Mechanisms Regulating Cytomegalovirus

人巨细胞病毒（HCMV）感染了超过一半的退伍军人，并威胁着那些患有巨细胞病毒的人的生命。 免疫系统受损HCMV是导致出生缺陷的主要传染性原因。没有HCMV 疫苗和抗病毒药物存在效力、毒性和耐药性问题。远程 这项研究的目的是确定病毒转录-DNA复制周期中的关键点， 作为治疗干预的新靶点。这一建议是基于这样一个前提，即我们在 病毒早期转录如何产生病毒DNA复制以及病毒DNA复制如何 病毒晚期转录的结果限制了我们设计用于病毒性疾病的新治疗方法的能力。 通过定制先进的技术和开发新的工具， 基因组学（dTag系统、PRO-Seq、ChIP-Seq、基因工程试验病毒和启动子功能 检测）以确定Pol II何时何地启动转录，鉴定病毒转录因子的位点， 结合全基因组，并量化来自单个启动子的Pol II新生转录物的变化， 核心启动子序列、转录因子丢失、感染阶段和病毒DNA复制。我们发现 病毒晚期转录有三种不同的途径其中两种途径涉及HCMV IE 2和晚期转录因子（LTF）组成员。3个不同IE 2中的每一个的单独作用 在病毒晚期转录中的同种型（IE 2 -86、IE 2 -60和IE 2 -40）是未知的。LTF的六成员集 与Pol II和基因启动子中的DNA序列标签结合，形成前起始复合物（PIC）， 驱动转录。序列特征模式的多样性可能决定了个体的数量。 启动子输出。LTF复合物何时以及如何在病毒启动子上组装， LTF装配如何在转录中与Pol II接合。我们使用一种新的高分辨率ChIP-Seq技术 和生物信息学管道来绘制核小体的基因组位置，以及IE 2和LTF PIC， 表明LTF PIC占据不被核小体占据的基因组区域。这个新的ChIP-Seq 这项技术将加强我们的综合功能基因组学方法，以进一步确定 与染色质结构相关的控制病毒启动子转录的机制。我们的初步数据 表明：1）早-晚转录转换滞后于病毒DNA复制的开始许多小时; 2)有活性的HCMV启动子群体成员因细胞类型和条件而异， 差异可能涉及IE 2和LTF功能;和3）HCMV启动子群体，其在IE 2和LTF过程中是有活性的。 NT 2模型中的病毒再活化不同于急性生产性感染中的病毒再活化。我们将检验这个假设 HCMV转录因子取代了宿主Pol II，Pol II引导了一个适合于 导致病毒晚期转录（目的1和2），并协调不同的病毒转录程序， 细胞类型（Aim 2）和支持NT 2中静止和再活化感染的细胞条件下 模型（目标3）。我们将对每个具体目标采用多方面的方法：1）阐明 早-晚转录开关的调节子，2）确定细胞类型差异的机制基础 在病毒转录中，以及3）确定静止感染的激活如何改变病毒转录。我们 该提案整合了Meier和Price实验室分别在病毒学和转录方面的专业知识。 我们将在这一富有成效的合作基础上完成拟议的研究计划。这些发现 来自这些研究将确定基因调控的一般特征， β-和γ-疱疹病毒亚科的成员，包括人疱疹病毒6和 致癌疱疹病毒、爱泼斯坦-巴尔病毒和卡波西肉瘤相关疱疹病毒。

Human cytomegalovirus (HCMV) infects over half of all Veterans and threatens the lives of those with impaired immune systems. HCMV is the leading infectious cause of birth defects. There is no HCMV vaccine, and the antiviral drugs have problems with potency, toxicity, and drug-resistance. The long-range goal of this research is to identify critical points in the viral transcription-DNA replication cycle that would serve as new targets for therapeutic intervention. This proposal is based on the premise that our gap in knowledge of how viral early transcription produces viral DNA replication and how viral DNA replication results in viral late transcription limits our ability to design new therapeutic treatments for the viral disease. By customizing advanced technologies and developing new tools, we have used integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, genetically engineered test viruses, and promoter function assays) to determine where and when Pol II initiates transcription, identify sites of viral transcription factor binding genome-wide, and quantify change in Pol II nascent transcripts from individual promoters in relation to core promoter sequences, transcription factor loss, stage of infection, and viral DNA replication. We find that there are three distinct pathways to viral late transcription. Two of these pathways involve the HCMV IE2 and late transcription factor (LTF) group members. The individual role of each of the 3 different IE2 isoforms (IE2-86, IE2-60, and IE2-40) in viral late transcription is unknown. The six-member set of LTFs bind to Pol II and a DNA sequence signature in gene promoters, forming a preinitiation complex (PIC) that drives transcription. Diversity in sequence signature pattern likely determines the amount of individual promoter output. It is unknown precisely when and how the LTF complex assembles on viral promoters and how the LTF assembly engages Pol II in transcription. Our use of a new high-resolution ChIP-Seq technique and bioinformatics pipeline to map genomic locations of nucleosomes, as well as IE2 and LTF PICs, suggests that LTF PICs occupy genome regions not occupied by nucleosomes. This new ChIP-Seq technique will strengthen our integrated functional genomics approach to further determining the mechanisms controlling viral promoter transcription in relation to chromatin structure. Our preliminary data indicate that: 1) the early-late transcription switch lags many hours behind the onset of viral DNA replication; 2) the HCMV promoter population members that are active differs by cell type and condition, and this difference may involve IE2 and LTF functions; and 3) the HCMV promoter population that is active during viral reactivation in the NT2 model differs from that in acute productive infection. We will test the hypothesis that HCMV transcription factors usurp host Pol II that navigates a modified chromatin environment suited to bring about viral late transcription (Aims 1 and 2) and to coordinate the viral transcription program in diverse cell types (Aim 2) and under cellular conditions supporting quiescent and reactivation infections in the NT2 model (Aim 3). We will apply a multifaceted approach to each of the specific aims to: 1) elucidate the regulators of the early-late transcription switch, 2) determine the mechanistic basis for cell type differences in viral transcription, and 3) determine how activation of quiescent infection changes viral transcription. Our proposal integrates the expertise of the Meier and the Price labs in virology and transcription, respectively. We will build on this productive collaboration to complete the proposed research plan. The discoveries coming from these studies will identify generalizable features of gene regulation that pertain to other members of beta- and gammaherpesvirus subfamilies, which include human herpesvirus 6 and the oncogenic herpesviruses, Epstein-Barr Virus and Kaposis sarcoma-associated herpesvirus.