Torahiko Tanaka

Marked sequence diversity in the putative envelope proteins of hepatitis C viruses

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent infection of hepatitis E virus transmitted by blood transfusion in a patient with T-cell lymphoma.

Aim:  With advent of reverse‐transcription (RT)/polymerase chain reaction (PCR) for detection of the hepatitis E viral genome, we carried out retrospective examinations.

Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.

We recently found that bovine lactoferrin (bLF), a milk glycoprotein belonging to the iron transporter family, prevented hepatitis C virus (HCV) infection in human hepatocyte PH5CH8 cells, that are susceptible to HCV infection, and demonstrated that the anti-HCV activity of bLF was due to the interaction of bLF and HCV. In this study we further characterized the anti-HCV activity of bLF and the mechanism by which bLF prevents HCV infection. We found that bLF inhibited viral entry to the cells by interacting directly with HCV immediately after mixing of bLF and HCV inoculum. The anti-HCV activity of bLF was lost by heating at 65 degrees C, and other milk proteins (mucin, beta-lactoglobulin and casein) did not prevent HCV infection, indicating that bLF prevented HCV infection in a rather specific manner. Furthermore, we found that bovine lactoferricin, a basic N-terminal loop of bLF that is an important region for antibacterial activity, did not exhibit any anti-HCV activity, suggesting that some other region is involved in anti-HCV activity. We confirmed that prevention of HCV infection by bLF was a general phenomenon, because bLF inhibited HCV infection with all five inocula examined, and bLF inhibited HCV infection in human MT-2C T-cells, that were susceptible to HCV infection. In addition, infection with hepatitis G virus, which is distantly related to HCV, was prevented also by bLF. In conclusion, lactoferrin is a natural glycoprotein which effectively protects against HCV infection in hepatocytes and lymphocytes by neutralizing the virus.

Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus

We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70-100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32 degrees C. Moreover, we demonstrated that interferon alpha had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.

Activation of the Interferon-Inducible 2′-5′-Oligoadenylate Synthetase Gene by Hepatitis C Virus Core Protein

ABSTRACT The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2′-5′-oligoadenylate synthetase (2′-5′-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2′-5′-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2′-5′-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2′-5′-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2′-5′-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2′-5′-OAS gene.

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.

Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b558 in the reaction.

Heterocyclic nitrogenous bases, such as pyridine and imidazole which bind to heme-iron in cytochromes, inhibited the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells. Inhibition was accompanied by a spectral change in reduced cytochrome b558 as judged by low-temperature spectroscopy at 77 K. The position and shape of the alpha-band of the cytochrome were significantly altered upon exposure to pyridine or some other bases. Both inhibition and spectral changes were reversible. The results are consistent with the view that cytochrome b558 is involved in the NADPH oxidase system in neutrophils.

Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector

SummaryProcessing of HCV viral precursor protein requires at least two viral proteinases, Cpro-1 and Cpro-2, in addition to cellular proteinases. The HCV polypeptide that covers the region for the two viral proteinase domains was expressed in insect cells using a baculovirus expression system. The two proteinase activities were demonstrated in the infected cells. The Cpro-1-dependent cleavage site was estimated from the amino acid sequence of the N-terminus of the processed product. Analyses of the susceptibilities of various mutants altered at position P 1 and P 1′ of the putative cleavage site suggested that amino acid residues at these positions is not essential for recognition and cleavage by Cpro-1-dependent activity.

Identification and characterization of a cytotoxic T cell epitope of hepatitis C virus presented by HLA-B*3501 in acute hepatitis

In order to clarify the role of cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) infection, an HLA-B35-restricted cytotoxic T cell epitope of HCV was identified using a strategy called reverse immunogenetics. Twenty-eight of 53 HCV peptides carrying two anchor residues were selected as HLA-B*3501 binding peptides. These peptides were used to induce the specific cytotoxic T cells in peripheral blood lymphocytes from a patient with acute hepatitis C. Six HLA-B*3501 binding peptides induced the peptide-specific CTL. One (HPNIEEVAL) of five peptides was confirmed as the epitope by the specific T cell clones. A sequence identical to the epitope was detected in isolates of the virus from the patient and a strong CTL response to this epitope was observed in the acute phase of hepatitis C but not in the recovery phase. The specific CTL for this epitope were not detected in peripheral blood lymphocytes from patients with chronic hepatitis C. Together these results suggest that the CTL specific for this epitope have an important role in the elimination of the virus in the patient.

In situ phage screening: A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 μm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-α) and one minor (α-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.