REGULATION OF HERPES SIMPLEX TYPE 1 INFECTION IN CORNEAL NEURONS

项目摘要/摘要 单纯疱疹病毒(HSV)1型是感染性角膜失明的主要原因。它会导致 通过角膜神经中潜伏的病毒储存库重新激活而导致的眼病。尽管广泛 研究表明，单纯疱疹病毒在神经元中感染的调节机制还没有得到很好的描述。一个 更好地理解这一点对于确定新的治疗策略至关重要。我们有 利用分离的成年小鼠三叉神经节(TG)开发了一种新的培养系统，用于 研究神经元中的HSV感染。来自我们实验室的初步数据表明，不同于它在 复制细胞，病毒即刻早期(IE)基因产物ICP27，限制了病毒的生产 感染神经元，特别是A5+神经节神经元，并促进病毒潜伏期。在 目前的建议我们将进一步表征ICP27的这一新功能，以及研究 ICP27完成这些功能的机制。在此的前两个具体目标中 提案中，我们将描述ICP27在限制生产性感染和 利用ICP27缺失突变体促进病毒潜伏，ICP27启动子突变体 表达动力学，以及ICP27不同功能区缺失的病毒突变体。 我们将通过使用ICP27进一步表征其在限制神经元感染中的作用 利用ICP27高效转导感觉神经元的新型AAV载体。我们的 初步数据还表明，在复制细胞中表达的VP16是一种晚期病毒蛋白 在TG神经元中，ICP27限制了VP16和ICP4的转录。 培养的三叉神经节神经元。在第三个具体目标中，我们将进一步描述ICP27介导的 抑制ICP4和VP16转录并检验有关其作用方式的假说 已实现。最后，我们将检验ICP27限制A5+中的生产性感染的假设 神经元，部分是通过将ICP4、VP16和HCF1限制在细胞质上，从而阻止 病毒IE基因的反式激活。这些概念和研究是创新的，是 能够直接研究神经元中的HSV感染，以及能够区分 单纯疱疹病毒潜伏期的主要部位A5+神经元感染的结果。这些措施的结果 研究应该对调节神经元HSV感染的机制产生新的见解； 开发新的治疗策略的第一步。

PROJECT SUMMARY/ABSTRACT Herpes simplex virus (HSV) type 1 is a leading cause of infectious corneal blindness. It causes eye disease by reactivation from a latent viral reservoir in corneal nerves. Despite extensive research, the mechanisms that regulate HSV infection in neurons are not well characterized. A better understanding of this is critical for identifying new therapeutic strategies. We have developed a novel culture system, using dissociated adult murine trigeminal ganglia (TG), for studying HSV infection in neurons. Preliminary data from our lab indicates that, unlike its role in replicating cells, the viral immediate early (IE) gene product, ICP27, restricts productive viral infection in neurons, especially in A5+ ganglionic neurons, and promotes viral latency. In the current proposal we will further characterize this novel function for ICP27, as well as study the mechanisms by which ICP27 accomplishes these functions. In the first two specific aims of this proposal, we will characterize the role that ICP27 plays in restricting productive infection and promoting viral latency, using ICP27 null mutants, an ICP27 promoter mutant with delayed kinetics of expression, and viral mutants with deletions in different ICP27 functional domains. We will further characterize the role of ICP27 in restricting infection in neurons through the use of novel AAV vectors for the efficient transduction of sensory neurons with ICP27. Our preliminary data also suggests that VP16, a late viral protein in replicating cells, is expressed very early in TG neurons, and that ICP27 restricts transcription of both VP16 and ICP4 in cultured TG neurons. In the third specific aim we will further characterize ICP27 mediated inhibition of ICP4 and VP16 transcription and test hypotheses about the way in which this is achieved. Finally, we will test hypotheses that ICP27 restricts productive infection in A5+ neurons, in part, by restricting ICP4, VP16 and HCF1 to the cytoplasm, thus preventing transactivation of viral IE genes. These concepts and studies are innovative, and are a result of being able to directly study HSV infection in neurons, as well as being able to differentiate the outcome of infection in A5+ neurons, the major site of HSV latency. The outcome of these studies should generate new insights into the mechanisms regulating HSV infection of neurons; the first step in developing new therapy strategies.