T98G细胞模型中人巨细胞病毒潜伏感染的激活机制

Congenital human cytomegalovirus infection is the most common cause of birth defects, primarily manifest as brain development disorders. Ten to fifteen percent (10%-15%) of subclinical infected newborns gradually develops the late-onset maldevelopment of brain, including mental retardation, sensorineural hearing loss, mental retardation, which is highly associated with HCMV latent infection. Latent infection and its reactivation of herpesviruses is a key scientific question. So far, latent infection and its reactivation in the nervous system haven’t been reported, and the treatments, conditions and mechanisms for HCMV reactivation from latency remain unclear. In the past a few years, we have found the HCMV latently infected neural cell model, T98G glioblastoma cell model, and confirmed that the infected cells maintain high copy numbers of HCMV genomes and last for a long time. In the study, we will utilize the HCMV latent infection T98G cells to specify the mechanism(s) for reactivation from latency. Different drugs and conditions will be applied to reactivate latent HCMV, and virus infection status will be monitored to confirm the reactivation. The differential expression of viral and cellular proteins will be investigated by comparative proteomics between the latent and reactivated T98G cells to dig out the key candidates of viral and cellular proteins. The function of the interested proteins which involved in the regulation of viral replication, cell cycle and cell differentiation will be further studied by overexpression and knockdown the targets. This project will serve to find out the key targets for clearance of latent HCMV, elucidate the mechanism(s) for reactivation of HCMV latency, and finally to help control congenital HCMV infection.

先天性人巨细胞病毒（HCMV）感染是导致出生缺陷最常见病因，表现为脑发育异常。10%-15%亚临床型感染儿在孩童时期逐渐出现神经发育迟缓、神经性耳聋等迟发性脑发育异常，与潜伏HCMV的激活密切相关。HCMV在神经系统中的潜伏与激活条件及其机制尚未见报道。前期我们发现T98G细胞可作为HCMV的潜伏感染模型，并证实感染细胞持续携带高拷贝的HCMV基因组。本项目拟在此细胞模型的基础上，用候选药物处理HCMV潜伏感染细胞，明确激活潜伏HCMV的药物和条件；通过比较蛋白质组学，分析HCMV潜伏与激活状态下差异表达的细胞和病毒蛋白，筛选影响HCMV感染状态的目标蛋白；进一步过表达或敲降目标蛋白，明确其对病毒复制及与复制相关的细胞特性的调控。旨在明确HCMV潜伏与激活的关键靶标并阐释其机制，为清除潜伏的HCMV提供关键药物靶点。对预防和干预先天性HCMV感染、降低出生缺陷和实施优生优育具有深远意义。

先天性人巨细胞病毒（HCMV）感染是导致出生缺陷最常见病因，表现为脑发育异常。10%-15%亚临床型感染儿在孩童时期逐渐出现神经发育迟缓、神经性耳聋等迟发性脑发育异常，与潜伏HCMV的激活密切相关。HCMV在神经系统中的潜伏与激活条件及其机制尚未见报道。前期我们发现T98G细胞可作为HCMV的潜伏感染模型，并证实感染细胞持续携带高拷贝的HCMV基因组。在此细胞模型的基础上，本项目为实时观察HCMV的潜伏激活情况，利用携带GFP标签的重组HCMV毒株（rHCMV），重建rHCMV潜伏感染的T98G细胞模型（T98G-LrV）。并筛选出cAMP/IBMX可诱导T98G-LrV中GFP信号的增多、病毒基因组复制、病毒裂解基因的表达、病毒粒子的释放等，证实T98G细胞可支持HCMV的潜伏与激活。.通过iTRAQ比较蛋白质组学分析T98G、T98G-LrV、T98G-LrV（cAMP/IBMX），共鉴定3092个蛋白质，定量分析2283个蛋白。将实验分为两组，“潜伏组”为T98G-LrV/T98G，“激活组”为T98G-LrV（cAMP/IBMX）/T98G-LrV。将p＜0.05，且比值大于1.3设为表达上调、小于0.77的为表达下调；共成功鉴定168个差异表达的蛋白，其中潜伏组89个（31个上调、58个下调）、激活组85个（63个上调、22个下调）。生物信息学分析发现，潜伏组差异蛋白主要涉及核糖体和昼夜节律通路，激活组差异蛋白主要涉及蛋白降解和吸附、ECM受体相互作用等。该项目初步揭示了T98G细胞中HCMV潜伏激活所涉及的信号通路，为揭示HCMV潜伏激活分子机制，清除潜伏HCMV寻找关键药物靶点奠定了基础。