HBV基因组A1846T变异对病毒复制力及核心启动子活性的上调作用研究

Up-regulation effect of hepatitis B virus genome A1846T mutation on viral replication and core promoter activity

目的评价乙肝病毒(HBV)基因组核苷酸(nt)A1846T变异对病毒体外复制力及核心启动子(CP)转录活性的影响。方法 385例研究对象包括116例轻中度慢性乙肝(CHB-M)患者,123例重度慢性乙肝(CHB-S)患者和146例慢加急性肝衰竭(ACLF)患者。从患者血清中提取HBV DNA,PCR扩增HBV全长基因组,统计A1846T变异的发生率。挑选代表性A1846T变异株HBV全长序列克隆至pGEM-Teasy载体中,并通过定点突变获得野生型对照。BspQⅠ/ScaⅠ双酶切HBV基因组,转染HepG2细胞,检测病毒复制力和HBsAg表达水平;用PCR分别扩增含nt1846变异型和野生型的HBVCP区片段,构建pGL3-CP双荧光素酶真核报告表达载体,转染HepG2细胞,分析检测A1846T变异对荧光素酶表达的影响。结果 HBV A1846T变异发生率随疾病程度加重依次增高,CHB-M、CHB-S和ACLF患者的变异检出率分别为31.03%、42.27%和55.48%(P<0.01)。A1846T变异株的复制力、分泌的HBsAg水平和核心启动子活性较野生株分别提高了320%、28%和85%,常见的CP区A1762T/G1764A双联变异株分别较野生株提高了67%、9%和72%,A1846T变异对提高病毒复制力的影响更强。结论 A1846T变异可显著增加HBV复制力,提高HBsAg表达水平,增强启动子活性,顺式激活下游基因转录表达,提示该变异可能与慢性乙肝重症化发生机制相关。

Objective To evaluate the influence of hepatitis B virus（HBV） genome nucleotide A1846T mutation on the viral replication capacity and the transcription activity of HBV core promoter（CP） in vitro.Methods A total of 385 patients with hepatitis B admitted to the 302 Hospital of PLA were enrolled in the study,including 116 with moderate chronic hepatitis B（CHB-M）,123 with severe chronic hepatitis B（CHB-S）,and 146 with acute-on-chronic liver failure（ACLF）.Serum HBV DNA was isolated and full-length HBV genome was amplified.The incidence of A1846T was analyzed.Full-length HBV genomes containing 1846T mutation were cloned into pGEM-T easy vector,and the counterpart wild-type 1846A plasmids were obtained by site-directed mutagenesis.The full-length HBV genome was released from recombinant plasmid by BspQ Ⅰ/Sca Ⅰ digestion,and then transfected into HepG2 cells.Secreted HBsAg level and intracellular HBV core particles were measured 72 hours post-transfection to analyze the replication capacity（a 1.0-fold HBV genome model）.1846 mutant and wild-type full-length HBV genomes were extracted to amplify the fragment of HBV CP region,and the dual luciferase reporter of the pGL3-CP was constructed.The luciferase activity was detected 48 hours post-transfection.Results The incidence of A1846T mutation gradually increased with the severity of hepatitis B,reaching 31.03%,42.27%,and 55.48% in CHB-M,CHB-S and ACLF patients respectively（P0.01）.The replication capacity of 1846T mutants,level of secreted HBsAg,and transcriptional activity of CP promoter were increased by 320%,28% and 85% respectively,compared with 1846A wild-type strains.While the more common double mutation A1762T/G1764A in CP region was increased by 67%,9% and 72% respectively,compared with its counterpart wild-type strains.A1846T had a greater influence on viral replication capacity in vitro.Conclusions A1846T mutation could significantly increase the replication capacity of hepatitis B virus,secretion of HBsAg and transcription activity of CP promoter,and cis-activate the downstream gene transcription.The finding indicates that HBV genome A1846T mutation might play a role in liver disease progression.