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Shinji Watanabe

Publications and source records attributed to Shinji Watanabe.

27 records · Page 2Linked to original sources
PubMed2003

Influenza B virus BM2 protein is transported through the trans-Golgi network as an integral membrane protein.

A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.

Animals↗
PubMed2003

Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses.

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.

Amino Acid Sequence↗
PubMed2003

[Significance of the visual evaluation method using the two-visit method (two-sample preference test suggested by Ferris)].

To increase the accuracy of the visual evaluation method, we studied the significance of the two-sample preference test using the two-visit method, as proposed by Ferris. Advantages of this method include not only its ability to estimate whether the image quality of the sample is statistically the same but also its ability to distinguish the difference between samples more clearly than when using the conventional two-sample preference test or paired-comparison method. However, a disadvantage is that this method involves two observations of the same sample. When using chest images, even if a difference between the samples is not recognizable by the conventional method, a significant difference can be elicited with this method. The data obtained by this method can be evaluated with the paired-comparison method, so some samples may be ranked as well. Therefore, we verified that the two-sample preference test, which Ferris formulated, is useful in visual evaluations.

Humans↗
PubMed2002

Influenza A virus with defective M2 ion channel activity as a live vaccine.

We propose a rational approach to the design of live virus vaccines against influenza infection by alteration of the influenza A virus M2 protein, which is responsible for ion channel activity. Previously we demonstrated that a mutant A/WSN/33 (H1N1) influenza virus with defective M2 ion channel activity did not show appreciable growth defects in cell culture, although its growth was attenuated in mice (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, 2001, J. Virol. 75, 5656-5662). Here, we show that this M2 ion channel defective mutant virus, the M2del29-31, protected mice against challenge with lethal doses of influenza virus, indicating the potential of incorporating this M2 alteration in a live influenza vaccine as one of the attenuating mutations.

Animals↗
PubMed2002

The FANCG Fanconi anemia protein interacts with CYP2E1: possible role in protection against oxidative DNA damage.

Fanconi anemia (FA) is a genetic disorder that leads to aplastic anemia and birth defects and predisposes to cancer. FA cells exhibit characteristic hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC), and FANCG is one of six known FA gene products. By immunocytochemical analysis of transfected cells, we discovered that although FANCG localized to both the nucleus and cytoplasm, there was an increase in cells with predominantly cytoplasmic staining after treatment with MMC. Concurrently, while searching by two-hybrid analysis for proteins that associate with FANCG, we identified a novel interaction between FANCG and cytochrome P450 2E1 (CYP2E1). A member of the P450 superfamily, CYP2E1 is associated with the production of reactive oxygen intermediates and the bioactivation of carcinogens. High constitutive levels of CYP2E1 were found in a FA-G lymphoblast cell line, whereas complementation of the FA-G line with wild-type FANCG was associated with decreased CYP2E1. These findings suggested that the interaction of FANCG with CYP2E1 might alter redox metabolism and increase DNA oxidation. Using a fluorescent assay, we found a dose-dependent increase in the oxidized DNA base, 8-oxoguanine (8-oxoG), after treatment of mutant FA-G cells with H(2)O(2) or MMC. Conversely, significantly lower levels of 8-oxoG were detected in FANCG-complemented FA-G cells. We conclude that the unknown function of FANCG involves at least transient interaction with cytoplasmic components, possibly including CYP2E1, and propose a role for FANCG in protection against oxidative DNA damage.

Blotting, Western↗
PubMed2002

Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.

Animals↗
PubMed2002

Immunogenicity and protective efficacy of replication-incompetent influenza virus-like particles.

Despite the success of influenza virus vaccines in reducing severe illness, their efficacy is suboptimal. We describe here the immunogenicity and protective capacity of replication-incompetent influenza virus-like particles (VLPs) which were generated entirely from cDNAs and lacked either the entire NS gene (encoding both the NS1 and NS2 protein) or only the NS2 gene. In mammalian cells infected with NS gene-deficient VLPs, the nucleoprotein, but not other viral proteins including hemagglutinin (HA) and neuraminidase (NA), was detected. In contrast, cells infected with VLPs expressing NS1 but not NS2 (NS2 knockout) expressed multiple viral proteins, including HA and NA. When challenged with lethal doses of an antigenically homologous mouse-adapted influenza virus, 94% of mice vaccinated with the NS2-knockout VLPs survived, compared with less than 10% of those given the NS-deficient VLPs. These results demonstrate the potential of replication-incompetent NS2-knockout VLPs as novel influenza vaccines and perhaps also as vectors to express genes from entirely unrelated pathogens.

Animals↗