Large hepatitis delta antigen activates STAT-3 and NF-κB via oxidative stress.

Hepatitis delta virus (HDV) coinfection or superinfection in hepatitis B virus (HBV)-infected patients results in a more aggressive liver disease, with more often fulminant forms and more rapid progression to cirrhosis and hepatocellular carcinoma. The mechanism(s) for this pejorative evolution remains unclear. To explore a specific HDV pathogenesis, we used a model of transient transfection of plasmids expressing the small (sHDAg or p24) or the large (LHDAg or p27) delta antigen in hepatocyte cell lines. We found that the production of reactive oxygen species was significantly higher in cells expressing p27. Consequently, p27 activated the signal transducer and activator of transcription-3 (STAT-3) and the nuclear factor kappa B (NF-κB) via the oxidative stress pathway. Moreover in the presence of antioxidants (PDTC, NAC) or calcium inhibitors (TMB-8, BAPTA-AM, Ruthenium Red), p27-induced activation of STAT-3 and NF-κB was dramatically reduced. Similarly, using a mutated form of p27, where the cysteine 211-isoprenylation residue was replaced by a serine, a significant reduction of STAT-3 and NF-κB activation was seen, suggesting the involvement of isoprenylation in this process. Additionally, we show that p27 is able to induce oxidative stress through activation of NADPH oxidase-4. These results provide insight into the mechanisms by which p27 can alter intracellular events relevant to HDV-related liver pathogenesis.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC): molecular mechanisms and novel paradigms.

Chronic hepatitis B (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Most HCCs complicate the evolution of an active or inactive cirrhosis. However, some tumors occur on livers with minimal histological changes; the prevalence of such cases varies from one geographical region to the other, being much higher in the Southern half of Africa (around 40% of HCCs) than in Asia, America and Europe, where at least 90% of HCCs are associated in the cirrhosis. This heterogeneity is probably a reflection of different environmental and genetic factors. This review will summarise the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis. It will show in particular how viruses can be viewed as tools to discover and dissect new cellular pathways involved in cancer development and emphasize the potential synergistic effects between HBV and hepatitis C virus (HCV), as well as between viral infections and other environmental factors, such as alcohol.

Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis.

As discussed in detail in other chapters of this review, chronic hepatitis B (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Most HCCs complicate the evolution of an active or inactive cirrhosis. However, some tumors occur on livers with minimal histological changes; the prevalence of such cases varies from one geographical region to the other, being much higher in the southern half of Africa (around 40% of HCCs) than in Asia, America and Europe, where at least 90% of HCCs are associated with the cirrhosis. This heterogeneity is probably a reflection of different environmental and genetic factors. This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis. It will show in particular how viruses can be viewed as tools to discover and dissect new cellular pathways involved in cancer development and emphasize the potential synergistic effects between HBV and hepatitis C virus, as well as between viral infections and other environmental factors, such as alcohol.

[Wide range antiviral activity of MxA protein]. / La protéine MxA, agent antiviral à large spectre.

Mx proteins are interferon-induced GTPases belonging to the dynamin superfamily of large GTPases.A unique property of Mx GTPases is their antiviral activity against a wide range of RNA viruses, including bunya-and orthomyxoviruses. The human MxA GTPase accumulates in the cytoplasm of interferon-treated cells, partly associating with the endoplasmic reticulum. In the cases of Thogoto and La Cross viruses, MxA appears to detect viral infection by sensing nucleocapsid-like structures. Indeed, MxA interacts directly with the viral nucleocapsid protein of these viruses. As a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new virus particles. In both cases, the GTP-binding and carboxy-terminal effector functions of MxA are required for target recognition. Moreover, human MxA possesses many biophysical properties of traditional dynamin and may act to inhibit viral replication through alterations in membrane organization or viral trafficking. This hypothesis still needs to be demonstrated.

Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues.

The hepatitis B virus (HBV) X protein (HBx) is a transcriptional transactivator that has been implicated in the development of HBV-related hepatocellular carcinoma. Mutations in the HBx open reading frame have been reported, but their general impact on the biological function of HBx remains unknown. To address this issue, we comparatively analyzed the structures and biological functions of HBx sequences isolated from sera and from tumor and nontumor tissues of patients with a HBV-related hepatocellular carcinoma. In addition to the HBx sequences derived from free HBV genomes, HBx from HBV integrants was also obtained from the tumor tissues by use of a HBx-Alu PCR-based approach. Sequence analysis showed that the HBx sequences derived from tumor tissues (6 of 7), particularly those isolated from HBV integrants (4 of 4), contained a deletion in the distal COOH-terminal region. Interestingly, most of the COOH-terminally truncated HBx sequences obtained from tumor tissues, in contrast to the full-length HBx isolated from the sera and nontumor tissues, lost their transcriptional activity and their inhibitory effects on cell proliferation and transformation. Importantly, although full-length HBx suppressed the focus formation induced by the cooperation of ras and myc oncogenes in primary rat embryo fibroblasts, COOH-terminally truncated HBx enhanced the transforming ability of ras and myc. Finally, by analyzing the artificial mutants, we were able to more precisely map the functional domains located at the COOH-terminal of HBx. Taken together, our results suggest a key role for the HBx COOH-terminal end in controlling cell proliferation, viability, and transformation. This study further supports the hypothesis that natural HBx mutants might be selected in tumor tissues and play a role in hepatocarcinogenesis by modifying the biological functions of HBx.

Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease and hepatocellular carcinoma (HCC). We demonstrate herewith that HCV core proteins encoded by sequences isolated from HCC tumor tissues, but not those derived from their non-tumor counterparts in the same liver, co-localise in vitro and in vivo and co-immunoprecipitate with PKR in hepatocytic Huh7 cells. We show that this association in fact augments the autophosphorylation of PKR and the phosphorylation of the translation initiation factor eIF2alpha, which are two markers of PKR activity. The present study therefore identifies a novel model of virus-cell interactions whereby a viral protein, the HCV core, activates PKR activity.

Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein.

Human MxA is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several RNA viruses. We had previously shown that overexpression of the hepatitis B virus (HBV) capsid led to selective downregulation of MxA gene expression, suggesting a mechanism by which the virus escapes from the host defense system (O. Rosmorduc, H. Sirma, P. Soussan, E. Gordien, P. Lebon, M. Horisberger, C. Brechot and D. Kremsdorf, J. Gen. Virol. 80:1253-1262, 1999). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two- to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed, using the well-characterized posttranscriptional regulation element (PRE) reporter system, we were able to demonstrate a marked reduction (three- to eightfold) in the nucleocytoplasmic export of unspliced RNA in MxA-expressing clones. In addition, MxA protein did not interact with HBV nucleocapsid or interfere with HBV nucleocapsid formation. Our results show an antiviral effect of MxA protein on a DNA virus for the first time. MxA protein acts, at least in part, by inhibiting the nucleocytoplasmic export of viral mRNA via the PRE sequence.

In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA.

Hepatitis B virus (HBV) is a small DNA virus with a compact genomic organization. All HBV proteins identified to date have been encoded by unspliced HBV RNAs. Spliced HBV RNAs have been described, but their functions are unknown. We show here that a singly spliced HBV RNA encodes a novel HBV protein in vivo. This HBV splice-generated protein (HBSP) corresponds to the fusion of a part of the viral polymerase and a new open reading frame that is created by the splicing event. In vivo, HBSP protein was found in HBV-infected liver samples, and anti-HBSP antibodies occurred in one-third of sera samples collected from chronic HBV carriers. In vitro, the ectopic expression of HBSP had no effect on viral DNA replication or transcription but induced cell apoptosis without a cell-cycle block. Overall, our results suggest that HBV has evolved a mechanism that directly modulates virus-cell interaction through RNA splicing.

Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx.

Chronic infection by HBV is the leading cause of hepatocellular carcinoma in man. Several lines of evidence suggest that the viral transactivator HBx plays a critical role in the molecular pathogenesis of HBV-related HCC. To study the actual impact of HBx and the mechanism of its action, we have recently cloned and characterized a set of X-sequences from HCC in patients with chronic infection by HBV. In the present study, we have compared the effects of HBx and its naturally arising mutants on cell growth and viability. We report that HBx inhibits clonal outgrowth of cells and induces apoptosis by a p53-independent pathway. Furthermore, HBx expression induced a late G1 cell cycle block prior to their counterselection by apoptosis. Importantly, mutations in the HBx-gene evolving in hepatocellular carcinoma abolished both HBx-induced growth arrest and apoptosis. Using a panel of engineered mutants we have mapped the growth suppressive effect of HBx to domains shown to be required for its transactivating function. Based on these results, we propose that abrogation of the anti-proliferative and apoptotic effects of HBx by naturally occurring mutations might render the hepatocytes susceptible to uncontrolled growth and contribute to multistep hepatocarcinogenesis associated with HBV-infection.

Direct association and nuclear import of the hepatitis B virus X protein with the NF-kappaB inhibitor IkappaBalpha.

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.

Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

Cytosol is the prime compartment of hepatitis B virus X protein where it colocalizes with the proteasome.

The hepatitis B virus X protein plays an important role in the regulation of viral genome expression and has also been implicated in the development of liver cancer associated with chronic viral infection. Several effects have been attributed to X but their biological relevance remains elusive. One of the confusing issues has been so far the uncertainty concerning its cellular location. To gain insight into the mechanism(s) how X exerts its effects, we have analysed its subcellular distribution and its dependency on the cell cycle. We used two complementary approaches namely, immunolocalization using a cell line stably expressing X, and characterization of the dynamics of X location in living cells by means of the reporter gene GFP. Our data clearly define the cytosol as the prime location of X, irrespectively of the cell cycle and show in addition the close attachment of a fraction of X to the nuclear membrane. However, X does not associate with any cytoplasmic vesicles and organelles so far tested. In contrast, our study provides strong evidence for the codistribution of X with the cytosolic fraction of proteasomes. In pulse-chase experiments, X decayed with a half-life of less than 30 min and proteasome-inhibitors did not modify its turnover, suggesting that X colocalization with the proteasome does not simply point to its degradation pathway. The proteolytic processing of the p105 precursor of the p50 subunit of the NF-kappaB transcription factor, which has been shown to be proteasome-dependent, is markedly slow down in the presence of X. These findings suggest that X modulates the processing rate of p105 by acting presumably at the level of the proteasome. Thus, targeting of proteasomes by X might be one of the pathways employed by this viral protein to subvert cellular functions.

Evidence for selection of hepatitis B mutants after liver transplantation through peripheral blood mononuclear cell infection.

BACKGROUND/AIMS: Despite anti-HBs immunoglobulin therapy, hepatitis B virus (HBV) infection recurs in a high proportion of patients transplanted for HBsAg positive and serum HBV DNA negative chronic liver disease. The contribution of HBV genetic variability to disease recurrence has not been yet thoroughly addressed. We have therefore undertaken a detailed comparison of preS/S and preC/C sequences in two selected patients with recurrence of HBsAg and HBV DNA after transplantation. METHODS: PreS/S and preC/C regions were amplified by PCR from the serum, peripheral blood mononuclear cell (PBMC) and liver tissues of two patients transplanted for end stage HBV-related cirrhosis. Samples were taken both pre- and post-transplantation. HBV-sequences from four to nine clones were determined and compared. RESULTS: A mixing of different HBV DNA molecules was observed within and between serum, liver and PBMC samples. Sequences from both patients showed mutations in the preC region which abolished HBeAg secretion, and in the preS2 initiation codon which prevented preS2 envelope protein production. In addition, for both patients, deletions in the preS2 domain (3 and 21 base pairs) led to the expression of modified preS1 envelope protein. For one patient, the predominant HBs protein sequence found in the PBMC before transplantation showed four specific mutations. One of these mutations was in the "a" determinant (codon 144, asparagine to glycine change) of the major envelope protein. These mutations were not detected, as predominant mutations, in the liver and serum pre-orthotopic liver transplant samples. In contrast, after liver transplantation, this was the major form identified in serum, liver and PBMC. CONCLUSIONS: Our results have shown the selection of different HBV DNA molecules in liver and mononuclear cells. In addition, they provide direct evidence for the role of PBMC in the infection of liver grafts and support the hypothesis that infection of PBMC might lead to selection of HBV variants which would escape immune therapy. Finally, we provide in vivo evidence for reinfection of the liver by HBV particles lacking preS2 envelope protein expression.

In vivo selection of a hepatitis B virus mutant with abnormal viral protein expression.

We have investigated the molecular basis for the in vivo selective advantage of a hepatitis B virus (HBV) mutant. We have determined the complete nucleotide sequences of the major HBV forms identified at the beginning (B1-83) and end (B1-89) of a 6 year follow-up of a chronically infected patient. The B1-89 sequence showed marked nucleotide rearrangements (a nucleotide divergence of 11.3 % compared with the adw2 subtype), but sequence comparison showed that both viral molecules were of common origin (62/138 mutations were found on both molecules, compared to adw2). In vitro transfection of Huh7 cells showed important modifications in B1-89 viral protein expression. We observed a decrease in B1-89 envelope protein expression associated with a modification of the migration pattern of the large envelope protein. For the B1-89 capsid protein, an insertion of 36 nucleotides at the 5' end of the C gene resulted in increased expression of a core-specific protein of abnormal size (24 kDa versus 22 kDa). Finally, our data also suggest an increase in the transcomplementation efficiency of the mutated B1-89 polymerase protein. Thus, we were able to demonstrate distinct intrinsic properties of HBV DNA molecules isolated from a chronic carrier with virus multiplication at different times during infection. Modifications of viral protein expression in the mutated form illustrate strategies used by the virus to prevent clearance and to contribute to viral persistence.

Effects of ethanol on hepatitis B virus Pre-S/S gene expression in the human hepatocellular carcinoma derived HEP G2 hepatitis B DNA positive cell line.

BACKGROUND/AIMS: Among the reported interactions between ethanol and hepatitis B virus (HBV), studies of transgenic mice have suggested an effect of ethanol on the secretion of viral envelope proteins. METHODS: We further investigated these interactions in vitro by determining HBs antigen levels and performing northern blots of viral mRNA in human cell culture (HepG2 HBV positive cells) exposed for 3 to 12 days to various concentrations of ethanol. RESULTS: In cultures exposed to 200 mM ethanol, HBs antigen concentrations increased in the medium (p < 0.05) after 3 days as Pre-S1 and Pre-S2 protein concentrations. This increase was not specific, as albumin and ferritin increased in the same proportions. Ethanol also increased the HBs antigen concentration in the cells (p < 0.05), whereas levels of viral mRNA encoding surface proteins were unaffected. CONCLUSIONS: These findings show that short-term ethanol exposure in vitro can induce HBs antigen overexpression via a post-transcriptional mechanism.

In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA.

The mechanisms involved in hepatitis B virus (HBV) persistence are still poorly understood. We have previously shown that the encapsidation of the singly spliced 2.2 kb-HBV RNA leads to the secretion of circulating HBV defective particles in patients with chronic hepatitis. We have now investigated the presence of the HBV defective particles in sera from patients with acute and chronic hepatitis, using polymerase chain reaction. These defective particles were detected in a larger amount in sera of patients with acute hepatitis that progressed to chronic hepatitis, or had already developed chronic hepatitis, as compared with those who recovered from acute hepatitis (the increase was estimated to be an average of 50-fold). In addition, we showed that the presence of these defective HBV particles is closely associated with the chronic course of hepatitis B virus infection and with viral multiplication. We also analyzed viral RNAs and proteins synthetized after in vitro transfection of Huh7 cell line with the corresponding defective hepatitis B virus DNA molecule. We showed that expression of the defective hepatitis B virus DNA alone leads to a marked intracellular accumulation of the major core protein (HBcAg) and to an increased secretion of hepatitis B e antigen (HBeAg). These observations may be consistent with a role of these defective hepatitis B virus (HBV) particles in viral persistence.

Evidence of non-A, non-B, non-C infection in chronic hepatitis by polymerase chain reaction testing for hepatitis B and C viruses.

BACKGROUND/AIMS: Although hepatitis C virus is clearly the major cause of non-A, non-B acute and chronic hepatitis, there is a group of patients with histologically documented chronic hepatitis with no serological marker of hepatitis B and C, nor any other risk factors for liver disease. METHODS: We have investigated 17 well-characterized patients with chronic active hepatitis. HBV-DNA and HCV-RNA were tested by polymerase chain reaction in 17 serum samples and in six liver biopsies. RESULTS: Four of the 17 patients had serum HCV-RNA detectable by polymerase chain reaction, while none had HBV-DNA detectable by polymerase chain reaction. Three of the six liver samples gave a positive signal by cyclin A and HLA, and only these were considered for the study. One of the three was HCV-RNA positive, while none was HBV-DNA positive. CONCLUSIONS: Our results, obtained through careful elimination of all known risk factors for liver disease, strongly suggest that non-A, non-B, non-C hepatotropic virus(es) could be involved in some cases of chronic active liver disease.

Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins.

The E1 and E2/NS1 genes, encoding the putative hepatitis C virus envelope proteins, show a high rate of sequence variations. We analyzed the degree and distribution of sequence heterogeneity in serum samples from hepatitis C virus-infected subjects. The mutations in the E1 region were mainly type-specific and the rate of variability was apparently not linked to the clinical phase of the infection. The sequence evolution of the E1 region during interferon treatment was low, regardless of the response to therapy. In contrast, an increased degree of variation, apparently related to the stage of viral replication, was present in E2 region derived from patients undergoing interferon treatment. These results are consistent with the hypothesis that the E2 protein represents a major target of the immune response.

Hepatitis C virus genotype 4 is highly prevalent in central Africa (Gabon).

Following a survey of hepatitis C virus (HCV) infection recently carried in central Africa (Gabon), we cloned and sequenced PCR products of the 5' non-coding and capsid-encoding regions of HCV RNA from three randomly selected HCV RNA-positive Gabonese subjects. In the capsid-encoding region, the identity between the three Gabonese isolates was 91 to 98%. The three Gabonese sequences showed a divergence of 11 to 17% from published HCV genotypes I to IV (1a, 1b, 2a and 2b) isolates and of 6 to 11% from HCV genotype 4 isolates. Thus the Gabonese isolates, termed HC-G, belong to HCV genotype 4. Based on the sequences of the three isolates, a specific probe (cpsG) was designed to detect the HC-G genotype in 30 randomly selected anti-HCV-positive Gabonese subjects, 14 of whom were HCV RNA-positive. Analysis with cpsG showed that 10 of 14 of the HCV RNA-positive subjects were infected by the HC-G genotype. HC-G is therefore highly prevalent in the HCV RNA-positive Gabonese population. The availability of these Gabonese sequences should facilitate the design of specific serological tests for African HCV isolates.