The role of latency-associated viral miRNAs during human cytomegalovirus latent infection

人巨细胞病毒（HCMV）在CD34+祖细胞及其单核细胞衍生物中建立潜伏感染。我们之前的研究表明，潜伏感染细胞中细胞基因表达的主要变化是由一组潜伏相关病毒基因编码的病毒蛋白表达引起的。虽然我们已经确定了潜伏相关病毒蛋白的许多功能，但对病毒编码的mirna或长链非编码rna在潜伏感染期间的作用知之甚少-然而，已知HCMV编码至少17个成熟mirna和3个长链非编码rna （lncRNA）。我们最近分析了潜伏感染期间的病毒miRNAome，并在使用原代骨髓细胞和已建立的骨髓细胞系的潜伏模型中发现了大量高水平表达的病毒miRNAs。其中之一是病毒miRNA UL22A。初步分析已经确定UL22A的一个靶点是磷酸肌肽3-激酶（PI3K）的p110delta亚基。本项目将分析ul22a介导的p110delta下调对潜伏感染的建立和维持的影响，以帮助确定病毒特异性靶向这种细胞激酶的基本原理。重要的是，对急性髓性白血病的研究表明，p110delta水平低的细胞对拓扑异构酶抑制剂（如依托泊苷）诱导细胞死亡的敏感性增加，这预示着这些抑制剂可能靶向潜伏感染的细胞。因此，该项目不仅旨在了解潜伏感染期间p100delta下调的作用，而且将这种病毒miRNA表达对细胞的影响转化为针对潜伏的新疗法-这是我们正在进行的研究的关键目标。同样，我们也将分析病毒4.9kb lncRNA的作用（如果有的话），该lncRNA被认为参与维持潜伏期间病毒裂解基因表达的抑制。我们将建立HCMV潜伏期模型，其中4.9kb lncRNA的表达被敲低，以确定缺乏该病毒基因潜伏载体的表达的影响。

Human cytomegalovirus (HCMV) establishes a latent infection in CD34+ progenitor cells and their monocyte derivatives. We have previously shown that major changes occur in cellular gene expression in latently infected cells due to expression of viral proteins encoded by a sub-set of latency-associated viral genes. Whilst we have identified a number of functions of latency-associated viral proteins, little is known about the role of virally encoded miRNAs or long non-coding RNAs during latent infection - yet, HCMV is known to encode at least 17 mature miRNAs and 3 long non-coding RNAs (lncRNA). We have recently analysed the viral miRNAome during latent infection and identified a number viral miRNAs expressed to high levels in latency models using both primary myeloid cells as well as established myeloid cell lines.One of these is the viral miRNA UL22A. Initial analyses have identified one target of UL22A to be the p110delta subunit of Phosphoinositide 3-kinase (PI3K).This project will analyse the effect of UL22A-mediated down-regulation of p110delta on the establishment and maintenance of latent infection to help identify the rationale for viral-specific targeting of this cellular kinase.Importantly, studies of acute myeloid leukaemia have shown that cells with low levels of p110delta display increased sensitivity to induction of cell death by topoisomerase inhibitors such as etoposide and this predicts that latently infected cells could be targeted by such inhibitors. Consequently, the project will not only aim to understand the role of p100delta down-regulation during latent infection but translate the effects of expression of this viral miRNA, on the cell, to novel therapies to target latency - a key aim of our ongoing research.Similarly, we also will analyse the role, if any, of the viral 4.9kb lncRNA which has been postulated to be involved in maintenance of repression of viral lytic gene expression during latency. We will generate models of HCMV latency in which expression of the 4.9kb lncRNA is knocked down to establish the effect of lack of expression of this viral gene latent carriage.