激活肝X受体抑制人巨细胞病毒颗粒成熟的机制研究

Human cytomegalovirus (HCMV) is a severe pathogen for immunocompromised individuals, such as AIDS patients and organ transplant recipients; and is also the most common and harmful agent causing congenital infection and birth defects..Our preliminary studies showed that HCMV growth was remarkably inhibited by the activation of liver X receptor (LXR). In human embryonic lung fibroblasts (HELF) treated with LXR agonist GW3965, viral titers were reduced over 1000 folds during late infection; however, viral DNA replication was only reduced several folds. Meanwhile, mRNA levels of viral genes, including UL122, UL77, UL82, UL86, UL99 and UL117, were sharply reduced in HCMV-infected HELFs treated with GW3965. Based on these results, it can be speculated that the activated liver X receptor may inhibit the HCMV packaging and maturation processes by regulating transcriptions of virus genes, which are closely related to virus maturation, and, hence reduce the amounts of infectious particles..To understand the inhibition mechanism of activated liver X receptor on maturation and infectivity of virus particles, we plan to study the effects of liver X receptor activated by its agonist, GW3965, on maturation and infectivity of virus particles, identify the viral target genes that are regulated by the activated liver X receptor, discover the inhibition mechanism of the activated liver X receptor on transcriptions of the virus target genes, and demonstrate the inhibition effects of virus target genes on virus titers. Finally, discover the inhibition mechanism of the activated liver X receptor on maturation and infectivity of virus particles..The accomplishment of this project is critical for understanding HCMV pathogenesis, and will provide a clearer view of host-virus interaction and a potential target for development of new anti-HCMV therapy.

人巨细胞病毒（HCMV）感染危害严重。预实验证实，激活肝X受体（LXR）显著抑制HCMV病毒滴度达千倍以上，但仅抑制病毒基因组拷贝数数倍，在激活LXR的感染细胞中HCMV UL122、UL77、UL82、UL86、UL99和UL117等在病毒颗粒组装成熟及感染能力方面起关键作用的病毒基因的mRNA含量显著降低。推测，激活的LXR可能通过转录调控机制调控与病毒颗粒组装成熟密切相关的病毒基因转录而抑制病毒颗粒的成熟过程，减少病毒颗粒的成熟数量和抑制病毒颗粒的感染能力。为验证这一假设，拟采用LXR激动剂GW3965激活LXR，研究激活LXR对病毒颗粒成熟数量及感染能力的影响；鉴定出受激活LXR转录调控的病毒靶基因；阐明激活LXR抑制病毒靶基因转录的机制；明确抑制病毒靶基因对于病毒滴度的影响；最终阐明激活LXR抑制HCMV病毒颗粒成熟的机制，为进一步研究以LXR为靶向的抗HCMV药物提供理论依据。

人巨细胞病毒（HCMV）感染危害严重。本项目预实验证实，激活肝X受体(LXR)显著抑制HCMV病毒滴度达千倍以上，但仅抑制病毒基因组拷贝数数倍。同时，在激活LXR的感染细胞中HCMV UL122、UL77、UL82、UL86、UL99和UL117等在病毒颗粒组装成熟及感染能力方面起关键作用的病毒基因的mRNA含量显著降低。推测，激活的LXR可能通过转录调控机制调控与病毒颗粒组装成熟密切相关的病毒基因转录而抑制病毒颗粒的成熟过程，减少病毒颗粒的成熟数量和抑制病毒颗粒的感染能力。为验证这一假设，拟采用LXR激动剂GW3965激活LXR，研究激活LXR对病毒颗粒成熟数量及感染能力的影响；鉴定出受激活LXR转录调控的病毒靶基因；阐明激活LXR抑制病毒靶基因转录的机制；明确抑制病毒靶基因对于病毒滴度的影响；最终阐明激活LXR抑制HCMV病毒颗粒成熟的机制，为进一步研究以LXR为靶向的抗HCMV药物提供理论依据。.本项目采用病毒滴定的方式证实了激活LXR的病毒感染细胞产生的子代病毒的滴度下降近千倍；通过电子显微镜技术发现激活LXR主要影响子代病毒的包膜化过程，产生大量的缺陷病毒；病毒的缺陷主要体现在病毒包膜的脂质含量显著减少，其表面的一些结构蛋白的含量也相对减少，导致病毒包膜结构异常、感染性下降。通过研究，证实了激活LXR对于子代病毒基因组DNA合成具有一定影响；通过双荧光素酶报告实验证实了激活的LXR对MIEP的启动活性具有一定的抑制作用，激活的LXR还抑制了包括UL83基因在内的一些病毒重要基因的转录，对于一些具有重要作用的病毒蛋白表达也具有一定影响。通过构建稳定表达带有标签LXR的细胞系，进而在LXR激活的条件下进行了co-IP和CHIP捕获，分别进行了LXR结合蛋白的飞行质谱分析和结合基因组DNA的测序分析。有关研究证实了激活LXR对HCMV子代病毒颗粒成熟数量和感染活性的影响和机制，基于现有工作的进一步研究将为建立有效低毒的抗病毒治疗方法提供重要的理论基础。