HBV前S缺失突变对病毒复制力及表面抗原启动子Ⅱ转录活性的下调作用

摘要

目的：分析HBV前S缺失突变病毒株体外复制力及其对表面抗原启动子（surface antigen promoter, SP）Ⅱ转录活性的影响。方法研究对象为119例解放军第三〇二医院的住院患者，包括38例慢性乙型肝炎（慢乙肝）轻中度、40例慢乙肝重度和41例慢加急性肝衰竭。从患者血清中提取HBV DNA，PCR扩增HBV全长基因组，统计前S缺失突变的发生率。挑选代表前S缺失突变株及其相应对照的HBV全长序列克隆至pGEM-Teasy载体中。用BspQⅠ/ScaⅠ双酶切1.0倍HBV基因组，转染HepG2细胞，72 h后检测病毒复制力；用PCR分别扩增含前S缺失突变型和野生型的HBV SPⅡ启动子片段，构建pGL3-SPⅡ双荧光素酶真核报告表达载体，转染HepG2细胞48 h后检测相对荧光素酶活性，分析前S1缺失突变对重叠的SPⅡ荧光素酶表达的影响。结果①HBV基因组前S缺失突变检出率在慢乙肝轻中度、慢乙肝重度和慢加急性肝衰竭3组中逐渐递增，分别为5.3%、12.5%和24.4%，差异有统计学意义（P<0.05）；②前S缺失突变病毒株的复制力较相应野生株降低69%；③与野生型相比，前S1缺失突变使重叠的SPⅡ转录活性降低了36%。结论 HBV前S缺失突变发生率随乙肝进展而升高，前S缺失突变株复制力降低，导致重叠的SPⅡ转录活性降低。%Objective To evaluate the effect of HBV genome pre-S deletion mutation on the viral replication capacity and the transcriptional activity of the overlapping surface antigen promoterⅡ(SPⅡ) in vitro. Methods A total of 119 patients admitted to 302 Hospital of PLA were enrolled in the study, including 38 patients with mild or moderate chronic hepatitis B (CHB-M), 40 patients with severe chronic hepatitis B (CHB-S), and 41 patients with acute-on-chronic liver failure (ACLF). Serum HBV DNA was isolated and full-length HBV genome was amplified. The incidence of pre-S deletion mutation was analyzed. The representative full-length HBV genomes containing pre-S deletion and the counterpart wild-type virus were cloned into pGEM-Teasy vector. The 1.0-ploid full-length HBV genome was released from reconstruction plasmid by BspQ I /Sca I double digestion, and then transfected into HepG2 cells. Intracellular HBV core particles were measured 72 hours post-transfection for analyzing the replication capacity. The overlapping HBV SPⅡregion with the deletion and the counterpart wild-type virus was amplified by PCR, respectively, and the dual luciferase reporter plasmids pGL3-SPⅡwas constructed. The relative luciferase activity was measured 48 hours post-transfection in HepG2 cells. The impact of the deletion in the overlapping SPⅡ on the expression of the downstream luciferase reporter gene was further analyzed. Results The incidence of pre-S deletion mutation was 5.3%, 12.5% and 24.4% in CHB-M, CHB-S and ACLF patients, respectively, which increased in a stepwise order with statistical significance (P<0.05). The replication capacity of pre-S deletion mutant and transcriptional activity of the overlapping SPⅡwere decreased by 69% and 36%, respectively. Conclusions The incidence of HBV pre-S deletion increases along with the disease progress. The mutant virus with pre-S deletion decreases viral replication capacity, and pre-S deletion in the overlapping SPⅡregion reduces the transcriptional activity of the SPⅡ.