Mechanisms Regulating Cytomegalovirus

人类巨细胞病毒(HCMV)感染了超过一半的退伍军人，并威胁到患有 免疫系统受损。人巨细胞病毒是导致出生缺陷的主要感染原因。没有人巨细胞病毒 疫苗，而抗病毒药物在效力、毒性和耐药性方面存在问题。长距离的 这项研究的目标是确定病毒转录-DNA复制循环中的关键点， 作为治疗干预的新靶点。这项提议是基于这样一个前提，即我们在 了解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这种病毒疾病的新治疗方法的能力。 通过定制先进技术和开发新工具，我们使用了集成的功能 基因组学(Dtag系统、Pro-Seq、ChIP-Seq、基因工程测试病毒和启动子功能 分析)以确定POL II何时何地启动转录，识别病毒转录因子的位点 全基因组结合，并量化单个启动子的POL II新生转录本的变化 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 病毒晚期转录有三条不同的途径。其中有两条途径涉及人巨细胞病毒 IE2和晚期转录因子(LTF)组成员。3种不同的IE2各自的作用 病毒晚期转录中的异构体(IE2-86、IE2-60和IE2-40)尚不清楚。LTFS的六人组 结合到POL II和基因启动子中的DNA序列签名，形成预起始复合体(PIC)，该复合体 推动转录。序列签名模式的多样性可能决定了个体的数量 促进剂输出。目前尚不清楚LTF复合体何时以及如何组装在病毒启动子和 LTF组件如何与POLII在转录中接合。我们使用了一种新的高分辨率芯片-序列技术 和生物信息学流水线，以绘制核小体的基因组位置，以及IE2和LTF PIC， 提示LTFPIC占据了未被核小体占据的基因组区域。这款新芯片--Seq 技术将加强我们的集成功能基因组学方法，以进一步确定 控制病毒启动子转录的机制与染色质结构有关。我们的初步数据 结果表明：1)病毒DNA复制的早期-晚期转录转换滞后于病毒DNA复制的开始； 2)激活的HCMV启动子群体成员因细胞类型和条件不同而不同，这 差异可能涉及IE2和LTF功能；以及3)HCMV启动子群体在 病毒在NT2模型中的重新激活与在急性生产性感染中的不同。我们将检验这一假设 HCMV转录因子篡夺宿主Pol II，该宿主导航适合于 导致病毒晚期转录(目标1和2)，并协调不同类型的病毒转录程序 细胞类型(目标2)以及在支持NT2中静止和重新激活感染的细胞条件下 模型(目标3)。我们将对每个具体目标应用多方面的方法：1)阐明 早期-晚期转录开关的调节，2)决定细胞类型差异的机制基础 3)确定静止期感染的激活如何改变病毒转录。我们的 Proposal分别整合了Meier和Price实验室在病毒学和转录方面的专业知识。 我们将在这种富有成效的合作的基础上完成拟议的研究计划。这些发现 这些研究将确定基因调控的可概括特征，这些特征与其他 贝塔和伽马疱疹病毒亚家族的成员，包括人类疱疹病毒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.