Hepatitis B virus core protein as a Rab-GAP suppressor driving liver disease progression.

Chronic hepatitis B virus (HBV) infection can lead to advanced liver pathology. Here, we establish a transgenic murine model expressing a basic core promoter (BCP)-mutated HBV genome. Unlike previous studies on the wild-type virus, the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age. Notably, agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose. As the BCP mutant exhibits a striking increase in HBV core protein (HBc) expression, we posit that HBc is actively involved in hepatocellular injury. Accordingly, HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15). HBc may also inhibit multiple Rab GTPase-activating proteins, including Rab7-specific TBC1D15 and TBC1D5, by binding to their conserved catalytic domain. In cells under mitochondrial stress, HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria. Moreover, sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation, which further hampers late-stage autophagy and substantially increases apoptotic cell death. Finally, we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury, independent of antigen-specific immune clearance. These findings reveal an unexpected cytopathic role of HBc, making it a pivotal target for HBV-associated liver disease treatment. The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies.

Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses.

Background & Aims: HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs. Methods: The replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs. Results: Patient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C. Conclusions: The infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening. Lay summary: The hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients.

AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes. METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed. RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ (2) = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ (2) = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively. CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Relationship between hotspot mutations in basic core promoter of hepatitis B virus and familial clustering of hepatocellular carcinoma in Guangxi / 实用医学杂志

Objective To explore the relationship between mutations in basic core promoter (BCP) of hepatitis B virus (HBV) and familial clustering of hepatocellular carcinoma (HCC) in Guangxi. Methods 153 pairs of members with HBsAg-positive were selected and matched from HCC high-incidence families and carcinoma-free families in Guangxi. The BCP genes were amplified and sequenced. Results The hotspot sites of the previous five mutations in BCP were T1762, A1764, G1775, V1753, G1803. In univariant analysis, HBV DNA≥105 copies/mL, T1762, A1764 and V1753 mutations were associated with the HCC high-incidence (P <0.05). The multivariate logistic analysis showed that HBV DNA≥105 copies/mL and A1764 were independent risk factors for it. Conclusion HBV DNA level, the mutations in BCP showed correlations with familial clustering of HCC in Guangxi.

The association of primary liver cancer with the mutations in basic core promoter and precore genes of hepatitis B virus / 中华临床感染病杂志

Objective To investigate the association of primary liver cancer(PLC)with the mutations of HBV precore and basic core promoter(BCP)genes.Methods The serum markers of hepatitis B and the quantities of serum HBV DNA were detected in 144 HBsAg-positive PLC patients.The precore and BCP gene mutations in patients with HBeAg-negtive and HBV DNA-positive were detected by real-time PCR.One hundred and twenty chronic hepatitis B(CHB)patients were randomly selected to serve as the conol.Results There were 46(3 1.94%)patients with HBeAg-positive and 98(68.06%)patients with HBeAg-negative.In 98 HBeAg-negative patients,56(57.14%)were HBV DNA-positive,in which 43 (76.79%)were with precore 1896 gene mutations,50(89.29%)were with BCP1762/1764 gene mutations.and 38(67.86%)were with both gene mutations.Precore 1896 and BCP1762/1764 gene mutation rates in PLC patients were much higher than those in CHB patients(χ2=9.36 and 5.77,P＜0.05).Conclusion PLC may be associated with the mutations of HBV precore anti BCP genes.

Correlation of Hepatitis B Virus Basic Promoter Mutation with IL-10,IL-12,TNF-?,IFN-? as well as HBV DNA Contents in Chronic Hepatitis / 中华医院感染学杂志

OBJECTIVE To study the correlation of hepatitis B virus(HBV)basic core promoter(BCP) mutation with the interleukins(IL-10,IL-12),tumor necrosis factor(TNF)-?,interferon(IFN)-?,as well as HBV DNA contents in patients with chronic hepatitis B virus infection.METHODS(1) Project subject: 176 patients(chronic hepatitis B with mild,moderate and severe degree;liver cirrhosis,chronic fulminant and hepatocellular carcinoma(HCC)) with chronic hepatitis B virus infection were studied.(2) Project methods: ① The A to T mutation at nucleotide 1762 and G to A mutation at nucleotide 1764 were determined by the method of polymerase chain reaction(PCR) microplate hybridization ELISA in these patients.② The serum cytokines(IL-10,IL-12,TNF-? and IFN-?) of these patients were measured by specific-ELISA.RESULTS The serum levels of cytokines(IL-10(80.96?30.86 vs 72.11?24.19 mg/L),IL-12(41.33?15.10 vs 35.98?14.47 mg/L),TNF-?(56.04?27.05 vs 38.01?10.49 mg/L),IFN-?(19.81?12.29 vs 16.55?8.99 mg/L))and HBV DNA contents(108.2478?0.9826 vs 105.8876?1.4822copies/ml) in BCP mutant group were significantly higher than that in non-mutant group(P

The association of hepatitis B virus precore/basic core promoter mutations with genotype and progression of liver disease / 中华消化杂志

Objective To study association of hepatitis B virus(HBV) precore (pre c)/basic core promoter(BCP) mutations with the genotype or the progression of liver disease. Methods The serum samples from 148 patients with HBV-relative diseases were collected, including 31 asymptomatic carriers, 32 with chronic hepatitis B (CHB), 40 with liver cirrhosis(LC) and 45 with hepatocellular carcinoma(HCC). The genes covering HBV pre c and BCP were amplified by nested polymerase chain reaction (nPCR). The PCR products were subjected to direct sequencing and the mutations in pre c 1896 and BCP 1762/1764 were determined by sequence analysis. HBV genotypes were also detected in the sera by restriction fragment length polymorphism based on S-gene PCR products. Results Of 148 serum samples of HBV, 128 were successfully genotyped and sequenced. There were 60 genotype B and 68 genotype C. The mutation in pre c (A1896) was significantly higher in genotype B than in genotype C (48.3% vs 29.34%, P

The influence of hepatitis B virus basic core promoter/precore mutations on adv ance of liver disease / 中国实用内科杂志

Objective To investigate the relationship between hepatitis B virus (HBV) basic core promoter(BCP)/precore(PreC) mutations and severity of liver dis ease. Methods In 113 patients chronically infected with HBV, do uble mutations in BCP(T1762/A1764) and PreC mutation(A1896) were determined by INNO-LiPA and HB V genotype was determined by S gene sequencing. Results Whether in all patients or in patients infected by single genotype C, compared with AsC, the prevalence of double mutations in BCP(T1762/A1764) was higher in patients with CHB, LC and HCC[(24.1% vs 2.8%,? 2=5.93, P0.05). Conclusions The doubl e mutations in BCP(T1762/A1764 ) may be related to progressive liver disease in patients with chronic HBV infec tion.

Detection of double-position mutation in the basic core promoter of hepatitis B virus with microplate sandwich hybridization / 中华传染病杂志

Objective To investigate 1762T、1764A double position mutation in the Basic Core Promoter(BCP) of hepatitis B virus and reveal its relation to clinical symptoms and HBeAg phenotype. Methods microplate sandwich hybridization technique was used to detect BCP double position mutation. One common capture probe and one mutant specific detector probe as well as one wild type detector probe were synthesized and hybridized with amplified HBV DNA from the sera of hepatitis B patients. The results of hybridization were exhibited with ELISA. Results 147 hepatitis B patients who had been confirmed HBV DNA positive were screened. 51 patients were BCP double position mutation, 42 of which were BCP single position mutation and, 9 were mix mutation(both mutation and wild type were positive). BCP mutant was detected in 36 of 117 with chronic hepatitis and, 8 of which were mix mutants. Moreover, BCP mutant was detected in 7 of 30 with acute hepatitis in 25 of 78 with HBeAg positive were mutant and in 26 of 65 with HBeAg negative were mutant.Conclusions (1) The rate of BCP double position mutation in chronic hepatitis B patients is higher than that in acute hepatitis B patients. (2) BCP mutation may impair HBeAg expression. (3) PCR microplate sandwich hybridization ELISA is a sensitive and efficient method for detecting gene mutations.