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Biomedical subjects

Akio Nomoto

Publications and source records attributed to Akio Nomoto.

At least 19 recordsLinked to original sources

Prevalence and phylogeny of Kakugo virus, a novel insect picorna-like virus that infects the honeybee (Apis mellifera L.), under various colony conditions.

We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To survey the prevalence of KV in worker populations engaged in various labors, we quantified KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was also detected from other worker populations in other colonies where the amount of KV detected in the workers was relatively high, suggesting that KV can infect various worker populations in the honeybee colonies. To investigate whether the KV strains detected were identical, phylogenetic analysis was performed. There was less than a 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, suggesting that all of the viruses detected were virtually the same KV. We also found that some of the KV-infected colonies were parasitized by Varroa mites, and the sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. KV strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus 1, respectively, suggesting that KV represents a viral strain closely related to, but distinct from, these two viruses.

Animals↗

Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141.

Natural killer (NK) cells are crucial in the control of cytomegalovirus infections in mice and humans. Here we show that the viral UL141 gene product has an immunomodulatory function that is associated with low-passage strains of human cytomegalovirus. UL141 mediated efficient protection of cells against killing by a wide range of human NK cell populations, including interferon-alpha-stimulated bulk cultures, polyclonal NK cell lines and most NK cell clones tested. Evasion of NK cell killing was mediated by UL141 blocking surface expression of CD155, which was previously identified as a ligand for NK cell-activating receptors CD226 (DNAM-1) and CD96 (TACTILE). The breadth of the UL141-mediated effect indicates that CD155 has a key role in regulating NK cell function.

Cells, Cultured↗

Blockade of the poliovirus-induced cytopathic effect in neural cells by monoclonal antibody against poliovirus or the human poliovirus receptor.

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2Apro, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2Apro was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2Apro is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.

Amino Acid Sequence↗

Ligand stimulation of CD155alpha inhibits cell adhesion and enhances cell migration in fibroblasts.

CD155 (poliovirus receptor) localizes in cell-matrix adhesions and cell-cell junctions, but its role in the regulation of cell adhesion and cell motility has not been investigated. We identified a conserved immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain of human CD155alpha. The ITIM was tyrosine-phosphorylated upon binding of anti-CD155 monoclonal antibody D171, poliovirus, and DNAM-1 (CD226) to human CD155alpha, and recruited SH2-domain-containing tyrosine phosphatase-2 (SHP-2). After CD155alpha stimulation with its ligands, cell adhesion was inhibited and cell motility was enhanced, effects that were associated with the phosphorylation of ITIM by Src kinases and accompanied by dephosphorylation of focal adhesion kinase and paxillin. These effects were abolished by introducing a point-mutation in Y398F into the ITIM of CD155alpha and by coexpression of a dominant negative SHP-2 mutant with CD155alpha. These results suggest that CD155alpha plays a role in the regulation of cell adhesion and cell motility.

Amino Acid Sequence↗

A poliomyelitis model through mucosal infection in transgenic mice bearing human poliovirus receptor, TgPVR21.

Transgenic mice bearing the human poliovirus receptor (TgPVR) are less susceptible to oral inoculation, although they are susceptible to parenteral inoculation. We investigated the susceptibility of TgPVR 21 line [Arch. Virol. 130 (1994) 351] to poliovirus through various mucosal routes. Intranasal inoculation of a neurovirulent Mahoney strain (OM1) caused flaccid paralysis with viral replication in the central nervous system at a dose of 10(6) cell culture infectious dose (CCID50), in contrast, no paralysis following oral or intragastric inoculation of the same dose. Intranasal inoculation of a vaccine strain, Sabin 1, at 10(6) CCID50, resulted in no paralysis. Initial replication of poliovirus in the nasal cavity was confirmed by virus isolation and detection of negative-stranded replicative intermediates by RT-PCR and viral antigens using a high-sensitive immunohistochemistry and genome/transcripts by in situ hybridization. Poliovirus-specific IgG antibodies were elevated in the sera of surviving TgPVR21. This model can be used as a mucosal infection model and for differentiation of neurovirulent and attenuated poliovirus strains.

Administration, Intranasal↗

Total viral genome copies and virus-Ig complexes after infection with influenza virus in the nasal secretions of immunized mice.

The kinetics of infectious virus (p.f.u.), total virus and virus-Ig complex formation following influenza A/PR8 (H1N1) viral infection was examined in the nasal secretions of naive mice and mice immunized with A/PR8, A/Yamagata (H1N1), A/Guizhou (H3N2) and B/Ibaraki influenza viruses. The total number of virus particles and the number within virus-Ig complexes, captured in advance using an anti-mouse Ig-coated plate, were determined on the basis of viral genome copy number using quantitative RT-PCR. The kinetics of infectious and total virus particle formation, the latter of which increased by 10(3)-10(4)-fold above infectious virus numbers, showed that virus elimination from the nasal area was earlier in A/PR8, A/Yamagata and A/Guizhou-X virus-immunized mice, in decreasing order, compared with naive mice. Early virus elimination correlated with the level of A/PR8 virus-reactive antibodies in immunized mice. Virus elimination coincided with the appearance of virus-Ig complexes shortly after infection. This result suggested that antibodies led to the formation of immune complexes in a dose-dependent manner together with a reduction in number of infectious virus particles. The fact that a large number of virus particles was observed in immune complexes for a wide range antibody levels made it difficult to detect slight differences in virus number within the immune complexes, depending on antibody level. These results suggested that the formation of virus-Ig complexes in virus-immunized mice shortly after infection is involved in early virus elimination, which is determined by the strength of protective immunity against challenge viruses.

Animals↗

Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome.

Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pull-down experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVR alpha, as well as a mutant hPVR alpha (hPVRM alpha) that had a reduced ability to bind Tctex-1, colocalized with Tctex-1. However, vesicles containing PV, dextran, and hPVR alpha had only retrograde motion, while those containing PV, dextran, and hPVRM alpha had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.

Amino Acid Sequence↗

Membrane binding properties and terminal residues of the mature hepatitis C virus capsid protein in insect cells.

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with alpha-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.

Amino Acid Sequence↗

Novel insect picorna-like virus identified in the brains of aggressive worker honeybees.

To identify candidate genes involved in the aggressive behavior of worker honeybees, we used the differential display method to search for RNAs exclusively detected in the brains of aggressive workers that had attacked a hornet. We identified a novel, 10,152-nucleotide RNA, termed Kakugo RNA. Kakugo RNA encodes a protein of 2,893 amino acid residues that shares structural features and sequence similarities with various picorna-like virus polyproteins, especially those from sacbrood virus, which infects honeybees. The Kakugo protein contains several domains that correspond to the virion protein, helicase, protease, and RNA-dependent RNA polymerase domains of various picorna-like virus polyproteins. When the worker bee tissue lysate was subjected to sucrose density gradient centrifugation, Kakugo RNA, except for the material at the bottom, was separated into two major peaks. One of the peaks corresponded to the position of Kakugo mRNA, and the other corresponded to the position of the poliovirus virion. These results suggest that the Kakugo RNA exists as an mRNA-like free RNA and virion RNA in the honeybee. Furthermore, injection of the lysate supernatant from the attacker heads into the heads of noninfected bees resulted in a marked increase in Kakugo RNA. These results demonstrate that Kakugo RNA is a plus-strand RNA of a novel picorna-like virus and that the brains of aggressive workers are infected by this novel virus. Kakugo RNA was detected in aggressive workers but not in nurse bees or foragers. In aggressive workers, Kakugo RNA was detected in the brain but not in the thorax or abdomen, indicating a close relation between viral infection in the brain and aggressive worker behaviors.

Aggression↗

Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells.

An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.

Animals↗

Characterization of the poliovirus 147S particle: new insights into poliovirus uncoating.

A Sabin 1 strain poliovirus (PV) mutant, S1(2Y-1I), carrying a Tyr at amino acid position VP2(142) and an Ile at position VP1(160), can establish persistent infections in HEp-2c cells. This mutant forms atypical 147S particles upon interaction at 0 degrees C with either cells expressing PV receptor (PVR) CD155, or PVR-IgG2a, a chimeric molecule consisting of an extracellular moiety of PVR and the hinge and Fc portion of a mouse IgG2a. Upon interaction with PVR at 37 degrees C, S1(2Y-1I), similar to the parental strain, forms both 135S A particles and 80S empty capsids. At 0 degrees C, surprisingly, at a concentration equal to or greater than 5 nM, PVR-IgG2a induced both the extrusion of VP4 from the capsid of S1(2Y-1I) and the formation of 80S particles. The same transitions were observed at 0 degrees C with the parental strain Sabin 1 at 40 nM PVR-IgG2a. Thus, the formation of 80S particles and VP4 extrusion, considered as one of the steps of PV uncoating, can be temperature-independent at high PVR concentration. This implies that structural changes of the PV capsid occurred following adsorption at low temperature.

Capsid↗

Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor.

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.

Animals↗

[Poliovirus].

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Diagnosis, Differential↗

Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant-combined A/Beijing/262/95 (H1N1) virus hemagglutinin or neuraminidase.

Antibody (Ab) responses and protection against influenza virus infection in mice immunized intranasally with hemagglutinin (HA) or neuraminidase (NA) purified from the A/Beijing/262/95 (A/Beijing) (H1N1) virus were compared among B10 congenic mouse strains. Mice were immunized intranasally with 0.1, 0.3 or 1microg of HA or NA together with the cholera toxin adjuvant, and then boosted intranasally with 0.3 microg of the adjuvant-combined HA or NA 4 weeks later. Two weeks after the second immunization, the mice were challenged by an infection of the upper respiratory tract with the homologous virus. After 3 days, nasal wash and serum specimens were collected for virus and Ab titration. The HA immunization induced HA-specific IgG Ab responses against A/Beijing HA, which depended on the H-2 haplotype of the strain: The B10.A (H-2(a)), B10.D2 (H-2(d)), B10.BR (H-2(k)) and B10 (H-2(b)) strains were the highest, high, intermediate and low responders, respectively. The nasal IgA responses were induced in the B10.A, B10.D2 and B10.BR strains, but not in the B10 strain. In parallel with Ab responses, the B10.A, B10.BR and B10.D2 strains were conferred significant protection at any dose of primary immunization, but the B10 strain was provided protection only at 1microg of HA. On the other hand, the NA immunization induced NA-specific Ab responses, which depended on the the H-2 haplotype of the strain: the B10.A, B10.D2, B10 and B10.BR strains were the highest, high, intermediate and low responders, respectively. In parallel with Ab responses, all the strains were conferred significant protection at any dose of primary immunization. These results indicate that the MHC-restricted responsiveness of mice to HA is different from that to NA, suggesting that the use of high-HA dose or NA as a component of the nasal influenza A (H1N1 subtype) virus vaccine improves the protective efficacy against influenza among low responder populations.

Adjuvants, Immunologic↗

Immunofluorescence analysis of poliovirus receptor expression in Peyer's patches of humans, primates, and CD155 transgenic mice: implications for poliovirus infection.

Oral transmission of poliovirus is restricted to humans and certain primate species. The expression of the human poliovirus receptor (CD155) within gastrointestinal-associated lymphoid tissues from species that are susceptible (human) or resistant (rhesus macaque and CD155 transgenic [Tg] mice) to oral poliovirus infection was examined. Sensitivity to oral infection correlated with CD155 expression not only in the intestinal epithelium, including the follicle-associated epithelium (FAE) and microfold (M) cells of Peyer's patches, but also in germinal centers within the Peyer's patches. CD155 expression in rhesus macaques was reduced in FAE and, significantly, absent in germinal centers. In CD155 Tg mice, CD155 expression was barely observable in the intestinal epithelium, absent in germinal centers, but prominent in the tunica muscularis. This suggests that productive poliovirus infection of the gut is dependent on the expression of CD155 within the FAE, including the M cells, and on cells within Peyer's patches, most likely within germinal centers.

Animals↗