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Biomedical subjects

K Shimotohno

Publications and source records attributed to K Shimotohno.

At least 109 records · Page 6Linked to original sources
PubMed1995

Increased protein tyrosine-phosphorylation in primary T-cells transduced with Tax1 of human T-cell leukemia virus type I.

Protein tyrosine-phosphorylation in primary human T-cells transduced with Tax1 of Human T-cell leukemia virus type I was investigated. In comparison with control T-cells, the level of protein tyrosine-phosphorylation after stimulation with anti-CD3 antibody increased significantly in Tax1-transduced T-cells. This enhancement in tyrosine-phosphorylation possibly accounted for the augmented proliferation response of these cells, which has been reported previously [J. Virol. 67 (1993) 1211-1217].

Cell Line, Transformed↗
PubMed1995

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 electron-dense 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.

Base Sequence↗
PubMed1995

Expression and processing of putative nonstructural proteins of hepatitis C virus in insect cells using baculovirus vector.

Processing of the putative nonstructural (NS) proteins, p70(NS3), p4(NS4A), p27(NS4B), p58/56(NS5A), and p66(NS5B), of Japanese type hepatitis C virus (HCV) in insect cells was analyzed by using a baculovirus expression system. Products processed by the HCV serine proteinase (Cpro-2) were essentially identical to those found in mammalian cultured cells transiently producing the NS region of the HCV precursor polyprotein. A series of internal and carboxy (C)-terminal deletion experiments coupled with epitope scanning analysis showed that efficient cleavage at the Cpro-2-dependent processing sites, except at the p4(NS4A)/p27(NS4B) site, is not significantly influenced by those mutations. Efficient cleavage at p4(NS4A)/p27(NS4B) required about 40% of the NS5A N-terminal region. Estimation of the processing sites by determination of the N-terminal amino acid sequences of the processed products revealed that all the Cpro-2-dependent cleavages occurred at essentially identical sites to those reported for another HCV genotype, suggesting that Cpro-2 is a possible target for the development of a strain-independent anti-HCV agent.

Amino Acid Sequence↗
PubMed1995

Novel genotypes of hepatitis C virus in Thailand.

We examined 24 C-type hepatitis specimens from Thailand and detected hepatitis C virus (HCV) RNA in all of them by RT-nested PCR for a portion of the HCV 5' non-coding (5' NC) region and a portion of the HCV core region. However, we failed to detect HCV RNA in 11 specimens by RT-nested PCR for a portion in the non-structural protein 5 (NS5) region that has been used commonly for HCV genotyping. We designed a new primer set for a separate portion of the NS5 region. Using this primer set, we succeeded in amplifying this portion in all 24 specimens. Two novel HCV genotypes, tentatively designated HCV-VII and HCV-VIII, were identified by sequencing these amplified regions. Our newly designed primers for RT-nested PCR may be useful for diagnosing infection as well as for genotyping unidentified HCV genomes.

Base Sequence↗
PubMed1995

Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.

A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.

Amino Acid Sequence↗
PubMed1995

NS3-4A of hepatitis C virus is a chymotrypsin-like protease.

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.

Base Sequence↗
PubMed1995

Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A.

Two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural region 5A (NS5A). Recently, we found that both proteins are phosphorylated at serine residues and that p58 is a hyperphosphorylated form of p56. Furthermore, hyper-phosphorylation depends on the production of an intact form of the HCV NS4A protein. To clarify the nature of NS5A phosphorylation, pulse-chase analysis was performed with a transient protein production system in cultured cells. The study indicated that basal and hyperphosphorylation of NS5A occurred after proteolytic production of NS5A was complete. In an attempt to identify the location of the hyperphosphorylation sites in p58, proteins with sequential deletions from the C-terminal region of NS5A and with mutations of possible phosphorylated serine residues to a neutral amino acid, alanine, were constructed. The deleted or mutated proteins were then tested for hyperphosphorylation in the presence of the NS4A product. Here, we report that serine residues 2197, 2201, and/or 2204 are important for hyper-phosphorylation. Important sites for basal phosphorylation were identified in the region from residues 2200 to 2250 and in the C-terminal region of the NS5A product. A subcellular localization study showed that most of the NS5A products were localized in the nuclear periplasmic membrane fraction.

Amino Acid Sequence↗
PubMed1995

The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A.

Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3.

Base Sequence↗
PubMed1995

Hepatitis C virus as a causative agent of hepatocellular carcinoma.

HCV infection has shown to be strongly linked to the development of hepatocellular carcinoma in epidemiological studies. However, the mechanism of carcinogenesis by HCV is poorly understood. Unlike other human oncogenic viruses, HCV is a typical RNA virus, and thus there is no integration of the viral genome or a piece of the genome into host chromosomes. Moreover, trans-acting transcriptional factors which are coded by other human oncogenic viruses and required primarily for virus replication and often involved in cell immortalization, may not be coded by HCV. Although regeneration of hepatocytes in an HCV-infected liver may be of primary importance in driving hepatocytes to the malignant stage, an additional unknown carcinogenic function of HCV may also exist. For this reason, clarification of the molecular mechanism of virus replication in hepatocytes should be emphasized.

Carcinoma, Hepatocellular↗
PubMed1995

Processing of the hepatitis C virus precursor protein.

Proteins of hepatitis C virus (HCV) are produced from a polyprotein precursor by post-translational processing. Production of HCV proteins was analyzed with in vitro translation, as well as plasmid-based transient gene expression, in mammalian cell lines. A minimum of three different processing pathways yielded at least 10 viral proteins from the polyprotein precursor. One pathway depended on signal protease processing, and the other two pathways utilized viral proteinases. The signal peptidase cleaved the viral structural proteins, and two viral activities broke up the viral nonstructural proteins. With staggered cleavages, the signal peptidase produced two E2 products from the E2 region, gp70 type A and type B, differing in the C-terminal structure. Two viral proteinases partially overlapped in the N-terminal region of NS3; the functional amino acid residues required for those two activities differed. Most of the processed viral proteins bound together; some of the associated proteins were membrane bound.

Amino Acid Sequence↗