人巨细胞病毒miR-UL112-1初级转录结构及转录调控机制的研究

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects a broad range of cell types in its human host, contributing to its complex and varied pathogenesis. It is one of the main pathogens causing congenital infection and birth defects. The pathogenic mechanism of HCMV congenital infection still remains unclear at present. microRNAs (miRNAs) are an abundant class of small non-coding RNA molecules that regulate gene expression at the posttranscriptional level. Recent studies have shown that HCMV is the only human β-herpesvirus found to express miRNAs. The important finding provides a new avenue of the investigation for HCMV pathogenic mechanism. .It is conceivable that HCMV encoded miRNAs are exploited during virus immune evasion.The major histocompatibility complex class 1-related chain B (MICB) has been identified as a functional target of HCMV miR-UL112-1. miR-UL112-1-mediated down-regulation of MICB perturbed MICB binding with NKG2D and reduced killing of HCMV infected cells by NK cells. .In our previous research, HCMV miR-UL112-1 has been furhter proven to play an important role in HCMV immune evasion by targeting mRNA sequences of interleukin 32 (IL-32) and NFkB activating protein. In addition, it was confirmed for the first time that HCMV miR-UL112-1 possessed alternative transcripts in different length. It is deduced from our results that the expression of HCMV miR-UL112-1 is regulated through different pathways according to its different transcript structures to maintain its immunological specificity and adaptability in host cells..Current miRNA researches are mainly focused on its biological functions and few has been done in its upper stream-the structure and transcriptional regulation of miRNA primary transcript.Our study will investigate the pathogenic mechanism of HCMV from a new level of virus-encoded miRNA primary transcript structure and transcriptional regulation. it will be of great significance in the prevention and therapy of HCMV infection.

人巨细胞病毒（HCMV）是引起先天感染和出生缺陷的主要病原体之一，可造成多系统多器官疾病和畸形，危害巨大。HCMV先天感染的致病机理尚不清楚。近年研究发现，HCMV是唯一具有编码并表达microRNA功能的人类β疱疹病毒，这一重要发现为我们对HCMV致病机理的深入研究提供了全新的思路。在前期研究中，我们在进一步证明HCMV miR-UL112-1具有调节宿主细胞多种免疫因子表达的重要功能的基础上，首次证实 miR-UL112-1具有不同片段长度的多种初级转录形式，由此提示miR-UL112-1的表达可因初级转录本结构的不同而存在多种不同的转录调控方式，以实现其在病毒特定感染时期的特异性表达及病毒对不同免疫状态宿主的适应性。本研究将从病毒编码microRNA的初级转录结构及转录调控机制这个全新的层面深入解读HCMV的致病机理，对预防及治疗HCMV感染，进而减少HCMV致畸儿出生具有重要意义。

人巨细胞病毒（HCMV）感染是引起出生缺陷和新生儿疾病的重要因素之一。HCMV编码的microRNA-UL112-1（miR-UL112-1）与病毒复制及免疫逃逸密切相关。本项目应用RACE、RT-PCR等方法对HCMV miR-UL112-1的转录情况进行研究。结果表明，miR-UL112-1在病毒感染即刻早期即进行转录，转录起始位点位于miR-UL112-1颈环结构上游148bp处。应用荧光素酶报告基因检测技术在miR-UL112-1初级转录结构上游DNA序列中确定了其转录的核心启动序列，增强子序列的范围。结合酵母单杂交技术及生物信息学预测，筛选并预测了与增强子序列结合的转录因子IRF3。应用电泳迁移率实验(EMSA)对增强子序列与转录因子IRF3的结合能力进行了鉴定，证明转录因子IRF3与增强子序列的体外结合能力。本研究从基因转录水平研究了HCMV miR-UL112-1的表达特点与调控机制，发现并证明了miR-UL112-1基因的增强子序列及其结合的转录调控因子，对进一步了解其转录调控特点及生物学功能具有重要意义。