Elucidation of HCMV latent viral genome maintenance/replication factors

描述（由申请方提供）：人巨细胞病毒（HCMV）是一种普遍存在的疱疹病毒，是免疫受损个体发病和死亡的主要原因。HCMV经历裂解性感染，其特征在于涉及整个基因组的转录激活，导致感染性病毒的产生。HCMV与所有疱疹病毒一样，与其宿主建立终身潜伏期。病毒潜伏期的标志是在没有立即早期基因表达的情况下产生有限数量的转录物，以及潜伏感染细胞中病毒基因组的持续存在。HCMV潜伏期显示发生在髓样细胞（CD 34+）和单核细胞（CD 14+）中。迄今为止，几乎没有什么是已知的机制控制HCMV潜伏期。虽然一些潜伏相关的转录本被确定，这些因素的关系，以维持和/或复制的病毒染色体在潜伏感染的细胞尚未建立。该提议的长期目标是鉴定直接有助于病毒1基因组的维持或复制的病毒编码因子，使得可以开发策略以从细胞中清除潜伏病毒。本申请的目的是定义介导病毒基因组维持/复制的顺式作用病毒序列，并阐明调节病毒基因组持久性的反式作用因子。中心假设是病毒顺式作用序列和反式作用因子介导病毒染色体在潜伏期期间的持久性。该假设是基于申请人实验室中生成的初步数据制定的。拟议研究的基本原理是，对病毒基因组维持机制的理解有可能导致更好的治疗HCMV相关疾病的疗法。在强有力的初步数据的指导下，该假设将通过追求3个具体目标进行检验：1）阐明IE 1和其他病毒和细胞因子在潜伏感染的CD 34（+）细胞中维持HCMV病毒基因组中的作用。我们最近发表的数据在实验和自然潜伏感染的CD 14（+）和CD 34（+）细胞中鉴定了潜伏相关转录本。这些转录本中的一些编码先前显示参与DNA复制和病毒DNA稳定性的因子。我们还发现，含有病毒基因组末端（TR）重复区的质粒在潜伏感染的CD 14（+）单核细胞以及CD 34（+）造血细胞中持续存在。2)开发瞬时复制试验，以确定维持TR元件所需的特定病毒DNA基序。亚克隆的TR元件将用于开发瞬时测定，以评价必需的顺式作用序列。第三个目标将探讨反式作用因子在病毒基因组维护中的作用，使用TR质粒作为报告维持/复制。该方法是创新的，因为它利用RNA-Seq和核小体消耗数据来鉴定有助于HCMV潜伏期的基本顺式和反式作用因子。这项研究意义重大，因为我们有望阐明HCMV潜伏期的机制，并可能导致治疗感染的新方法。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is a major cause of morbidity and mortality in immune compromised individuals. HCMV undergoes a lytic infection, which is characterized by transcriptional activation that involves the entire genome leading to the production of infectious virus. HCMV, like all herpesviruses, establishes a life-long latency with its host. Viral latency is marked by the production of a limited number of transcripts in the absence of immediate early gene expression and the persistence of the viral genome in latently infected cells. HCMV latency was shown to occur in myeloid cells (CD34+) and monocytes (CD14+). To date, almost nothing is known about the mechanisms controlling HCMV latency. Although a few latency-associated transcripts were identified, the relationship of these factors to maintenance and/or replication of the viral chromosome in latently infected cells are not established. The long-term goal for this proposal is to identify viral encoded factors that directly contribute to maintenance or replication of the virl genome such that strategies can be developed to purge the latent virus from cells. The objective for this application is to define the cis acting virus sequence(s) that mediate maintenance/replication of the virus genome, and elucidate the trans acting factors that regulate viral genome persistence. The central hypothesis is that viral cis acting sequences and trans acting factors mediate the persistence of the viral chromosome during latency. This hypothesis was formulated based on preliminary data generated in the applicants' laboratory. The rationale for the proposed research is that the understanding of the mechanism(s) involved in maintenance of the viral genome has the potential to lead to better therapies for the treatment of HCMV-related disease. Guided by strong preliminary data, this hypothesis will be tested by pursuing 3 specific aims: 1) Elucidate the role of IE1 and other viral and cellular factors in maintenance of the HCMV viral genome in latently infected CD34 (+) cells. Our recently published data identified latency associated transcripts in experimentally and naturally latently infected CD14 (+) and CD34 (+) cells. Some of these transcripts encode factors previously shown to be involved in DNA replication and viral DNA stability. We also show that a plasmid containing the terminal (TR) repeat region of the virus genome persists in latently infected CD14 (+) monocytes as well as CD34 (+) hematopoietic cells. 2) Develop transient replication assays to define specific viral DNA motifs required for maintenance of the TR element. The subcloned TR element will be used to develop a transient assay to evaluate essential cis acting sequences. The third aim will explore the role of trans acting factors in viral genome maintenance using the TR plasmid as a reporter for maintenance/replication. The approach is innovative because it utilizes RNA-Seq and nucleosome depletion data to identify essential cis and trans acting factors that contribute to HCMV latency. The proposed research is significant because it is expected that we will elucidate the mechanism involved in HCMV latency and potentially lead to novel approaches to treat infection.