HSV latency and reactivation and the novel neuronal regulation of VP16 in vivo.

描述（由申请人提供）：世界范围内的大多数人群已被单纯疱疹病毒感染。在最初的裂解感染后，HSV在感觉神经元内建立永久性潜伏感染。潜伏病毒的再活化不仅导致病毒性疾病（新感染、失明和脑炎），而且还导致HIV感染、糖尿病、心血管和神经退行性疾病。没有有效的疫苗可用，也没有消除潜伏期或防止再激活的治疗方法。该项目的长期目标是通过定义控制HSV-1潜伏期的建立和再激活的机制来找到复发性HSV发作的干预措施。 HSV-1裂解性感染期间的基因表达级联始于病毒体蛋白VP 16与宿主因子Oct-1和HCF-1激活立即早期（IE）基因转录。与此相反， 延迟的定义仍然很差。该提议的中心假设是VP 16表达和活性的调节是潜伏期的建立和从潜伏期再激活的基础。这两个水平的控制涉及多个阳性和阴性输入，以允许或抑制体内感觉神经元中的病毒复制。 目标1。本项目将在体内研究VP 16基因在感觉神经元中的激活和沉默机制。工作假设是HSV-1基因组中的VP 16基因可以通过神经元特异性和应激反应性启动子元件和相应的转录因子的作用来调节，以允许或抑制初始感染时的裂解复制或退出潜伏期。在感染和潜伏期的小鼠模型中使用重组病毒，我们发现VP 16的表达对于触发潜伏期的退出是必要的和充分的，并且我们鉴定了VP 16基因的神经元特异性启动子。我们将测试该启动子是否控制进入裂解期感染在急性感染和再激活过程中。我们将确定预测的转录因子是否与该启动子的各种元件结合，以正向或负向调节VP 16基因表达，无论是单独、组合还是竞争。 目标2.该项目将定义VP 16辅激活因子相互作用的VP 16依赖退出从潜伏期和确定机制，在体内调节这些相互作用。我们的数据有力地表明，HSV从潜伏期的退出是由CK 2介导的磷酸化调节的，并且这种磷酸化也可能是由O-GlcNAc酰化（一种调节响应于营养和应激的信号传导的PTM）竞争性调节的。我们的目标是阐明这些PTM中的每一个在病毒潜伏期中的功能，并确定它们的串扰在通过VP 16反式激活调节病毒蛋白的立即早期基因表达中的作用。这项工作的结果将确定转录因子或蛋白质修饰 这些酶可能是未来开发HSV再活化治疗干预的靶点。

DESCRIPTION (provided by applicant): Most of the human population world-wide has been infected by herpes simplex viruses. Following the initial lytic infection, HSVs establish permanent latent infections within sensory neurons. Reactivation of latent virus not only results in viral disease (new infections, blindness, and encephalitis) but also contributes to HIV infection, diabetes, cardiovascular and neurodegenerative diseases. No effective vaccine is available and no therapy eliminates latency or prevents reactivation. The long-term goal of this project is to find interventions for recurrent HSV episodes by defining mechanisms that control establishment and reactivation of HSV-1 latency. The gene expression cascade during HSV-1 lytic infection begins with activation of immediate-early (IE) gene transcription by the virion protein VP16 with host factors Oct-1 and HCF-1. In contrast, the initial events in the reactivation from latency are still poorly defined. The central hypothesis of this proposal is that regulation o both VP16 expression and activity underlie the establishment of latency and reactivation from latency. These two levels of control involve multiple positive and negative inputs to allow or inhibit viral replication in the sensory neuron in vivo. Aim 1. This project will determine the mechanism of de novo VP16 gene activation and silencing in sensory neurons in vivo. The working hypothesis is that the VP16 gene in the HSV-1 genome can be regulated by action of neuron-specific and stress-responsive promoter elements and corresponding transcription factors either to allow or inhibit lytic replication upon initial infection or exit from latency. Uing recombinant viruses in a mouse model of infection and latency, we found that expression of VP16 is both necessary and sufficient to trigger the exit from latency and we identified a neuron-specific promoter for the VP16 gene. We will test whether this promoter controls the entry into lytic phase infection during acute infection and during reactivation. We will determine whether the predicted transcription factors bind to the various elements of this promoter to positively or negatively regulate VP16 gene expression, whether singly, in combination, or in competition. Aim 2. This project will define the VP16 coactivator interactions essential for VP16-dependent exit from latency and identify mechanisms regulating these interactions in vivo. Our data strongly suggest that the exit from latency by HSV is regulated by CK2 mediated phosphorylation and that this phosphorylation may be also competitively regulated by O-GlcNAcylation (a PTM that regulates signaling in response to nutrients and stress). Our goal is to elucidate the functions of each of these PTMs in viral latency and to define the roles of their crosstalk in regulating immediate early gene expression of viral proteins through VP16 transactivation. The outcomes of this work will identify transcription factors or protein modifying enzymes that could be targets for future development of therapeutic interventions for HSV reactivation.