Corneal HSV-1: LAT Blocks Apoptosis in Rabbit TG

描述(申请人提供)：复发性单纯疱疹病毒1型 L型单纯疱疹病毒感染是病毒性致盲的主要原因。理解 单纯疱疹病毒L潜伏期-再激活周期的分子机制应导致 开发一种减少HSV-I诱导的发病率的方法 失明。后来，唯一在潜伏期内大量转录的单纯疱疹病毒L基因是 对于有效的自发再激活是必不可少的。然而，潜在的 机制仍不清楚。我们最近发现，表达LAT的质粒 阻断组织培养中的细胞凋亡。此外，在兔三叉神经节(TG) 眼部感染LAT突变体后第7天出现广泛的细胞凋亡 感染(从裂解感染向潜伏感染过渡期间)。相比之下， 兔眼部感染LAT+病毒后三叉神经节细胞几乎无细胞凋亡 或得救的记分员)。这种LAT抗细胞凋亡功能可以增强自发性 通过促进急性感染后神经元的存活而重新激活，从而 使更大的潜伏感染神经元池可用于重新激活。 这一建议中要检验的主要假设是：LAT增强自发性 通过阻断细胞凋亡而重新激活。 具体目标是： 1.精细定位兔TG中负责阻断细胞凋亡的LAT区域。 我们现有的LAT突变体文库将被分析用于抗细胞凋亡 兔甘油三酯体内活性。递增表达的更多突变体 将构建更小的LAT区域并分析其抗细胞凋亡 活跃性和自发性再激活。如果LAT的抗凋亡功能共同映射 借助LAT的自发重新激活功能，它将有力地支持 假设LAT通过阻断细胞凋亡来增强再激活。 2.确定LAT的重启功能是否可以用替代方案替代 抗细胞凋亡基因。LAT调控下的多种抗细胞凋亡基因 启动子将被单独插入不阻断的LAT突变体 低水平甘油三酯急性感染兔的细胞凋亡 自发重新激活。If阻断细胞凋亡，增强自发性 重新激活恢复到野生型水平，这将证实LAT 通过阻断细胞凋亡来增强再激活作用。

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.