DMSO促进HepG2细胞感染乙型肝炎病毒后核心蛋白入核

Dimethyl sulphoxide facilitates the nuclear entry of hepatitis B virus core protein in infected HepG2 cells

目的:探讨DMSO诱导处理的HepG2细胞被HBV感染的作用机制.方法:将HepG2分为DMSO处理组和对照组,分别用添加或不添加2%DMSO的DMEM培养基培养4 d.将HBV病毒颗粒(1000拷贝/细胞)加入培养基中于37℃感染12h.以蛋白免疫印迹,免疫荧光染色及共聚焦显微镜观察HBcAg在细胞内的定位.选择性PCR检测HBV cccDNA,流式细胞分析仪检测细胞周期.结果:对照组HepG2细胞在感染HBV阳性血清后,HBcAg在细胞质内分布,核内呈阴性,至感染后2 d细胞内核心蛋白消失,未检出明显的cccDNA信号.2%DMSO处理后的HepG2细胞感染后12 h细胞质内HBcAg水平明显高于对照组,感染后24 h HBcAg在核周浓集.在感染后24 h和48 h核内HBcAg明显增高,且cccDNA结果阳性.流式分析结果显示DMSO处理后处于G_1/G_0和G_2/M期的HepG2比例升高,处于有丝分裂期的HepG2细胞内HBcAg弥散分布于全细胞.结论:HepG2细胞感染HBV后核心颗粒不能穿过核孔携带HBV DNA进入细胞核可能是其抵制HBV感染的重要原因.DMSO可促进HBV核心颗粒进入细胞核而有助于感染.

AIM： To study the effect of diInethyl sulphoxide on the nuclear entry of hepatitis B virus core protein in infected HepG2 cells. METHODS： In the DMSO and control groups, HepG2 cells were cultured in DMEM with or without 2% DMSO for 4 d. Then the cells were incubated with HBV （1000 copies per cells） for 12 hours at 37℃. Hepatitis B core antigen （HBcAg） in the infected HepG2 cells was stained by fluorescent immunocytochemistry and observed by confocal microscopy. HBcAg in the cytoplasm and nucleus was respectively extracted and analyzed by Western blotting. HBV covalently closed circular DNA （cccDNA） was detected by ploymerase chain reaction, and cell cycle was analyzed by flow cytometry. RESULTS： In control HepG2 cells, HBcAg was mostly expressed in cytoplasm and almost absent from the nucleus, and it disappeared from the cells at 2 days post infection （PI）. No obvious sign of cccDNA was detected in the control group at 2 days. After DMSO administration, compared with that of control group, a higher level of HBcAg was detected in the cytoplasm at 12 hours PI. HBcAg was accumulated around the nucleus at 24 hours PI. The level of HBcAg in the nucleus obviously increased and cccDNA was present at 24 and 48 hours PI. Flow cytometry showed that DMSO increased the ratio of G1/G0 and G2/M phases of HepG2 cells. HBcAg was distributed throughout the cells in mitosis phase HepG2 cells. CONCLUSION： In HepG2 cells, blocking the entry of core particles to the nucleus might be an important reason for the cells being refractory to HBV infection. DMSO promotes nuclear entry of core protein/core particles and then facilitates HBV infection.