Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

Role of Hepatitis C virus core protein in viral-induced mitochondrial dysfunction.

Hepatitis C virus (HCV) infection results in several changes in mitochondrial function including increased reactive oxygen species (ROS) production and greater sensitivity to oxidant, Ca(2+) and cytokine-induced cell death. Prior studies in protein over-expression systems have shown that this effect can be induced by the core protein, but other viral proteins and replication events may contribute as well. To evaluate the specific role of core protein in the context of viral replication and infection, we compared mitochondrial sensitivity in Huh7-derived HCV replicon bearing cells with or without core protein expression with that of cells infected with the JFH1 virus strain. JFH1 infection increased hydrogen peroxide production and sensitized cells to oxidant-induced loss of mitochondrial membrane potential and cell death. An identical phenomenon occurred in genome-length replicons-bearing cells but not in cells bearing the subgenomic replicons lacking core protein. Both cell death and mitochondrial depolarization were Ca(2+) dependent and could be prevented by Ca(2+) chelation. The difference in the mitochondrial response of the two replicon systems could be demonstrated even in isolated mitochondria derived from the two cell lines with the 'genome-length' mitochondria displaying greater sensitivity to Ca(2+) -induced cytochrome c release. In vitro incubation of 'subgenomic' mitochondria with core protein increased oxidant sensitivity to a level similar to that of mitochondria derived from cells bearing genome-length replicons. These results indicate that increased mitochondrial ROS production and a reduced threshold for Ca(2+) and ROS-induced permeability transition is a characteristic of HCV infection. This phenomenon is a direct consequence of core protein interactions with mitochondria and is present whenever core is expressed, either in infection, full-length replicon-bearing cells, or in over-expression systems.

Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

Structural basis of the oligomerization of hepatitis delta antigen.

BACKGROUND: The hepatitis D virus (HDV) is a small satellite virus of hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe liver disease in humans. The small 195 amino-acid form of the hepatitis delta antigen (HDAg) functions as a trans activator of HDV replication. A larger form of the protein containing a 19 amino acid C-terminal extension inhibits viral replication. Both of these functions are mediated in part by a stretch of amino acids predicted to form a coiled coil (residues 13-48) that is common to both forms. It is believed that HDAg forms dimers and higher ordered structures through this coiled-coil region. RESULTS: The high-resolution crystal structure of a synthetic peptide corresponding to residues 12 to 60 of HDAg has been solved. The peptide forms an antiparallel coiled coil, with hydrophobic residues near the termini of each peptide forming an extensive hydrophobic core with residues C-terminal to the coiled-coil domain in the dimer protein. The structure shows how HDAg forms dimers, but also shows the dimers forming an octamer that forms a 50 A ring lined with basic sidechains. This is confirmed by cross-linking studies of full-length recombinant small HDAg. CONCLUSIONS: HDAg dimerizes through an antiparallel coiled coil. Dimers then associate further to form octamers through residues in the coiled-coil domain and residues C-terminal to this region. Our findings suggest that the structure of HDAg represents a previously unseen organization of a nucleocapsid protein and raise the possibility that the N terminus may play a role in binding the viral RNA.

Cellular response to conditional expression of the hepatitis B virus precore and core proteins in cultured hepatoma (Huh-7) cells.

BACKGROUND: The expression of the hepatitis Be antigen (HBeAg) is one of several strategies used by hepatitis B virus (HBV) to ensure persistence. The HBeAg may function as a toleragen in utero and has been shown to regulate the host's immune response. AIM: The aim of this study was to examine the effect of the HBV precore and core protein on cellular gene expression in the hepatoma cell line Huh-7. STUDY DESIGN: Huh-7 cells with tight regulated expression of the HBV core or precore protein were produced using the Tet-Off tetracycline gene expression system. Changes in cellular gene expression in response to core/precore expression compared to Huh-7 cells not expressing the proteins were determined using a commercial high-density oligonucleotide array (Affymetrix Hu95A GeneChip) containing probes for 12,626 full-length human genes. RESULTS: Analysis of differential mRNA gene expression profiles at 7 days post precore and core expression revealed 45 and 5 genes, respectively, with mRNA changes greater than three-fold. The most striking feature was in Huh-7 cells expressing the precore protein in which 43/45 genes were downregulated 3-11-fold. These included genes that encoded products that regulate transcription/DNA binding proteins, cell surface receptors, cell-cycle/nucleic acid biosynthesis and intracellular signalling and trafficking. The only known gene, which was upregulated encoded a cytoskeletal protein. For the core cell line, 4/5 genes were downregulated 3-15-fold upon core induction and included genes that encoded products that affect intermediary metabolism, cell surface receptors and intracellular signalling. The one gene, which was upregulated was a cytokine gene. CONCLUSION: The results of this study show that HBV precore protein has a much greater effect on cellular gene expression in comparison to the core protein, suggesting that core and precore proteins may have diverse effects on cellular functions and equally different roles in modulating HBV pathogenesis.

Analysis of hepatitis A virus translation in a T7 polymerase-expressing cell line.

Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.

Why do human hepatitis viruses replicate so poorly in cell cultures?

The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays.

Hepatitis A virus (HAV), a non-cytopathic picornavirus, has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridization. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

A replication-deficient adenovirus enhances liposome-mediated nucleic acid transfer into a stable cell line expressing T7 RNA polymerase.

Liposome-mediated transfer of nucleic acids into a cell line expressing bacteriophage T7 RNA polymerase was enhanced by addition of a replication-deficient adenovirus (Ad5-259A) to transfection mixtures. Increasing quantities of Ad5-259A resulted in a dose-related (up to 30-fold) enhancement of reporter gene activity expressed in BT7-H cells transfected with plasmid DNA containing the reporter sequence fused to the internal ribosome entry site of encephalomyocarditis virus. Similarly, Ad5-259A enhanced reporter gene expression 7-fold following transfection of DNA containing the reporter sequence under transcriptional control of the Rous sarcoma virus long terminal repeat. Addition of Ad5-295A to transfection mixtures increased the proportion of cells staining positively for reporter gene activity, from 2 to 25% when the reporter was expressed via the T7 polymerase and from 20 to 50% when the reporter was under the control of a eucaryotic promoter. Thus, Ad5-259A enhanced reporter protein activities expressed by cytoplasmic T7-directed transcription and cap-independent initiation of translation, or nuclear transcription and cap-dependent translation. Transfection enhancement was blocked by neutralizing antibody to Ad5, and is most likely related to the endosome-disrupting activities of the virus. Adenovirus enhancement of liposome-mediated transfection provides a useful method for efficient nucleic acid transfer into eucaryotic cells.

Hepatitis A virus and blood products: virus validation studies.

As there have been reports of the transmission of hepatitis A virus by single units of blood, virus validation studies were carried out to determine the fate of the virus at four major steps (cryoprecipitation, solvent/detergent inactivation, DEAE chromatography and lyophilization) involved in the production of solvent-detergent inactivated factor VIII products. If the reduction in infectivity is additive over the four steps, the total reduction would be between 8.6 and 9.3 log10. It is unlikely that such a reduction would provide a sufficient safety margin, particularly if the original plasma pool from which the FVIII was prepared, was obtained from a population in which hepatitis A virus was not rare. In such a situation, an additional method of effectively reducing hepatitis A virus infectivity should preferably be included in the production process.

Virus-specific mechanisms of carcinogenesis in hepatitis C virus associated liver cancer.

The development of hepatocellular carcinoma (HCC) in persons who are persistently infected with hepatitis C virus (HCV) is a growing problem worldwide. Current antiviral therapies are not effective in many patients with chronic hepatitis C, and a greater understanding of the factors leading to progression of HCC will be necessary to design novel approaches to prevention of HCV-associated HCC. The lack of a small animal model of chronic HCV infection has hampered understanding of these factors. As HCV is an RNA virus with little potential for integration of its genetic material into the host genome, the mechanisms underlying HCV promotion of cancer are likely to differ from other models of viral carcinogenesis. In patients persistently infected with HCV, chronic inflammation resulting from immune responses against infected hepatocytes is associated with progressive fibrosis and cirrhosis. Cirrhosis is an important risk factor for HCC independent of HCV infection, and a majority of HCV-associated HCC arises in the setting of cirrhosis. However, a significant minority arises in the absence of cirrhosis, indicating that cirrhosis is not a prerequisite for cancer. Other lines of evidence suggest that direct, virus-specific mechanisms may be involved. Transgenic mice expressing HCV proteins develop cancer in the absence of inflammation or immune recognition of the transgene. In vitro studies have revealed multiple interactions of HCV-encoded proteins with cell cycle regulators and tumor suppressor proteins, raising the possibility that HCV can disrupt control of cellular proliferation, or impair the cell's response to DNA damage. A combination of virus-specific, host genetic, environmental and immune-related factors are likely to determine the progression to HCC in patients who are chronically infected with HCV. Here, we summarize current knowledge of the virus-specific mechanisms that may contribute to HCV-associated HCC.

Type A viral hepatitis: epidemiology, diagnosis, and prevention.

Hepatitis A virus (HAV) infection occurs worldwide and is an important cause of acute viral hepatitis in the US. In this review, I cover the epidemiology, course of infection, clinical manifestations, serological responses, and prevention of this infection. Although most patients completely recover from this disease, elderly patients have a substantial mortality risk. Recently licensed vaccines are highly efficacious.

Immunologic approaches to assessing the response to inactivated hepatitis A vaccine.

Natural immunity to hepatitis A virus (HAV) is complex and likely to involve several distinct arms of the immune system. There is evidence that natural killer cells, human leukocyte antigen (HLA)-restricted cytotoxic T cells, and antibody-secreting cells of B-cell lineage all play roles in the immune response to infection with HAV. However, antibody alone is sufficient to provide a high level of protection against clinical disease. A comparison of the serum levels of antibody to HAV (anti-HAV) following administration of immune serum globulin and hepatitis A vaccine may provide a useful estimate of vaccine efficacy. Such comparisons may be accomplished using solid-phase immunoassays for detection of anti-HAV. However, tests which measure antibody capable of neutralizing virus in vitro are generally more sensitive than solid-phase immunoassays. The use of endogenously labelled virus in radioimmunoprecipitation assays shows promise of providing an equally sensitive means of measuring antibodies which are reactive with HAV particles.

The natural history of hepatitis A: the potential for transmission by transfusion of blood or blood products.

A study of the natral history and risk factors for hepatitis A can shed light on the potential for contamination of plasma concentrate with hepatitis A virus (HAV). According to the long-term Sentinel Counties Study conducted by Alter and colleagues at the Centers for Disease Control and Prevention in Atlanta, the most frequently reported risk factors for HAV infection are living with a patient who has hepatitis, homosexual activity, and close contact with young children. International travel to hepatitis A endemic areas and illicit parenteral drug use were less frequently documented risk factors, although illicit injectable drug use has been considered more significant in other hepatitis A studies. Approximately 40% of patients with hepatitis A reported no apparent risk factors. Hepatitis A occurs most often today in the 5- to 30-year-old age group. Young adults, who are also eligible donors, are thus at risk of infection. The natural history of hepatitis A was studied in New World owl monkeys. Fecal shedding of infectious virus was detected by 4 days after intravenous injection of infectious material and peaked at almost 10 million infectious particles per gram of feces just prior to onset of chemical evidence of liver disease. Viremia of substantial magnitude occurred throughout most of the 4-week incubation period and was maximal during the prodromal stage, prior to the development of clinical, chemical, or serologic manifestations of infection. Although the magnitude of hepatitis A viremia has not been well documented in humans, it is likely to reach levels of 10(4)-10(6) infectious particles per milliliter of blood.(ABSTRACT TRUNCATED AT 250 WORDS)