Effect Of Hepatitis B Virus Basal Core Promoter Mutations On The Transcriptional Cis-activating Function And Virus Replication Capacity

Hepatitis B is a worldwide infectious disease caused by hepatitis B virus (HBV) and is closely related to the development of the hepatic failure, liver cirrhosis and hepatocelluar carcinoma. HBV is the highly variable virus because the polymerase/reverse transcriptase(RT) is error prone in replicating its virus genome. Conservative internal basal core promoter(BCP) sequence of HBV genome is essential for maintaining viral replication and mutations in BCP region would influence viral replicative capacity and reduce the synthesis of the hepatitis B e antigen(HBeAg), leading to different clinical outcomes. Currently, the hot mutations in BCP regions contain T1753A/C, A1762T, G1764A, C1766T etc. The most common is double mutations, such as A1762T/G1764A and C1766T/T1768A, as well as multi-mutation A1762T/G1764A/C1766T and T1753C/A1762T/G1764A/C1766T. In this study, we investigated the effect of the BCP mutations on promoter activity and HBV genome replication capacity through statistical analysis of HBV genome sequnces from 40 patients with chronic hepatitis B(CHB) and 40 patients with acute-on-chronic liver failure(ACLF) enrolled in PLA 302 Hospital, and screened samples containing G1764A/C1766T/T1768A triple mutations. The main results of this study are as follows:Firstly, the clinical features analysis showed that HBeAg positive rate and HBV DNA load were significantly higher and total bilirubin(TBIL) level was significantly lower in CHB patients than those in ACLF patients (P<0.05), indicating that the above factors may associated with the severity of hepatitis B.Secondly, the full-length genome cloning analysis from 80 clinical samples and frequency analysis of the hot mutations in BCP region demonstrated that the frequency of G1764A/C1766T/T1768A triple mutation was significantly higher in ACLF patients (5/40, 12.5%) than that in CHB patients (0/40, 0.0%) (P<0.05), suggesting the possible correlation of the triple mutation with the severity of hepatitis B.Thirdly, HBV genotyping was performed based on phylogenetic tree analysis according to HBV complete sequence by MEGA 4.0 software. 9 cases out of 40 CHB patients was infected with HBV/B viruses (9/40, 22.5%); 30 cases with HBV/C viruses (30/40, 75.0%) and one case with HBV/D virus (1/40, 2.5%). 8 cases out of 40 ACLF patients was infected with HBV/B viruses (8/40, 20.0%); 31 cases with HBV/C viruses (31/40, 77.5%) and one case with HBV/D virus (1/40, 2.5%). There was no significant difference in genotype distribution between CHB and ACLF patients.Fourthly, the recombinant luciferase reporter vectors pGL3-BCP containing the BCP double and triple mutants were successfully constructed. Then site-directed mutagenesis was performed to generate counterpart wild-type virus. The results of dual-luciferase assay showed that the expressions of luciferase in HepG2 cells transfected with BCP A1762T/G1764A double mutants and BCP G1764A/C1766T/T1768A triple mutants were 1.67-fold and 1.43～1.80-fold higher than those with BCP counterpart wild-type viruses on average, respectively,suggesting that the BCP double and triple mutation could up-regulate promoter activity.Lastly, 1.0-fold HBV genome with BCP double and triple mutations and counterpart wild-type virus generated by site-direct mutagenesis was introduced into HepG2 cells. Then the real-time quantitation PCR was performed and the results showed that HBV DNA replication in HepG2 cells transfected with BCP A1762T/G1764A double mutants and BCP G1764A/C1766T/T1768A triple mutants were 1.56-fold and 1.54～2.49-fold higher than those with BCP counterpart wild-type viruses on average, respectively, suggesting that the double and triple mutation can increase the level of HBV DNA replication.Taken together, we cloned and analyzed complete HBV genome sequences and found BCP G1764A/C1766T/T1768A triple mutations might be associated with severity of hepatitis B which could upregulate the promoter activity and virus replication capacities. The findings will contribute to understand the characteristics and clinical significance of HBV BCP triple mutation and clarify the molecular virological mechanism of severity of hepatitis B.