人巨细胞病毒潜伏感染相关基因UL138及LUNA转录调控机制研究

Infection of human cytomegalovirus (HCMV) in immunocompromised patients is a significant cause of morbidity and mortality. HCMV represents a major cause of disease in transplant patients, AIDs sufferers and newborns. The main source of HCMV infection in these patients is from reactivation of latent virus during special period, such as pregnancy. Knowledge about the mechanism of formation, maintenance and reactivation of latent HCMV infection, and establishment of powerful methods for inhibiting HCMV reactivation are key steps for prevention of HCMV infection. In 2007, Felicia Goodrum et al firstly reported that HCMV UL138 ORF was required for HCMV to establish and/or maintain a latent infection in hematopoietic progenitor cells infected in vitro [Blood, 2007, 110: 937-945]. In 2005, Mariana Bego et al reported a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product [J. Virol., 2005, 79(17); 11022-11034]. And the latent gene transcript UL81-82ast was named as latency unique natural antigen (LUNA) [Arch Virol. 2011, 156:1847-1851]. So far, it has been confirmed that the UL138 and LUNA genes were highly expressed during HCMV latent infection, and the expressions were closely associated with formation and maintenance of HCMV latent infections [J. Virol., 2009, 83(11); 5615-5629; PLoS Pathogens, 2011, 7(12); e1002444; J. Virol., 2011; 85(21): 11409-11421; J. Virol. 2012; 86(16): 8653-62; J. Gen. Virol., 2010; 91: 599-604]. Although two reports have discussed the putative promoter of UL138 and LUNA during productive infection[J. Virol., 2010, 84(18): 9472-9486; J.Virol., 2010, 84(14); 7185-7194;], and one recent report has demonstrated that LUNA is regulated by the myeloid transcription factor GATA-2[J. Virol. 2013, PMID: 23365437], it is still unclear about the transcriptional mechanism of the UL138 and LUNA genes. In this study, we plan to analyze the transcriptional regulation elements of the UL138 and LUNA genes, such as promoter, enhancer and inhibitor, identify the related transcription factors and transcriptional regulation factors corresponding to the transcriptional regulation elements of the UL138 and LUNA genes, and detect the different distributions of these factors between virus sensitive cells (human embryo lung fibroblast) and latently infected cells (CD34+ hematopoietic progenitor cells, HPCs, or Kasumi-3 cells) [J. Virol, 2012; 86(18): 9854-9865]. The aim of this study is to find the UL138 and LUNA related transcription factors and transcriptional regulation factors, which have determinant role on HCMV latency in latently infected cells, and to study the effects of expression changes of these factors on virus infection stages (productive or latent infections). This project will accumulate data for understanding the mechanism of HCMV latent infection, and help to establish a powerful prevention means for inhibiting HCMV reactivation. It should have both theological and actual meanings for HCMV preventions.

人巨细胞病毒先天感染危害巨大，并引起免疫抑制患者致死性感染。其危害主要来自于潜伏病毒在孕期等特定时期的再激活。搞清病毒潜伏状态维持及再激活机制并建立有效的病毒再激活控制手段是预防人巨细胞病毒危害的关键。目前，已经证明人巨细胞病毒潜伏感染过程中UL138及LUNA基因处于高表达状态，并与病毒潜伏感染的形成与维持密切相关。但UL138及LUNA基因在潜伏感染过程中高表达的转录调控机制尚不清楚。本项目拟通过分析UL138及LUNA基因的转录调控元件、相关转录因子及转录调控因子、以及这些因子在病毒敏感细胞与潜伏感染细胞中分布的差异，找出在潜伏感染细胞中针对病毒潜伏感染形成与维持起决定作用的UL138及LUNA基因的转录因子及转录调控因子，进一步研究这些因子在不同细胞中的表达改变对病毒感染状态改变的作用。本项目将为阐明人巨细胞病毒潜伏感染机制、建立有效的潜伏病毒再激活干预手段积累资料，具有重要意义。

人巨细胞病毒（HCMV）先天感染危害巨大，并可引起免疫抑制患者致死性感染。其危害主要来自于孕期和免疫抑制期等特定时期潜伏病毒的再激活。搞清病毒潜伏状态维持及再激活机制并建立有效的病毒再激活控制手段是预防HCMV危害的关键。目前，已经证明HCMV潜伏感染过程中UL138及LUNA基因处于高表达状态，并与病毒潜伏感染的形成与维持密切相关。但UL138及LUNA基因在潜伏感染过程中高表达的转录调控机制尚不清楚。本项目拟通过分析UL138及LUNA基因的转录调控元件、鉴定参与基因转录调控的相关转录因子、分析相关转录因子在病毒敏感细胞与潜伏感染细胞中分布的差异、进一步研究调控UL138及LUNA基因的转录因子对病毒感染状态的作用。通过研究，本项目鉴定出了HCMV UL138基因上游调控区启动子、增强子和抑制子序列；明确了在病毒感染过程中转录因子AP-1参与了UL138基因的转录调控。证实了在HELF细胞中LUNA基因不存在强的启动子，在转录起始位点上游133bp内有一个弱启动子，在523-653bp内有一个弱增强子。在293细胞中LUNA基因在转录起始位点上游393-523bp存在一个正调控序列。通过人工染色体技术成功构建了替换LUNA 转录起始端33bp序列的LUNA基因转录缺失的病毒株；证实了在HCMV可形成潜伏感染的THP-1细胞和产毒性感染的HELF细胞中存在REL，NFATC2，MEF2D，IRF5，ELF1等5种表达量差异显著的转录因子。进而，本项目尝试了在THP-1细胞中进行HCMV感染潜伏模型的建立、并成功进行了THP-1细胞的诱导分化和在人外周血中成功分离了CD34+和CD14+的HCMV潜伏感染细胞。有关研究明确了UL138和LUNA基因的转录调控元件，鉴定了相关转录因子及其对UL138转录的影响。为全面了解HCMV潜伏感染维持和潜伏再激活的发生机制积累了资料，有助于进一步针对HCMV潜伏感染和潜伏再激活建立治疗和控制手段。