Corneal HSV-1: LAT Blocks Apoptosis in Rabbit TG

描述（由申请人提供）：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。理解 HSV-1潜伏-再激活循环的分子机制，应该导致 涉及开发一种降低HSV-1诱导的 失明LAT是唯一在潜伏期大量转录的HSV-1基因， 对于有效的自发性再激活至关重要。然而，底层 机制仍然未知。我们最近发现表达LAT的质粒 在组织培养中阻断细胞凋亡。在家兔三叉神经节（TG）中， 眼部感染LAT突变体后第7天发生广泛的细胞凋亡 感染（从溶解性感染到潜伏性感染的转变）。与此相反， LAT+病毒（WT）感染兔眼后，TG细胞凋亡较少 或标记获救）。这种LAT抗凋亡功能可以增强自发性 通过促进急性感染后的神经元存活而重新激活，从而 使得更大的潜伏感染神经元池可用于重新激活。 本提案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.精细定位负责阻断兔TG中细胞凋亡的LAT区域。 我们现有的LAT突变体库将被分析用于抗细胞凋亡 在兔TG中的体内活性。进行性表达的其他突变体 将构建较小的LAT区域并分析其抗凋亡作用 活性和自发再活化。如果LAT的抗凋亡功能与 由于LAT的自发再激活功能，它将有力地支持 假设LAT通过阻断凋亡增强再激活。 2.确定LAT的重新激活功能是否可以由替代功能替代 抗凋亡基因LAT控制下的各种抗凋亡基因 启动子将被单独插入到LAT突变体中，所述LAT突变体不阻断 在兔TG急性感染过程中细胞凋亡， 自发再激活如果阻断细胞凋亡， 恢复到野生型水平，这将证实LAT 通过阻断细胞凋亡增强再激活。

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.