潜伏相关基因UL138在人巨细胞病毒潜伏－激活感染中的调控作用

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Transplacental transmission during pregnancy or neonatal infection of premature newborns can lead to neurological damage. Primary infection of healthy individuals with HCMV is usually asymptomatic and results in the establishment of a lifelong latent infection of the host. Both primary and latency-activation infection of HCMV during gestation pose a risk of intrauterine transmission. Given the extremely high seropositive rate in pregnant women, latency-activation infection seems to play a more important role in congenital HCMV infection. UL138 located in HCMV ULb' region is a newly-found latency-associated gene expressing during both experimental and natural latency. However, the definite mechanisms by which UL138 contributes to HCMV latency-activation infection remain poorly understood. In this study, a model of HCMV latency based on CD14+ monocyte will be constructed for exploring the regulatory functions of UL138 in latency-activation. The transcription and expression levels of UL138 in this model will be compared to those in human embryonic lung fibroblast with lytic infection of HCMV. Furthermore, the UL138 expression level will be up-regulated or down-regulated by the transfection of a vector expressing recombinant UL138 protein or small interfering RNAs specifically targeting UL138 cDNA. The levels of HCMV immediate-early antigens IE1 and IE2 as the hallmarks of reactivation will be detected after the up/down-regulation of UL138 expression to determine the transformation of HCMV infectious states. Simultaneously, the changes of cellular morphology, differentiation antigens, and expression levels of TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), IL-1β (interleukin-1β), and TGFβ1 (transforming growth factor β1) in culture supernatants from the latency model will be observed to evaluate the effect of UL138 expression on monocyte differentiation. The regulatory function of UL138 in HCMV latency-activation revealed in this study will be helpful to the further exploration of the pathogenesis and treatment of congenital HCMV infection.

人巨细胞病毒（HCMV）是先天性感染最常见的病原体，其潜伏－激活的感染特性可能在先天性感染发病机制中起关键作用。UL138是HCMV重要的潜伏相关基因，但其在潜伏－激活中的确切调控作用和机制还不清楚。本项目拟首先建立实验室HCMV潜伏感染模型，并以增殖性感染细胞为对照，研究该模型中UL138基因的转录和表达情况。然后通过转染UL138蛋白表达载体上调或特异性siRNA下调UL138的表达水平，观察HCMV感染状态的变化，阐明UL138表达在HCMV潜伏－激活过程中的确切调控作用；同时监测UL138表达上/下调后，潜伏感染细胞形态、分化抗原、以及与细胞分化相关的细胞因子TNF-α、IL-6、IL-1β和TGFβ1表达水平的变化，评价UL138对潜伏感染细胞分化的影响。本项目将有助于深入阐明HCMV潜伏－激活感染的发生机制，为先天性HCMV感染的临床防治奠定基础。

人巨细胞病毒（HCMV）是先天性感染最常见的病原体，其潜伏-激活的感染特性可能在先天性感染发病机制中起关键作用。UL138 是HCMV重要的潜伏相关基因，但其在潜伏-激活中的确切调控作用和机制还不清楚。本项目在建立实验室HCMV潜伏感染的THP-1细胞模型的基础上，通过转染UL138基因过表达或表达干扰的慢病毒载体调控UL138的表达水平，监测UL138表达水平对HCMV感染状态的影响，从而阐明UL138在HCMV潜伏-激活感染中的确切作用；同时检测HCMV潜伏感染的THP-1细胞的形态、分化抗原以及分化相关细胞因子TNF-α、IL-6、IL-1β、TGF-β1水平等方面，揭示UL138对潜伏感染的单核细胞分化的影响。研究结果显示：(1) 与不转染慢病毒载体的对照组比较，UL138表达水平上调后THP-1细胞中的HCMV DNA拷贝数减少，病毒复制的关键基因IE表达降低；UL138表达水平下调后THP-1细胞中的HCMV DNA拷贝数增加，IE基因表达升高，表明UL138基因对HCMV潜伏-激活感染的调控作用是促进或维持病毒的潜伏感染。(2) 与不转染慢病毒载体的对照组比较，UL138表达水平上调后THP-1梭形细胞增多，标志单核细胞未向树突细胞分化的细胞表面抗原CD14表达下降，TGF-β1蛋白水平升高；UL138表达水平下调后标志单核细胞分化的细胞表面抗原CD45表达下降，表明UL138基因表达与HCMV潜伏感染的单核细胞的分化呈正相关，并可能通过TGF-β1信号通路起作用。本项目如期、圆满完成所有研究计划，研究成果包括已发表的SCI收录论文2篇、已接收待发表的中文综述1篇、全国性学术会议口头报告论文1篇和壁报交流论文2篇，培养博士研究生1名。本项目为HCMV潜伏-激活感染机制的深入阐明以及先天性HCMV感染的临床防治奠定了基础。