Down-regulation effect of HBV genome pre-S deletion mutation on viral replication capacity and the transcriptional activity of the overlapping surface antigen promoterⅡ

MIAO Fu-zhen; XU Zhi-hui; LIU Yan; LI Qi; LI Xiao-dong; YAO Wei-ming; XIN Shao-jie; LI Jin; XU Dong-ping

Abstract：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.