A double-spliced defective hepatitis B virus genome derived from hepatocellular carcinoma tissue enhanced replication of full-length virus.

In hepatitis B virus (HBV) replication cycle, pregenomic RNA undergoes splicing and the reverse transcribed defective genomes can be packaged and released. Various types of spliced defective HBV genomes have been isolated from the sera and liver tissues of viral hepatitis B patients. To explore the functions of a 2.2 kb double spliced HBV variant, a 3.2 kb full-length HBV isolate (#97-34) and its 2.2 kb double-spliced HBV variant (#AP-12) from the tumor tissue of a patient with hepatocellular carcinoma (HCC) were amplified and cloned. Sequencing results showed that #AP12 had deletions in pre-S2, part of pre-S1, S genes, part of the spacer, and part of the reverse transcriptase gene, while the X gene was intact. When this defective double-spliced genome and its full-length counterpart genome were co-transfected into HepG2 cells, the former was shown to enhance the replication of the latter, both by real-time PCR and Southern blotting. When a replication incompetent clone 97-34G1881A was used to co-transfect with #AP12, #AP12 DNA was increased, indicating that replication of the wild-type virus was not the only factor involved in this observation. However, the replication enhancing competency of #AP12 was shown to require an intact HBV X expression cassette. The double-spliced defective variant might contribute to persistent HBV replication in a subpopulation of HCC patients.

Mutational analysis revealed that conservation of hepatitis B virus reverse transcriptase residue 306 (rtP306) is crucial for encapsidation of pregenomic RNA.

Hepatitis B virus (HBV) is a DNA virus which replicates via reverse transcription. The structure and function of the reverse transcriptase play important roles in HBV replication. We have previously reported that when proline at residue 306 in HBV reverse transcriptase was substituted by other amino acids, most of the mutants showed decreased replicative competency. To explore the mechanisms for this decrease in replicative competency, constructs with substituted amino acid residues at rtP306 were used to transfect Huh-7 cells, and replication competencies, transcription levels and encapsidation efficiencies of the mutants and the parental viral strain were compared. Decreased replication competency was found with many of the mutants and confirmed by trans-complementation between each mutant and a replication-defective replicon. No change in transcriptional level was detected between all mutated constructs. The encapsidation competencies of these constructs were studied by assaying pregenomic RNAs in intracytoplamic core particles from transfected cells, which were normalized for the amount of HBV core protein by Western blotting using anti-core antibodies. Impaired encapsidation was found in several mutants substituted at residue 306, thereby demonstrating for the first time that conservation of proline at this residue is crucial for efficient encapsidation of pregenomic RNA.

Replicative efficiency and pathogenicity of hepatitis B virus e-minus precore variant.

To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti-hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild-type virus infection, A1896 variant infection and dual infection. Higher titre of anti-HBe was found in patients with no virus replication and in patients coinfected with the wild-type virus and A1896 variant, which suggest that anti-HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site-directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.

Role of hepatitis B surface antigen in the development of hepatocellular carcinoma: regulation of lymphoid enhancer-binding factor 1.

BACKGROUND: There are around 350 million of hepatitis B surface antigen (HBsAg) carriers worldwide, and among them, high risk of developing hepatocellular carcinoma (HCC) has been identified by epidemiological studies. To date, the molecular role of HBsAg in HCC development has not been fully studied. We have previously reported that in cell cultures, HBsAg up-regulated the expression of lymphoid enhancer-binding factor 1 (LEF-1), a key component of the Wnt pathway. In this study we aimed to study this effect of HBsAg on LEF-1 in the development of HCC. METHODS: Expression of HBsAg, LEF-1 and its downstream effector genes were compared among 30 HCCs, their peritumor tissue counterparts and 9 normal control liver tissues by quantitative real-time PCR. In addition, immunohistochemical staining studies on HBsAg and LEF-1 expression were conducted among these samples. RESULTS: The expression of LEF-1 was compared between 13 HBsAg positive HCC tissues and 17 HBsAg negative HCC tissues. Simultaneous detection of LEF-1 and HBsAg was observed in HBsAg positive HCC tissues and, additionally, the simultaneous detection of HBsAg and LEF-1 was more pronounced in peritumor tissues, compared to that in the tumor tissues. The distribution of cellular LEF-1 in peritumor tissues was predominantly in the cytoplasm; while LEF-1 in the tumor tissues was located either exclusively in the nucleus or both in the nucleus and cytoplasm. By real-time PCR, the expression levels of LEF-1 downstream effector genes cyclin D1 and c-myc were higher in peritumor cells compared to that of the tumor cells. However, a 38 kDa truncated isoform of LEF-1, rather than the 55 kDa wild-type LEF-1, was significantly elevated in the HBsAg positive tumor cells. CONCLUSION: Data indicate that deregulation of the Wnt pathway by HBsAg occurred in HBV-associated HCCs, but was more pronounced in the peritumor cells. It is speculated that HBsAg could stimulate proliferation and functional modification of hepatocytes via LEF-1 through the Wnt pathway at the pre-malignant stage.

Gene-expression profiles of a hepatitis B small surface antigen-secreting cell line reveal upregulation of lymphoid enhancer-binding factor 1.

The genome of hepatitis B virus (HBV) consists of four open reading frames, encoding the envelope proteins (Pre-S/S), the core proteins (Pre-C/C), the polymerase (P) and the transactivating X protein (X). In the sera of HBV-infected patients, hepatitis B surface antigen (HBsAg) particles without the viral genome can outnumber virions by more than 1000-fold. To analyse the interactions between HBsAg and host cells, global gene-expression profiles of a small HBsAg (SHBs)-secreting stable cell line (HepG2-S-G2) and its counterpart control cell line (HepG2-Neo-F4) were compared. Marked upregulation of lymphoid enhancer-binding factor 1 (LEF-1), a transcription factor in the Wnt pathway, was found in SHBs-expressing cells and was confirmed by interference experiments with small interfering RNA. However, compared with the control cells, HepG2-S-G2 did not show higher proliferative competence in culture or increased tumorigenesis in nude mice. A possible mechanism to explain the discrepancy between the upregulation of LEF-1 and the lack of increased tumorigenesis is SHBs expression resulting in altered expression and distribution of LEF-1 protein in cell compartments and upregulation of LEF-1 isoforms that could suppress, rather than enhance, the Wnt pathway.

A putative new domain target for anti-hepatitis B virus: residues flanking hepatitis B virus reverse transcriptase residue 306 (rtP306).

Previous work showed that conservation of proline at residue 306 (rtP306) of hepatitis B virus (HBV) reverse transcriptase (RT) is crucial for virus replication and encapsidation of pregenomic RNA (pgRNA). In this study, the functions of residues flanking rtP306 in HBV RT (rtG304, rtY305, rtA307, rtL308 and rtL311) are presented. Alanine or phenylalanine was used to substitute these residues by constructing site-directed mutants which were used to transfect Huh-7 cells. Replication competencies and encapsidation efficiencies were compared between the mutants and the parental viral strain. Substitutions at these residues resulted in marked decrease of replication competency, which was confirmed by Southern blot hybridization of HBV DNA isolated from intracytoplasmic core particles, and trans-complementation between a non-replicative defective mutant and corresponding RT mutants. Impaired pgRNA encapsidation efficiency of these mutants was shown as the major mechanism for decreased replication efficiency. Since residues from rt304 to rt311 are highly conserved among genotypes A-H HBV strains, results suggest that rt304 to rt311 in HBV RT may serve as a putative anti-HBV new target domain.

High replicative full-length lamivudine-resistant hepatitis B virus isolated during acute exacerbations.

During chronic HBV infections, exacerbations of disease usually occur without clearly understood mechanisms. In this study, full-length HBV genomes were analyzed from four chronic hepatitis B patients who developed resistance to lamivudine [-2'-deoxy-3'-thiacytidine, LMV] accompanied by acute exacerbation of disease. Paired full-length HBV isolates were cloned from the sera of patients prior to LMV treatment and after drug resistant breakthrough isolates emerged with exacerbation. Compared to the isolates before treatment, isolates from all four patients during exacerbation showed marked increase in replicative competence by cell transfection study. Viral genome amplification and direct sequencing was used further to study the sequence differences between the dominant species and the clones used for functional analysis. Apart from mutations at the YMDD motif, no shared mutations were shown between all isolates. The isolates from the one patient who recovered from the exacerbation showed a lower number of mutations, and in particular, lacked basal core promoter (BCP) mutations at 1762/1764. In contrast, BCP mutations were found in isolates from the other three patients. Thus, in patients with acute exacerbation, high replicative strains might be selected from the total HBV quasispecies during treatment, and amongst these strains, those with core promoter mutations were most likely to be associated with severe clinical exacerbations.

Substitution of proline 306 in the reverse transcriptase domain of hepatitis B virus regulates replication.

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the alpha-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1.96 and 4.51% of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9.4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.

Replication efficiency and sequence analysis of full-length hepatitis B virus isolates from hepatocellular carcinoma tissues.

Prolonged replication of hepatitis B virus (HBV) in liver tissues of hepatitis B patients has been considered as an important risk factor for the development of malignancy. Few studies on full-length HBV sequencing in association with the replication efficiency of isolates from HCC tissues have been reported. To study the structural and functional genomics of HBV isolates from Chinese hepatocellular carcinoma (HCC) patients, full-length HBV genomes were amplified from 6 HBV-marker positive HCC tissues and used to transfect HepG2 cells. Five of 6 isolates showed high replicative efficiency. All isolates were of genotype C and "hot-spots" mutations were detected in the B cell and T helper (Th) cell epitopes of the envelope and the core region. In addition, the X region of 2 isolates contained a stop-codon mutation that was predicted to result in a truncated X protein. High replicative HBV immune escape mutants that persist in infected hepatocytes could be 1 of the important factors to initiate pathological processes for the development of HCC in Chinese patients.