Site Of Hepatitis Research Articles

Extrahepatic and intrahepatic replication and expression of hepatitis C virus.

In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531 bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minus-strand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.

Lipophilic Derivatization of the Antiviral Drug 9-(2-Phosphonylmethoxyethyl)adenine and Its Incorporation into a Lactosylated Lipid Carrier to Improve Its Liver Uptake

Lipophilic derivatization of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with lithocholic acid-3-oleate and its subsequent incorporation into a lactosylated lipid carrier was found to substantially increase uptake of the drug by the liver. Competition experiments with asialofetuin point to a major role of the parenchymal liver cell, the main site of hepatitis B virus infection.

Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome

Abstract Background & Aims: The process and the site of hepatitis C virus (HCV) particle formation in cells after infection remain unknown. The aim of this study was to create an in vitro model for the study of HCV particle formation. Methods: HeLa G cells were transfected with the full-length sequence of the HCV genome. Viral protein expression was analyzed using immunoblotting. The cells were examined using immunoelectron and conventional electron microscopy. Results: Core, E2, NS3, NS5a, and NS5b proteins were identified using immunoblotting. Immunoelectron microscopy showed that the core antigen was located along the membrane of the endoplasmic reticulum (ER) and occasionally in its cisternae. Core antigen-positive particles of 30 nm in diameter were found in the cytosol and in the cisternae of the ER. The particles in the cisternae were coated with an outer membrane that was connected to the ER membrane. Conventional electron microscopy revealed particles of 45 nm in diameter with electrondense cores in the cisternae of the ER. The outer membrane of the particles was occasionally connected to the ER membrane. Conclusions: The findings suggest that HCV core proteins are synthesized and assembled into particles in the cytosol and that they bud into the cisternae of the ER to form coated particles.

Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome

Background & Aims: The process and the site of hepatitis C virus (HCV) particle formation in cells after infection remain unknown. The aim of this study was to create an in vitro model for the study of HCV particle formation. Methods: HeLa G cells were transfected with the full-length sequence of the HCV genome. Viral protein expression was analyzed using immunoblotting. The cells were examined using immunoelectron and conventional electron microscopy. Results: Core, E2, NS3, NS5a, and NS5b proteins were identified using immunoblotting. Immunoelectron microscopy showed that the core antigen was located along the membrane of the endoplasmic reticulum (ER) and occasionally in its cisternae. Core antigen-positive particles of 30 nm in diameter were found in the cytosol and in the cisternae of the ER. The particles in the cisternae were coated with an outer membrane that was connected to the ER membrane. Conventional electron microscopy revealed particles of 45 nm in diameter with electrondense cores in the cisternae of the ER. The outer membrane of the particles was occasionally connected to the ER membrane. Conclusions: The findings suggest that HCV core proteins are synthesized and assembled into particles in the cytosol and that they bud into the cisternae of the ER to form coated particles.

Structure and expression of the cyclin A gene in human primary liver cancer. Correlation with flow cytometric parameters

Background/Aims: The cyclin A gene plays an important role in both the S and G2-M phases of the cell cycle, and has been identified at a site of hepatitis B virus DNA integration in a human liver cancer. We analyzed tumorous and non-tumorous samples from patients with primary liver cancer to determine whether a) the cyclin A gene is rearranged in liver tumors and b) the cyclin A transcript level correlates with the percentage of proliferating cells. Methods: Samples from 43 patients were analyzed by Southern blot. Cyclin A RNA accumulation was evaluated in 18 cases by slot blot and correlated with the percentage of cells in S plus G2-M phases defined by flow cytometry. Results: No rearrangement of the cyclin A gene was found in tumorous compared to non-tumorous tissue. A very strong positive correlation was found between the cyclin A RNA level and the cumulative percentage of cells in S plus G2-M phases (r=0.99; p<0.0001). Conclusions: This in vivo study shows that the expression of cyclin A RNA correlates with the percentage of proliferating cells in primary liver cancer. Thus, cyclin A is a new potential liver tumor cell proliferation index.

Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage.

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.

Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: evidence for viral replication in peripheral blood mononuclear cells.

Hemophiliacs who have been exposed to unheated and/or dry heated pooled clotting factor concentrates are at high risk of chronic hepatitis C. Although the mechanism and site of hepatitis C virus (HCV) replication are not yet known, HCV is thought to replicate through a complementary negative RNA strand, as has been shown for flaviviruses. The detection of negative RNA strands has therefore been regarded as a marker of replication. We investigated the prevalence of HCV-RNA and of negative HCV-RNA strands in peripheral blood mononuclear cells (PBMC) and plasma of hemophiliacs. Forty-three of 47 patients studied (91%) had anti-HCV antibodies and in 36 patients HCV-RNA was detectable in PBMC. In one group of 20 patients negative HCV-RNA strands were present in PBMC and 10 of these patients also had negative HCV-RNA strands in plasma. In another group of nine patients HCV-RNA was detected in PBMC, although cDNA synthesis was carried out in the absence of primers. Only in two of these nine patients negative and positive HCV-RNA strands were demonstrated specifically in PBMC using a modified reverse transcription step. If the presence of negative HCV-RNA strands can be considered as marker of viral replication, the findings indicate that HCV can replicate in PBMC. Furthermore, in certain patients it is impossible to use the currently available technique to detect selectively positive or negative HCV-RNA strands.

Low frequency of allelic loss in the cyclin A gene in human hepatocellular carcinomas: a study based on PCR.

The cyclin A gene was first identified at a site of hepatitis B virus DNA integration in a primary liver cancer (PLC). It has now been mapped to 4q27, in the proximity of a chromosomal locus (4q32) which is frequently rearranged and deleted in PLC. We took advantage of the TaqI polymorphism recently described in the cyclin A gene to search for allelic loss in this gene by means of PCR. Tumorous and non-tumorous tissue from 50 patients with PLC was analyzed: 27 samples (54%) were homozygous for the A1 type allele (i.e. the allele bearing the TaqI restriction site), three (6%) were homozygous for the A2 type allele (without the TaqI site) and 20 (40%) were heterozygous. Comparison of tumorous and non-tumorous patterns showed that only one (5%) tumorous tissue out of 20 heterozygous patients was homozygous, indicating the loss of an allele at this locus. We conclude that allelic loss in the cyclin A gene is a rare event in patients with PLC, and that PCR is rapid and reliable for the detection of allelic loss in the cyclin A gene when only small amounts of DNA are available.

Immunocytochemical and Electron Microscopic Study of Hepatitis B Virus Antigen and Complete Particle Production in Hepatitis B Virus Dna Transfected Hepg2 Cells

The relationship between the presence of hepatitis B virus antigens, their localization and hepatitis B virus replication was studied in different clones of cultured HepG2 hepatoblastoma cells transfected with cloned hepatitis B virus DNA. Intracellular hepatitis B virus antigens were detected by immunofluorescence. The production of these antigens was evaluated in the culture media by enzyme-linked immunoassay. Hepatitis B virus DNA was detected using dot-blot hybridization. Three types of HBcAg staining were observed in transfected HepG2 cells: (a) cells with nuclear HBcAg, (b) cells with cytoplasmic HBcAg and (c) cells with both nuclear and cytoplasmic HBcAg. Cell types b and c also expressed hepatitis B virus DNA in their culture media. Our results suggest that cytoplasmic HBcAg may be more involved than nuclear HBcAg in hepatitis B virus replication. The site of hepatitis B virus formation in hepatocytes was studied by electron microscopic examination of a specific hepatitis B virus producer clone, thereby allowing detection of intracellular Dane particles more easily than liver biopsy samples from infected patients. Dane particles and HBsAg filaments were found in large, dilated structures probably related to the endoplasmic reticulum. Budding of core particles into cisternae of endoplasmic reticulum-related structures appears to be a possible mechanism for hepatitis B virus formation; our results suggest that the exocytosis of cisternae to extracellular spaces may be a mechanism for release of hepatitis B virus particles.

The pre S1 domain is the attachment site of hepatitis B virus to host cell membranes

The pre S1 domain is the attachment site of hepatitis B virus to host cell membranes

Expression of the hepatitis B virus genome in chronic hepatitis B carriers and patients with hepatocellular carcinoma.

We examined the methylation status of CCGG sites in hepatitis B virus (HBV) DNA to determine whether methylation could be responsible for the selective expression of the HBV surface gene in chronic hepatitis B infection and hepatocellular carcinoma. Infected liver tissue from patients with low levels of viral replication was analyzed for HBV DNA copy number per haploid cell genome. Total cellular DNA, with sufficient HBV DNA, was digested with the restriction endonucleases Msp I and Hpa II, to determine whether the HBV DNA was methylated, or HindIII, to determine whether the HBV DNA was integrated or episomal. The cleavage fragments were analyzed by Southern blotting and hybridization to 32P-labeled HBV DNA. In replicative chronic hepatitis B, hypomethylation of the HBV genome correlated with HBV expression in both virions and infected tissue. In carriers with nonreplicative infection, it was difficult to ascertain the role of methylation as copy number was low. HBV DNA copy number was also low in 17 out of 29 of the tumor tissues tested and as many as 14 out of 16 of the adjacent non-neoplastic tissues tested. Integrated sequences were hypermethylated in the PLC/PRF/5 cell line and in six of the tumor tissues suggesting that methylation plays a role in HBV gene repression. However, since DNA from five other tumors was hypomethylated, the belief that methylation per se is an absolute determinant of HBV core gene repression does not hold for human hepatocellular carcinoma tissue. Additional factors, such as gene rearrangements, therefore, must influence HBV expression in hepatocellular carcinoma.

Chromosomal site of hepatitis B virus (HBV) integration in a human hepatocellular carcinoma-derived cell line.

The single site of integration of hepatitis B virus in the human hepatocellular carcinoma cell line Hep 3B 2-1/7 was found to segregate with human chromosome 12 in somatic cell hybrids. Analysis of metaphase spreads of Hep 3B 2-1/7 following in situ hybridization with pHBV revealed integration at 12q13----q14, a location that coincides with a fragile site, fra (12q13). The possible significance of this location to the development of hepatocellular carcinomas is discussed.