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In vitro expression of precore proteins of hepatitis B virus subgenotype A1 is affected by HBcAg, and can affect HBsAg secretion.

ABSTRACT: HBeAg, a non-particulate protein of hepatitis B virus (HBV), is translated from the precore/core region as a precursor, which is post-translationally modified. Subgenotype A1 of HBV, which is a risk factor for hepatocellular carcinoma (HCC), has unique molecular characteristics in the basic core promoter/precore regions. Carriers of A1 exhibit early HBeAg loss. We sought to further characterize the precore proteins of A1 in vitro. HuH-7 cells were transfected with subgenomic constructs expressing individual precore proteins. Western blot analysis using DAKO anti-core antibody showed the expected sizes and a 1 kDa larger band for P22, P20 and P17. Using confocal microscopy, a cytoplasmic accumulation of HBeAg and precursors was observed with P25-expressing plasmid, whereas P22 localized both in the cytoplasm and nucleus. P20 and P17, which lack the carboxy end of P22 showed strong nuclear accumulation, implicating a nuclear localization signal in the N-terminal 10 amino acids. G1862T, unique to subgenotype A1, is frequently found in HBV from HCC patients. P25 with G1862T showed delayed and reduced HBeAg expression/secretion. Knock-out of core in the replication competent clones led to precore protein accumulation in the cytoplasm/perinuclear region, and decreased HBeAg secretion. Knock-out of precore proteins increased HBsAg secretion but intracellular HBsAg expression was unaffected. Over-expression of precore proteins in trans led to decreased HBsAg expression and secretion. Intracellular trafficking of HBV A1 precore proteins was followed. This was unaffected by the CMV promoter and different cell types. In the viral context, precore protein expression was affected by absence of core, and affected HBsAg expression, suggesting an interrelationship between precore proteins, HBcAg and HBsAg. This modulatory role of HBeAg and its precursors may be important in viral persistence and ultimate development of HCC.

SUBMITTER: Deroubaix A

PROVIDER: S-EPMC8046783 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimTo molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.MethodsSera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05.ResultsIrrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D.ConclusionKerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.

Project description:AIM:Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-alpha), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS:HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS:We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind III and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION:These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Project description:Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities.IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

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In vitro expression of precore proteins of hepatitis B virus subgenotype A1 is affected by HBcAg, and can affect HBsAg secretion.

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