Chimeric constructs between two hepatitis B virus genomes confirm transcriptional impact of core promoter mutations and reveal multiple effects of core gene mutations.

Hepatitis B virus (HBV) clone 4B replicated much more efficiently than clone 2A of the same genotype. Introduction of its T1753C, A1762T, G1764A, and C1766T core promoter mutations into the 2A genome greatly enhanced genome replication and suppressed HBeAg expression. Here we show that these effects are mediated by transcriptional up regulation of pregenomic RNA and suppression of precore RNA. Analysis of chimeric constructs suggested that the 5' end of the 2A core gene conferred higher level of pregenomic RNA, but less core protein and genome replication relative to the 4B sequence. Genome maturity of secreted virions was reduced by mutations present in the core protein of the 2A genome but enhanced by mutations found in the 4B core protein. The 4B core protein migrated faster than that of clone 2A. The possible links among the various phenotypes and the responsible mutations remain to be established.

Expression of apolipoprotein C-IV is regulated by Ku antigen/peroxisome proliferator-activated receptor gamma complex and correlates with liver steatosis.

BACKGROUND/AIMS: We previously reported that hepatitis C virus (HCV) core protein up regulated transcription of apolipoprotein C-IV (ApoC-IV, 10.7-fold increase), a member of the apolipoprotein family implicated in liver steatosis. Here, we identified host transcription factors regulating the ApoC-IV gene expression. METHODS: Transcriptional regulators were identified by DNA affinity purification and steatosis was detected by oil red O staining and triglyceride assay. RESULTS: We defined a 163-bp ApoC-IV promoter as a core protein responsive element, and identified Ku antigen complex (Ku70 and Ku80) as well as nuclear receptors PPARgamma/RXRalpha as key regulators of ApoC-IV gene expression. Both Ku70 overexpression and PPARgamma agonist significantly increased ApoC-IV promoter activity; conversely, Ku70 silencing or mutation of PPARgamma binding site diminished the ApoC-IV promoter activity. Interestingly, transient transfection of ApoC-IV cDNA into a human hepatoma cell line was able to trigger moderate lipid accumulation. In agreement with this in vitro study, ApoC-IV transcript level was increased in HCV infected livers which correlated with triglyceride accumulation. CONCLUSIONS: ApoC-IV overexpression may perturb lipid metabolism leading to lipid accumulation. HCV core protein may modulate ApoC-IV expression through Ku antigen and PPARgamma/RXRalpha complex.

Evaluation of clinical usefulness of second-generation HCV core antigen assay: comparison with COBAS AMPLICOR HCV MONITOR assay version 2.0.

BACKGROUND: Hepatitis C virus (HCV) is an important etiologic agent for chronic liver diseases. METHODS: The aim of this study was to evaluate the clinical usefulness of second-generation HCV core antigen assay by comparing the results of the assay with those of the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0). RESULTS: HCV core antigen was detectable by this assay in 142/149 (95.3%) of serotype 1 (3821+/-322 fmol/l; mean+/-SD), in 56/58 (96.6%) of serotype 2 (2589+/-449 fmol/l), and in 6/6 (100%) of serotypes 1+2 (1240+/-548 fmol/l). The HCV core antigen levels measured by this assay correlated well with the HCV RNA levels by COBAS v2.0 (r=0.848, P<0.0001). In relation to the outcome of interferon monotherapy, the pretreatment HCV core antigen levels of sustained and non-sustained virological responders were 659+/-189 and 4904+/-376 fmol/l in serotype 1, 1993+/-740 and 3145+/-519 fmol/l in serotype 2. The cutoff values with the best accuracy for HCV core Ag levels to discriminate between sustained and non-sustained virological response were 699 fmol/l for serotype 1 and 292 fmol/l for serotype 2, respectively, by receiver operating characteristic curve analysis. CONCLUSION: This new assay was considered to be useful in evaluating the HCV levels in patients with chronic hepatitis C.

Lower incidence of hepatic failure than hepatocellular carcinoma in Japanese patients with chronic hepatitis C.

BACKGROUND: Previous studies have shown that the development of hepatic failure was found more frequently than that of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C in the United States and European countries. We investigated the status in Japan in a retrospective cohort study. METHODS: The incidences of HCC and hepatic failure were accessed in 459 patients with biopsy-proven C-viral chronic liver disease with a mean follow-up period of 8.9+/-3.2 years and the cause of death was also analyzed in the cohort. RESULTS: HCC developed in 63 patients, 46 of 355 interferon (IFN)-treated and 17 of 104 untreated patients. In contrast, the development of hepatic failure was found in 18 patients, 12 of 355 IFN-treated and six of 104 untreated patients. HCC developed in four of 116 with sustained virological response (SVR), and hepatic failure developed in one of them. Thirty-two of 63 patients developing HCC and eight of 18 patients developing hepatic failure died. CONCLUSIONS: Development of hepatic failure was less frequent than that of HCC in Japan. It is important for a favorable prognosis of patients with C viral chronic liver disease to achieve a higher SVR and thus inhibit the development of HCC in Japan.

Effect of lamivudine in HBeAg-positive chronic hepatitis B: discordant effect on HBeAg and HBV DNA according to pretreatment ALT level.

AIM: To clarify differences in antiviral effect of the drug in patients with different ALT levels, we examined the changes in HBV markers in patients with high or low ALT levels with or without lamivudine treatment. METHODS: Thirty-seven HBeAg-positive patients were studied. Ten patients with ALT levels higher than 200 IU/L (group 1) and 8 patients with ALT below 200 IU/L (group 2) were treated orally with 100 mg/d of lamivudine. As untreated control, 9 patients with ALT above 200 IU/L (group 3) and 10 patients with ALT below 200 IU/L (group 4) were examined. ALT level, HBeAg/HBeAb status, and HBV DNA level were examined monthly for 11.9+/-0.4 mo. RESULTS: The ALT level normalized in all 10 patients of group 1, 7/8 of group 2, 4/9 of group 3, and 1/10 of group 4 within 6 mo (groups 1 vs 2, P = NS; groups 1 vs 3, P = 0.002; groups 1 vs 4, P<0.0001). HBV DNA fell below the detection limit in all 10 patients of group 1, 7/8 of group 2, 0/9 of group 3, and 0/10 of group 4 within 6 mo (groups 1 vs 2, P = NS). HBeAg became seronegative in 7/10 patients of group 1, 1/8 of group 2, 3/9 of group 3, and 0/10 of group 4 within 12 mo (groups 1 vs 2, P = 0.02; groups 1 vs 3, P = NS). CONCLUSION: Our data suggest that HBeAg-positive patients with higher ALT levels can be considered good candidates for lamivudine therapy, probably because lamivudine accelerates the natural seroconversion of HBeAg, accompanied by HBV DNA loss, in these patients.

Hepatitis C virus core protein stimulates hepatocyte growth: correlation with upregulation of wnt-1 expression.

Hepatitis C virus (HCV) core protein has been implicated in the development of human hepatocellular carcinoma (HCC). Here we report that expression of HCV core protein by transient transfection increased cell proliferation, DNA synthesis, and cell cycle progression in Huh-7 cells, a human HCC-derived cell line. Culture supernatant from transfected cells also harbored a growth-promoting effect. Moreover, a full-length HCV replicon, but not a subgenomic replicon devoid of the core gene, significantly stimulated growth of transiently transfected Huh-7.5 cells. However, growth of the subgenomic replicon-containing Huh-7.5 cells could be stimulated by secondary transfection with core gene but not other structural genes present in the full-length replicon. Microarray analysis revealed threefold or more transcriptional changes in 372 of 12,500 known human genes in core protein expressing Huh-7 cells, with most genes involved in cell growth or oncogenic signaling, being upregulated rather than downregulated. Of particular interest is the marked upregulation of both wnt-1 and its downstream target gene WISP-2. Indeed, small interfering RNA against wnt-1 blunted growth stimulation by core gene, whereas transfection of Huh-7 cells with the wnt-1 gene sufficed to promote cell proliferation. Consistent with secretion of the wnt-1 protein, conditioned medium from wnt-1 transfected cells accelerated cell growth. In conclusion, HCV core protein induces Huh-7 cell proliferation whether alone or in the context of HCV replication, which is at least partly mediated by transcriptional upregulation of growth-related genes, in particular wnt-1.

Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion.

Hepatitis B virus (HBV) variants with impaired expression of e antigen (HBeAg) frequently arise at the chronic stage of infection, as exemplified by precore and core promoter mutants. Since an intramolecular disulfide bond maintains the secondary structure of HBeAg, we explored effect of missense mutations of either cysteine codon. Consistent with earlier reports, substitution of each cysteine rendered HBeAg nearly undetectable. With underlying nucleotide changes at the loop of pregenome encapsidation signal, the C-7 mutants were severely impaired in pregenomic RNA packaging and hence DNA replication. Although none of the missense mutations at C61 reduced DNA replication, replacement with arginine, but not alanine, aspartic acid, phenylalanine, or serine, blocked virion secretion. Consistent with the detection of C61R genome from a patient serum, secretion block of the C61R mutant could be overcome by co-expression of wild-type core protein. In conclusion, point mutations of the C61 codon may generate viable HBeAg-negative variants.

High sustained virologic response rate after interferon monotherapy in Japanese hepatitis C patients with a low HCV RNA titer and/or HCV genotype 2. A prospective study.

OBJECTIVE: Hepatitis C virus (HCV) RNA titer and HCV genotype are considered to be major determinants of the outcome of interferon monotherapy. To clarify whether interferon monotherapy is really effective in patients with the appropriate viral parameters, we prospectively examined these parameters and treated the patients with interferon monotherapy. METHODS: Sixty-four patients with an HCV RNA titer <100 kIU/ml and/or HCV genotype 2 were enrolled in the study. Eighteen patients with an HCV RNA titer >100 kIU/ml and genotype 1 were also enrolled as controls. All patients were treated with 10 megaunits of interferon-alpha2b every day for 2 weeks and then 3 times a week for 24 weeks. RESULTS: Of the 64 patients with either HCV RNA <100 kIU/ml and/or genotype 2, seven dropped out from the study. Of the remaining 57 who completed the treatment, 48 (84%) showed a virologic sustained response. In contrast, only 4 of the 18 patients (22%) with HCV RNA >100 kIU/ml and genotype 1 were virologic sustained responders (p < 0.001). CONCLUSION: Our current study showed that the patients with HCV RNA <100 kIU/ml and/or HCV genotype 2 are good candidates for interferon monotherapy.

A case of severe acute hepatitis of unknown etiology treated with the Chinese herbal medicine Inchinko-to.

A prolonged severe hepatitis of unknown etiology was treated with Inchinko-to, a Chinese herbal medicine, and this case is herein described. Inchinko-to was given with ursodeoxycholic acid (UDCA) and glycyrrhizin. The improvement in the patient's liver function seemed to be accelerated after the treatment, especially after stopping the administration of kanamycin sulfate which might possibly inhibit the conversion of geniposide, one of the constituents of Inchinko-to, to an active ingredient through the suppression of the bacterial growth in intestinal flora, suggesting the usefulness of Inchinko-to for treatment of severe hepatitis.

Sequence variation upstream of precore translation initiation codon reduces hepatitis B virus e antigen production.

BACKGROUND & AIMS: Most South African hepatitis B virus strains harbor point mutations immediately upstream of the precore AUG codon. The aim of this study was to determine their effect on hepatitis B e antigen expression. METHODS: The hepatitis B virus DNA sequence around the precore region was determined from sera of 45 black South Africans. The South African mutations were introduced into hepatitis B virus dimers of the same genotype, and hepatitis B e antigen was quantified from culture medium of transfected HepG2 or Huh7 cells. RESULTS: The South African sequence changes were easily detectable in the acute, hepatitis B e antigen-positive phase of infection, suggesting that they were stable traits and were not selected by immune pressure. Triple mutations at the -5, -3, and -2 positions of the AUG codon severely impaired hepatitis B e antigen expression (P < 0.001). The frequent double mutation at the -5 and -2 positions moderately reduced hepatitis B e antigen levels (P < 0.001) to an extent comparable to that of the common core promoter mutations (1762(T)1764(A)). The presence of both South African and core promoter mutations diminished hepatitis B e antigen expression in an additive manner. It is interesting to note that the triple South African mutations enabled core protein translation from precore messenger RNA, which could rescue the replication defect of a hepatitis B virus genome with an ablated core gene. CONCLUSIONS: We have identified a novel class of hepatitis B e antigen variants with reduced hepatitis B e antigen translation by a ribosomal leaky scanning mechanism. Reduction in hepatitis B e antigen expression may contribute to accelerated seroconversion from hepatitis B e antigen to its antibody in black South Africans infected with hepatitis B virus very early in life.

Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants.

The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.

Evaluation of the clinical usefulness of COBAS AMPLICOR HCV MONITOR assay (ver2.0): Comparison with AMPLICOR HCV MONITOR assay (ver1.0) and HCV core protein level.

The quantitation of serum levels of hepatitis C virus (HCV) RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of interferon (IFN) therapy. The AMPLICOR HCV MONITOR version 1.0 (AMPLICOR v1.0) assay is widely used for the evaluation of the HCV level. A new generation assay called the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0) assay, which is semiautomated and modified to amplify all genotypes equally, has been developed. The aim of this study was to evaluate the clinical relevance of the COBAS v2.0 assay in comparison with the AMPLICOR v1.0 assay and HCV core protein assay in patients with chronic hepatitis C before IFN therapy. HCV RNA was detectable in 230 cases (97.5%) and undetectable in 6 cases (2.5%) by the COBAS v2.0 assay. The RNA levels measured by the AMPLICOR v1.0 assay correlated significantly with those measured by the COBAS v2.0 assay, and the sensitivity of the new version 2.0 assay was better than that of version 1.0, especially in serotype 2. In relation to the outcome of IFN therapy, HCV RNA levels from virologically sustained responders by the AMPLICOR v1.0 assay were 82.3 +/- 22.9 kcopies/ml in serotype 1 and 36.9 +/- 13.4 kcopies/ml in serotype 2, and those from virologically nonsustained responders were 525.2 +/- 48.6 kcopies/ml in serotype 1 and 76.7 +/- 19.5 kcopies/ml in serotype 2. The rates of sustained response to <100 kcopies/ml were 34/63 (54.0%) in serotype 1 and 24/48 (50.0%) in serotype 2. A statistically significant virological response was seen in serotype 1 (P < 0.0001), but not in serotype 2. In contrast, the levels in virologically sustained responders by the COBAS v2.0 assay were 88.2 +/- 20.5 KIU/ml in serotype 1 and 136.8 +/- 40.1 KIU/ml in serotype 2, and those in virologically nonsustained responders were 608.8 +/- 48.4 KIU/ml in serotype 1 and 328.3 +/- 62.8 KIU/ml in serotype 2. The rates of sustained response to <100 KIU/ml were 33/60 (55.0%) in serotype 1 and 21/35 (60.0%) in serotype 2. Statistical significance in virological response was seen in both serotype 1 (P < 0.0001) and serotype 2 (P < 0.05). Although the sensitivity of the HCV core protein assay was lower than that with the COBAS v2.0 assay, the HCV core protein levels also correlated well with the results of the COBAS v2.0 assay. The HCV core protein levels of virologically sustained responders were 37.6 +/- 12.0 pg/ml in serotype 1, 81.3 +/- 37.0 pg/ml in serotype 2, and those of virologically nonsustained responders were 289.9 +/- 23.5 pg/ml in serotype 1, 191.4 +/- 32.1 pg/ml in serotype 2. This assay could predict the outcome of IFN therapy in both serotype 1 (P < 0.0001) and serotype 2 (P < 0.05). Thus, both the COBAS v2.0 assay and the HCV core protein assay showed that the viral load was an indicator of virologically sustained response in serotype 2 and in serotype 1.