Molecular Switch Regulating Human Cytomegalovirus Replicative and Latent States

总结 人巨细胞病毒（HCMV）在造血祖细胞中建立潜伏感染。分化 潜伏感染的细胞诱导HCMV裂解周期的重新激活，随后病毒扩散到多个器官 和纸巾。我们知识上的一个重大差距是我们对控制HCMV的机制的理解有限 重新激活了解HCMV从潜伏期重新激活的机制对发展中国家的免疫系统至关重要。 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 移植接受者HCMV潜伏期的建立和再激活的关键步骤是表达 主要的立即早期蛋白（MIE）IE 1和IE 2，它们是病毒裂解周期的关键反式激活因子。 在裂解性感染过程中，主要立即早期启动子（MIEP）驱动IE 1和IE 2的表达， 单一的选择性剪接的mRNA。在潜伏期，MIEP沉默，IE 1和IE 2不表达。 HCMV再活化诱导IE 1和IE 2再表达，因此推测HCMV再活化 需要激活MIEP以允许IE 1和IE 2的重新表达。然而，我们的初步结果显示， 当潜伏感染的细胞暴露于再激活刺激时，MIEP保持沉默。这些数据提高了 有趣的问题是：如果MIEP保持不活动，HCMV如何表达MIE转录本， 重新激活？通过一系列的合作研究，我们发现，替代MIE启动子，我们最近 在第一个MIE内含子内鉴定的基因在再激活期间被激活到高水平。这些抄本不同于 MIEP衍生的转录物仅在其5'非翻译区中，这是由于选择性转录起始位点的使用。 重要的是，每个新的转录本编码全长IE 1（72-kDa）或IE 2（86-kDa）蛋白。此外，我们的 数据显示，新的MIE启动子在IE 1和IE 2的再激活后是IE 1和IE 2再表达所必需的， HCMV潜伏期的实验模型，但不是在成纤维细胞复制期间。此外，我们还发现， 在分化过程中诱导的特异性宿主转录因子（TF）也激活内含子MIE 发起人。我们假设HCMV从潜伏期的再激活需要新的MIE内含子的激活， 启动子通过与骨髓分化相关的宿主TF。在目标1中，我们将定义启动子元件 在细胞中使用重组病毒使HCMV从潜伏期再活化期间需要IE 1和IE 2再表达 系和原代人造血祖细胞。在目标2中，我们将定义特定宿主因子的作用 需要分化依赖性再激活的IE 1/2的再激活。我们提出的研究提供了令人兴奋的 对控制疱疹病毒再激活机制的范式转变见解。通过合作，这 多PI研究具有独特的优势，可以使我们对疱疹病毒的理解取得重大进展 我们预计这项工作将广泛地告知疱疹病毒再激活的重要机制， 开发预防HCMV疾病的新策略。

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.