The Study For The Impact Of Hepatitis B Virus Pre-S1Deletion On The Viral Biological Activity

Objective: This study aimed to1) investigate the incidence of hepatitis Bvirus (HBV) pre-S deletion mutation in patients with mild or moderate chronichepatitis B (CHB-M), severe chronic hepatitis B (CHB-S) and acute-on-chronicliver failure (ACLF)；2) evaluate the influence of HBV genome pre-S deletionmutation on the viral replication capacity, secreted HBsAg levels, HBV RNAlevels, pre-S1expression levels and the transcriptional activity of the overlappingsurface promoterⅡ (SPⅡ) of HBV in vitro. Methods: A total of119patients whoadmitted to our hospital from August2005to May2008were enrolled in the study,including38patients with CHB-M,40patients with CHB-S, and41patientswith ACLF. HBV DNA was isolated from serum samples, and the incidence ofpre-S deletion mutation was calculated based on full-length HBV sequencing ofthe amplified HBV DNA. The representative full-length HBV genomes containingpre-S1deletion and their wild-type counterparts were cloned into pGEM-Teasyvector. The1.0-ploid full-length HBV genome was released from BspQ I/Sca Idouble digestion the recombinant plasmid, and then transfected into HepG2cells.Intracellular HBV core particles were measured72hours post-transfection foranalyzing the in vitro replication capacity and secreted HBsAg levels. HBV RNAlevels in transfected HepG2cells were analyzed using the relative fluorescencequantitative assay72hours post-transfection.1.1-ploid pTriEX-HBV (C) expre- ssion vector was constructed and transfected into HepG2cells. Pre-S1antigenexpression was measured by ELISA72hours post-transfection. The overlappingHBV SPⅡ regionwith the deletion and the wild-type counterpart was amplified,respectively, and the dual luciferase reporter plasmids pGL3-SPⅡ was constructed.The relative luciferase activity was measured48hours post-transfection in HepG2cells. The impact of the deletion in the overlapping SPⅡ on the expression of thedownstream luciferase reporter gene was further analyzed. Results:1) Theincidence of pre-S deletion was5.3%,12.5%and24.4%in CHB-M, CHB-S andACLF patients, respectively, which increased in a stepwise order with statisticalsignificance (P <0.05).2) The replication capacity and secreted HBsAg level ofpre-S1deletion mutant and transcriptional activity of the overlapping SPⅡ weredecreased by69%,23.7%, and36%compared to wild-type virus without deletion,respectively. The levels of the3.5kb pregenome RNA,2.4kb mRNA, and the2.1kb mRNA reduced by87.67%，91.40%，and85.94%of the wild-type HBVrespectively.3) Compared to wild-type HBV without deletion, pre-S1deletionleaded to loss of pre-S1antigen expression.4) Compared to wild-type HBVwithout deletion, pre-S1deletion mutant reduced the transcriptional activity of theoverlapping SP II promoter by36%in HepG2cells. Conclusion: The incidence ofHBV pre-S deletion increased along with the disease progression. The mutantvirus with pre-S deletion had decreased viral replication capacity, secreted HBsAgand RNA levels, causing the loss of pre-S1antigen, as well as reducing thetranscriptional activity of the overlapping SPⅡ. The resμlts suggest that pre-S1deletion of HBV had significance both in clinic and in virology.