MOLECULAR ASPECTS OF CYTOMEGALOVIRUS LATENCY

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目的长期目标是了解巨细胞病毒（CMV）在宿主中持续存在的细胞和分子机制。CMV是种特异性病毒，在初次感染后在宿主中建立持续/潜伏感染。这种能力需要避免免疫检测并限制对携带病毒的细胞的细胞病变效应。疱疹病毒通过限制大部分病毒基因组的表达来实现这一点，从而减少急性复制并最小化抗原蛋白呈递的可能性，同时保持在后期再活化的能力。疱疹病毒限制自身基因表达的过程，从而促进持续或潜伏状态，还不清楚，但可能涉及细胞和病毒因素。最近发现的RNA干扰和microRNA的广泛表达揭示了一个新的转录后基因调控层，这是以前未知的。许多研究表明，很大比例的转录本可能是由miRNA靶向和调节的，表明小RNA的调节可能代表了基因表达控制中的关键机制。我们自己的小组和其他人的研究已经确定了由多种DNA病毒编码的70多个miRNA基因，其中大多数已经在疱疹病毒家族中被确定。疱疹病毒的所有三个亚组，α，β和γ，已被证明在急性复制和潜伏期期间表达miRNA。虽然对病毒编码的miRNA的功能知之甚少，但它们调节多种转录物的潜在能力和缺乏免疫原性应答使miRNA成为促进和维持持续或潜伏病毒基因表达谱的理想候选物。此外，该提案中提出的初步数据表明至少一种HCMV miRNA在限制病毒急性复制中可能起作用。在生物信息学研究之后，我们已经鉴定了HCMV miRNA UL 112 -1的许多潜在的病毒靶转录物。这些靶点包括尿嘧啶DNA糖基化酶基因，其与UL 112 -1直接反义，并被miRNA直接切割，以及主要反式激活蛋白IE72，其通过信使RNA的3' UTR内的靶序列进行转录后调节。在这个提议中，我们将扩展HCMV UL 112 -1在急性感染和持续感染期间的表征。此外，我们将确定和表征其他病毒编码的miRNA的HCMV基因靶点，并分析它们在急性感染期间对病毒调控的贡献。最后，我们将研究HCMV miRNA在单核细胞、巨噬细胞和持续感染的内皮细胞中的表达和功能。阐明miRNA调控HCMV基因表达的机制将为疱疹病毒及miRNA研究领域的整体研究提供重要贡献。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of cytomegalovirus (CMV) persistence in the host. CMV is a species-specific virus that establishes a persistent/latent infection in the host after primary infection. This ability requires avoidance of immune detection and limitation of cytopathic effect on cells harboring virus. Herpesviruses achieve this by restricting expression of the majority of the viral genome, thereby reducing acute replication and minimizing the potential for presentation of antigenic proteins, while maintaining the ability to reactivate at a later stage. The process by which herpesviruses restrict their own gene expression, thereby promoting a persistent or latent state, is not well understood, but is likely to involve both cellular and viral factors. The recent discovery of RNA interference and the widespread expression of microRNAs has uncovered a new layer of post-transcriptional gene regulation that was previously unknown. A number of studies have suggested that a large percentage of transcripts may be targeted and regulated by miRNAs, indicating that regulation by small RNAs may represent a crucial mechanism in the control of gene expression. Studies by our own group and by others have identified over 70 miRNA genes encoded by multiple DNA viruses, the majority of which have been identified within the herpesvirus family. All three subgroups of herpesviruses, alpha, beta and gamma, have been shown to express miRNAs during both acute replication and during latency. Although relatively little is known about the function of virally encoded miRNAs, their potential ability to regulate multiple transcripts and the lack of an immunogenic response make miRNAs ideal candidates for the promotion and maintenance of a persistent or latent viral gene expression profile. Furthermore, preliminary data presented in this proposal suggests a possible role for at least one of the HCMV miRNAs in restricting acute replication of the virus. Following bioinformatics studies we have identified a number of potential viral target transcripts of the HCMV miRNA UL112-1. These targets include the uracil DNA glycosylase gene, which resides directly antisense to UL112-1 and is directly cleaved by the miRNA, and the major transactivating protein IE72, which is regulated post-transcriptionally via a target sequence within the 3' UTR of the messenger RNA. In this proposal we will extend the characterization of HCMV UL112-1 during acute infection and during persistent infection. In addition we will identify and characterize the HCMV gene targets of the other virally encoded miRNAs and analyze their contribution to regulation of the virus during acute infection. Lastly we will examine the expression and function of the HCMV miRNAs in monocyte macrophages and persistently infected endothelial cells. Elucidating the mechanisms of miRNA regulation of HCMV gene expression will provide an important contributions herpesviruses and miRNA field as a whole.