Molecular Switch Regulating Human Cytomegalovirus Replicative and Latent States

摘要 人巨细胞病毒(HCMV)在造血祖细胞中建立潜伏感染。差异化 潜伏感染的细胞诱导重新激活HCMV裂解周期，随后病毒扩散到多个器官 还有纸巾。我们知识中的一个重大差距是我们对控制人类巨细胞病毒的机制的了解有限。 重新激活。了解人巨细胞病毒潜伏期重新激活的机制对发展至关重要。 限制和控制免疫受损个体中危及生命的疾病的新策略，例如 移植受者。在建立和重新激活HCMV潜伏期的关键步骤是表达 主要的即刻早期蛋白(MIE)IE1和IE2，它们是病毒裂解周期的关键反式激活因子。 在裂解感染过程中，主要的即刻早期启动子(MIEP)驱动IE1和IE2的表达。 单个、选择性剪接的mRNA.在潜伏期，MIEP被沉默，IE1和IE2不表达。 HCMV再激活可诱导IE1和IE2的重新表达，因此推测HCMV再激活 需要激活MIEP以允许IE1和IE2的重新表达。然而，我们的初步结果表明， 当潜伏感染的细胞暴露在重新激活的刺激下时，MIEP保持沉默。这些数据提高了 耐人寻味的问题是：如果MIEP仍然处于不活跃状态，人巨细胞病毒是如何在 重新激活？通过一系列合作研究，我们发现，我们最近推出的替代MIE促进剂 在第一个MIE内含子中识别的基因在重新激活期间被激活到高水平。这些成绩单与 由于选择性转录起始点的使用，MIEP来源的转录本仅在其5‘非翻译区。 重要的是，每个新的转录本都编码全长IE1(72 KDa)或IE2(86 KDa)蛋白。此外，我们的 数据表明，新的MIE启动子是IE1和IE2重新激活后重新表达所必需的 人巨细胞病毒潜伏期的实验模型，但在成纤维细胞的复制过程中不存在。此外，我们已经确定 在分化过程中诱导的特定宿主转录因子(TF)也激活内含子酶 推动者。我们假设从潜伏期重新激活hcmv需要激活新的mie内含子。 宿主转录因子的启动子与髓系分化相关。在目标1中，我们将定义启动子元素 在细胞内使用重组病毒从潜伏期重新激活HCMV期间IE1和IE2重新表达所需的 细胞系和原代人类造血祖细胞。在目标2中，我们将定义特定宿主因素的作用 在重新激活过程中，IE1/2的分化依赖重新激活所需。我们提议的研究提供了令人振奋的 对控制疱疹病毒重新激活的机制的范式转换的见解。通过协作，这 多PI研究在我们对疱疹病毒的理解方面具有独特的地位 重新激活，我们预计这项工作将广泛地告知疱疹病毒重新激活的机制，对 开发预防巨细胞病毒病的新策略。

SUMMARY Human cytomegalovirus (HCMV) establishes latent infection in hematopoietic progenitor cells. Differentiation of latently infected cells induces reactivation of the HCMV lytic cycle, and subsequent viral spread to multiple organs and tissues. A significant gap in our knowledge is our limited understanding of the mechanisms controlling HCMV reactivation. Understanding the mechanisms by which HCMV reactivates from latency is critical to developing new strategies to limit and control life-threatening disease in immune compromised individuals, such as transplant recipients. A critical step in the establishment of and reactivation from HCMV latency is the expression of the major immediate early proteins (MIE) IE1 and IE2, which are crucial transactivators of the viral lytic cycle. During lytic infection, the major immediate early promoter (MIEP) drives the expression of IE1 and IE2 from a single, alternatively spliced mRNA. During latency, the MIEP is silenced, and IE1 and IE2 are not expressed. HCMV reactivation induces IE1 and IE2 re-expression, therefore it has been presumed that HCMV reactivation requires activation of the MIEP to allow for re-expression of IE1 and IE2. However, our preliminary results show that the MIEP remains silent when latently infected cells are exposed to reactivation stimuli. These data raise the intriguing question: If the MIEP remains inactive, how does HCMV express MIE transcripts during reactivation? Through a series of collaborative studies, we find that alternative MIE promoters we recently identified within the first MIE intron are activated to high levels during reactivation. These transcripts differ from MIEP-derived transcripts solely in their 5’ untranslated regions due to alternative transcription start site usage. Importantly, each of the novel transcripts encodes full length IE1 (72-kDa) or IE2 (86-kDa) proteins. Further, our data show that the novel MIE promoters are necessary for IE1 and IE2 re-expression upon reactivation in experimental models of HCMV latency, but not during replication in fibroblasts. Further, we have identified specific host transcription factors (TFs) induced during differentiation that also activate the intronic MIE promoters. We hypothesize that HCMV reactivation from latency requires activation of novel MIE intronic promoters by host TFs associated with myeloid differentiation. In Aim 1, we will define the promoter elements required for IE1 and IE2 re-expression during HCMV reactivation from latency using recombinant viruses in cell lines and primary human hematopoietic progenitors cells. In Aim 2, we will define the role of specific host factors required for differentiation-dependent reactivation of IE1/2 in reactivation. Our proposed studies offer exciting paradigm-shifting insights into the mechanisms that control herpesvirus reactivation. Through collaboration, this multi-PI study is uniquely positioned to make significant advances in our understanding of herpesvirus reactivation and we anticipate this work will broadly inform mechanisms of herpesvirus reactivation important to developing novel strategies to prevent HCMV disease.