment with doxycycline or Tamoxifen. The reactivation phenotype of these mutant viruses will be determined when LAT is expressed only during establishment of latency compared to when LAT is expressed only during reactivation from latency.
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