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

描述(申请人提供)：世界上大多数人口都感染了单纯疱疹病毒。在最初的裂解性感染之后，HSV在感觉神经元内建立永久的潜伏感染。潜伏病毒的重新激活不仅会导致病毒性疾病(新感染、失明和脑炎)，还会导致艾滋病毒感染、糖尿病、心血管和神经退行性疾病。没有有效的疫苗可用，也没有治疗方法可以消除潜伏期或防止重新激活。该项目的长期目标是通过定义控制HSV-1潜伏期的建立和重新激活的机制，找到对复发HSV发作的干预措施。HSV-1裂解感染过程中的基因表达级联始于病毒粒子蛋白VP16与宿主因子Oct-1和HCF-1共同激活即刻早期(IE)基因转录。相比之下，重新激活中的初始事件 起始延迟的定义仍然不明确。这一提议的中心假设是，对VP16表达和活性的调节是建立潜伏期和从潜伏期重新激活的基础。这两个水平的控制包括多个积极和消极的输入，以允许或抑制体内感觉神经元中的病毒复制。目的1.本课题将在体内研究VP16基因在感觉神经元中的激活和沉默机制。工作假说是，HSV-1基因组中的VP16基因可以通过神经元特异性和应激反应启动子元件和相应的转录因子的作用来调节，从而在最初感染或退出潜伏期时允许或抑制裂解复制。利用重组病毒在感染和潜伏期的小鼠模型中，我们发现VP16的表达是触发潜伏期退出的必要条件和充分条件，并且我们确定了VP16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在急性感染和重新激活期间进入裂解期感染。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VP16基因的表达，无论是单独、组合还是竞争。目的2.本项目将定义VP16共激活子相互作用对VP16依赖的潜伏期的退出至关重要，并确定体内调节这些相互作用的机制。我们的数据有力地表明，HSV退出潜伏期是由CK2介导的磷酸化调节的，这种磷酸化也可能受到O-GlcN酰化(一种调节信号对营养和胁迫的反应的PTM)的竞争性调节。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VP16反式激活来调节病毒蛋白的即刻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 这些酶可能成为未来单纯疱疹病毒再激活治疗干预措施发展的目标。

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.