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)感染是病毒性致盲的主要原因。阐明HSV-1潜伏期-再激活周期背后的潜在分子机制，将有助于开发一种降低HSV-1致盲发生率的方法。HSV-1 LAT基因是野生型高度激活表型所必需的，这似乎与LAT的抗凋亡活性有关。我们计划确认LAT作为一种RNA发挥作用，LAT阻断多条凋亡途径的能力是其增强再激活能力的关键，以及LAT在再激活时发挥着重要作用。我们的具体目标是： 1.确认LAT通过其RNA而不是通过LAT蛋白来阻止细胞凋亡的假设。我们将：a)敲除LAT功能区内所有4个潜在的LAT ORF，并确认这些质粒仍然有效地阻止细胞凋亡，并且将这些敲除基因引入病毒中并不会减少重新激活的表型；b)确认LAT ORF的表达(每个在一个单独的质粒中)不会单独或共同阻止细胞凋亡；c)证明即使在蛋白质合成被阻止的情况下，LAT也可以阻止细胞凋亡。 2.证实LAT通过多个步骤和/或途径阻断细胞凋亡而增强细胞再激活表型的假说。我们将用替代的抗凋亡基因来取代病毒中的LAT，这些基因可以阻断特定的凋亡途径。我们预测，只有阻断多个凋亡步骤的替代抗凋亡基因才能替代LAT，恢复高度再激活的表型。我们还将研究LAT RNA阻止细胞凋亡的机制。 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突变体进行改造，使特定的转基因小鼠在用多西环素或三苯氧胺治疗后，LAT的表达将被打开或关闭。当LAT仅在潜伏期建立期间表达时，将确定这些突变病毒的重新激活表型，而当LAT仅在潜伏期重新激活期间表达时，将确定其重新激活表型。

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.