卡波氏肉瘤相关疱疹病毒感染神经元细胞的特点与致病性研究

The disease caused by Kaposi’s Sarcoma-associated Herpesvirus (KSHV) has become the major cause of death for AIDS. Xinjiang is a province that has both high infection rate of KSHV and HIV. We have found that KSHV can infect the neuron cells of patients. The difference with the other cells that can be successfully infected by KSHV is viruses in neuron cells can active lytic state by themselves. These cells may become the reservoir pool of KSHV in central nervous system (CNS) and cause the persistently infection of neuron cells. The relative characteristics and pathogenicity need to do further research. We plan to sort the neuron cells with lytic viruses and compare them with the neuron cells with latent viruses as well as uninfected cells. Use RNA-sequence method to analysis the character genes for lytic state of neuron cells. Analyze the supernate KSHV copy number and titration, cell number, KSHV copy number in cells and expression level of LANA,RTA, K8.1 in different time point. At the same time detect the combine ability of LANA and RBP-Jκ, methylation level of RTA promoter in the cells. Use the RNA interference to silence the expression of the character genes. And then use KSHV virus to infect the neuron cells again. Try to detect the difference of lytic rate and other characters. At last, use patch clamp to analyze the electrophysiology characters of the cells. These researches will help to understand the effection of KSHV CNS infection, and lay the foundation for better diagnosis and treatment of the KSHV infected nervous system diseases.

卡波氏肉瘤相关疱疹病毒(KSHV)感染引发的疾病已成为艾滋病最常见的致死原因。新疆是我国KSHV和HIV的高感染区。我们发现KSHV可感染患者神经元细胞，与其他细胞不同的是此类细胞可自发激活潜伏态病毒进入裂解态，这些细胞可能形成中枢神经系统KSHV的储存池造成神经元持续感染，其特征和致病性需深入研究。我们拟分选裂解态神经元细胞，与潜伏态和未感染细胞作对比，进行转录组测序，分析裂解态细胞的特征基因，并检测不同时间细胞培养液病毒载量、滴度、细胞数目、细胞病毒载量和LANA、RTA、K8.1表达等特点，同时检测LANA与RBP-Jκ结合能力、RTA启动子甲基化水平；干扰神经元细胞特征基因表达后，再测病毒感染后裂解态细胞比例和特点变化；最后通过全细胞膜片钳检测细胞动作电位变化，说明KSHV感染对神经元细胞的影响。本研究将为揭示KSHV感染对中枢神经系统造成的影响提供依据，也为此类疾病诊疗奠定基础。

卡波氏肉瘤相关疱疹病毒 (KSHV) 感染引发的疾病是艾滋病（AIDS）最常见的死因。AIDS 患者易感 KSHV且常出现神经系统症状，我们前期研究发现患者脑组织中有KSHV感染。为进一步研究KSHV感染神经细胞的特点、确定特征基因并进行功能与调控机制分析，我们开展了此研究。我们采用KSHV感染了神经细胞SH-SY5Y，通过流式细胞技术分选了裂解态和潜伏态细胞，发现自发裂解的细胞占细胞总数的1.2%，分选后不同时间点检测细胞培养液病毒载量、细胞增殖、细胞内病毒载量和病毒基因LANA、RTA和K8.1的表达，发现裂解态细胞可存活并能产生有感染性的病毒，感染滴度为5.12×103 TCID50/ml，该细胞既能表达裂解态基因K8.1、RTA，也能表达潜伏态基因LANA，并循环往复的出现裂解态和潜伏态交替，感染细胞的增殖能力增强，我们提出KSHV感染的神经细胞以循环裂解和潜伏的方式逃避杀伤并持续释放病毒的理论。进一步通过转录组测序分析特征基因，发现Notch1通路在裂解感染细胞中异常活化，转录因子c-Fos表达上调，免疫共沉淀实验证明Notch1通路的RBP-Jκ(CSL)与病毒潜伏态蛋白LANA结合互作，c-Fos与裂解态分子开关RTA结合互作，RTA启动子区甲基化程度降低，这些涉及病毒裂解态激活的机制。干扰特征基因Notch1、c-Fos表达使病毒载量下降，病毒基因LANA、RTA、K8.1表达抑制，细胞增殖能力下降，通过Notch1抑制剂、激活剂及双荧光素酶报告基因实验证实Notch1下游基因Hes1通过c-Fos启动子区调控其表达。通过转录组测序分析上游调控miRNA，发现miR-34a-5p表达下调且是调控c-Fos的重要非编码RNA，过表达miR-34a-5p、同时过表达c-Fos的Rescue实验和裸鼠动物模型证明miR-34a-5p通过干预c-Fos间接调控病毒载量、基因表达及细胞增殖。全细胞膜片钳检测发现KSHV感染后细胞神经冲动动作电位发放频率上升。这些研究说明Notch1和c-Fos在KSHV感染的神经细胞中发挥重要作用，miR-34a-5p是控制KSHV感染及抑制致病性的候选小分子核酸药物，进一步研究将有重要应用前景，这对于今后开展抗KSHV研究具有重要意义。