Corneal HSV-1: LAT's Anti-Apoptosis Activity and Latency

描述：复发性单纯疱疹病毒1型（HSV-1）感染是病毒性失明的主要原因。阐明HSV-1潜伏-再激活周期背后的潜在分子机制，将导致开发一种降低HSV-1诱导的失明发生率的方法。HSV-1 LAT基因对于高野生型再活化表型是必需的，这似乎与LAT的抗凋亡活性有关。我们计划确认LAT作为RNA发挥作用，LAT阻断多种凋亡途径的能力对于其增强再激活的能力至关重要，并且LAT在再激活时起着重要作用。 我们的具体目标是： 1.证实LAT通过其RNA而不是通过LAT蛋白阻断细胞凋亡的假设。我们将：a）敲除LAT功能区内的所有4个潜在LAT ORF，并确认这些质粒仍然有效地阻断细胞凋亡，并且将这些敲除引入病毒中不会降低再活化表型; B）确认LAT ORF的表达（各自在单独的质粒中）不单独或一起阻断细胞凋亡; c）表明LAT可以阻断细胞凋亡，即使蛋白质合成被阻断。 2.确认LAT通过阻断多个步骤和/或途径的凋亡增强再激活表型的假设。我们将用阻断特定凋亡途径的抗凋亡基因替代病毒中的LAT。我们预测，只有阻断多个凋亡步骤的替代性抗凋亡基因才能够取代LAT并恢复高再活化表型。我们还将研究LAT RNA阻断细胞凋亡的机制。 3.验证LAT的抗凋亡活性在再激活时起重要作用的假设。将对HSV-1突变体进行工程改造，使得在用多西环素或他莫昔芬治疗后，在特定的转基因小鼠中LAT表达将被打开或关闭。当LAT仅在潜伏期建立期间表达时，与LAT仅在潜伏期再活化期间表达时相比，将确定这些突变病毒的再活化表型。

DESCRIPTION: 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.