Roscovitine抑制HBV复制的机制研究  

Mechanism of Roscovitine in inhibiting HBV replication

作　　者：胡杰[1] 陈彦猛[1] 乔淼[1] 袁琳[2] 周星[1] 胡源[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,400016 [2]重庆医科大学基础医学院,400016

出　　处：《中华微生物学和免疫学杂志》2017年第7期487-491,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（81471945）;重庆市科委自然科学基金项目（cstc2014jcyjA10075）;重庆市教委科学技术研究项目（KJ1600205）

摘　　要：目的研究周期蛋白依赖性蛋白激酶（cyclin．dependentkinase，CDK）抑制剂Roscovi-tine抑制HBV复制的分子机制。方法首先通过构建SAMHDI蛋白dNTPase活性位点及T592磷酸化位点的突变体研究磷酸化负调控抑制作用；接着在肝癌细胞Huh7．0细胞中加入不同浓度的Rosco-vitine．通过MTr法检测了Roscovitine化合物对细胞毒性的影响；转染HBV复制质粒后，提取病毒核心颗粒DNA．通过southeITIblot检测了Roscovitine对HBV的抑制作用，Western blot检测SAMHD1蛋白的磷酸化水平。结果在增殖细胞中，SAMHD1蛋白dNTPase活性突变体D207N丧失了对HBV的抑制作用；T592位点的去磷酸化增强了SAMHD1对HBV的抑制作用；Roscovitine的半数毒性浓度（TC50）为11．20μmoL／L，Roscovitine可以显著抑制SAMHD1的磷酸化和HBV的复制。结论在增殖细胞中，SAMHD1蛋白T592位点的去磷酸化增强了其对HBV的抑制作用，Roseovitine可以通过抑制SAMHD1的磷酸化后抑制HBV的复制。Objective To investigate the mechanism of Roscovitine, an inhibitor of cyclin-de- pendent kinase （CDK）, in inhibiting HBV replication. Methods Recombiant expression plasmids of SAMHD1 （ sterile alpha motif and histidine/aspartie acid domain-containing protein 1 ） mutants that were de- fective in dNTPase （deoxynucleoside triphosphate triphosphohydrolase ） activity and phosphorylation at the threonine （T） 592 residue were constructed. HuhT. 0 cells were respectively co-transfected with different SAMHDI mutants in combiantion with HBV replication plasmid to analyze whether the retroviral restriction ability of SAMHD1 was regulated by phosphorylation. The cytotoxicity of Roscovitine to Huh7 cells was eval- uated by MTY assay. HBV core-associated DNA levels and phosphorylation of SAMHD1 in transfected Huh7.0 cells which were treated with different concentrations of Roscovitine were measured by Southern blot and Western blot assays. Results The SAMHD1 mutant that was defective in the dNTPase active site of D207N lost its ability to restrict HBV replication. Dephosphorylation of SAMHD1 at T592 enhanced its re- striction on HBV. The median toxic concentration （ TC50 ） of Roscovitine was 11.20 μmol/L. Both the HBV core-associated DNA levels and the phosphorylation of SAMHD1 were down-regulated by Roscovitine. Con- clusion The anti-HBV function of SAMHD1 in dividing cells is regulated by phosphorylation. Roscovitine can inhibit the replication of HBV through reducing the phosphorylation of SAMHD1.

关 键 词：ROSCOVITINE 乙型肝炎病毒 SAMHD1 磷酸化作用 抑制 

分 类 号：R373[医药卫生—病原生物学]