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Biomedical subjects

A Nomoto

Publications and source records attributed to A Nomoto.

At least 55 records · Page 3Linked to original sources
PubMed1997

Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor.

The transgenic (Tg) mice carrying the human gene for poliovirus receptor (PVR) are susceptible to poliovirus intravenously (i.v.) inoculated as well as intracerebrally or intraspinally inoculated. Thus, i.v.-inoculated poliovirus may invade the central nervous system (CNS) through the blood-brain barrier (BBB). To know the contribution of PVR to tissue distribution and BBB permeability of i.v.-inoculated polioviruses, these dissemination processes were investigated and compared between the Tg mice and non-Tg mice. Distribution profile of i.v.-inoculated poliovirus in various tissues of the Tg mice is similar to that in non-Tg mice. The data suggest that tissue distribution of the virus occurs independently of the transgene for PVR. The amount of poliovirus delivered to the CNS suggested the existence of a specific delivery system of the virus to the CNS. Virus accumulation in the CNS of the Tg mice was measured up to 7.5 hr after the i.v. inoculation. The viruses, regardless of whether the virulent or attenuated strain, seem to accumulate at a constant rate of approximately 0.2 microliter/min/g tissue. Similar phenomena were observed when the viruses were inoculated into non-Tg mice. The rates of the virus accumulation in the CNS are more than 100 times higher than that of albumin, which is considered not to permeate through the BBB via a specific transport system, and only three times lower than that of monoclonal antibody against transferrin receptor (OX-26), which is a potential candidate as a drug delivery vehicle specific to the CNS. These data suggest that polioviruses permeate through the BBB at a fairly high rate, independently of PVR and virus strains.

Animals↗
PubMed1997

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.

Animals↗
PubMed1997

Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.

Base Sequence↗
PubMed1995

Studies on neurovirulence in poliovirus-sensitive transgenic mice and cynomolgus monkeys for the different temperature-sensitive viruses derived from the Sabin type 3 virus.

We have studied methods for testing the neurovirulence of live poliovaccine viruses by intraspinal inoculation into mice carrying the human poliovirus receptor gene (Tg mice). A comparison of the neurovirulence of Sabin type 3 vaccine virus and related viruses using the 50% paralysis dose determined after intraspinal inoculation into the Tg mice as an index revealed a close correlation between the results of the paralysis dose in Tg mice, the neurovirulence expressed by the histopathological lesions core in monkeys, and the temperature sensitivity of the viruses. The results of experiments in the Tg mice also showed a good correlation with the number of mutations at position 472 from U to C in the 5' noncoding region in the genomes of the viruses tested. These results strongly suggest that the neurovirulence test for oral poliomyelitis vaccine using the Tg mice is an excellent method and may be used in place of the test using monkeys.

Animals↗
PubMed1995

Poliovirus-specific CD4+ Th1 clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor.

The current understanding of the function of CD4+ T helper (Th) cells in immunity to infectious diseases is that Th1 cells, which secrete interleukin (IL)-2 and interferon-gamma, induce cellular immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, provide helper function for humoral immunity. We have used a panel of poliovirus-specific murine CD4+ T cell clones and mice transgenic for the human poliovirus receptor to evaluate the role of Th cell subpopulations in protective immunity to poliovirus. The majority of T cell clones, as well as polyclonal T cells generated from mice infected or immunized with poliovirus, secreted IL-2 and interferon-gamma, but not IL-4, IL-5, or IL-10, a profile typical of Th1 cells. The Th1 clones displayed major histocompatibility complex class II-restricted cytotoxic T lymphocyte activity against specific poliovirus peptide-pulsed target cells, but also provided help for antipoliovirus neutralizing antibody production. To examine the mechanism of immunity in vivo, we have used poliovirus receptor-transgenic mice on a BALB/c (H-2d) background. These animals developed a poliomyelitis-like disease when challenged intravenously with a virulent wild-type strain of poliovirus, but not with an attenuated vaccine strain. Furthermore, mice immunized with the vaccine strain were protected against a subsequent challenge with wild-type virus. Using an adoptive transfer technique, we demonstrated that it was possible to confer protection with primed B cells in the presence of polyclonal poliovirus-specific T cells, but not when transgenic mice received either B cells or T cells alone. Furthermore, protection was observed when mice received primed B cells in the presence of a VP4-specific Th1 clone. The findings demonstrate that Th1 cells can mediate a protective immune response against poliovirus infection in vivo through helper activity for humoral immunity and that CD4+ T cells, specific for the internal poliovirus capsid protein, VP4, can provide effective help for a protective antibody response directed against surface capsid proteins.

Animals↗
PubMed1995

Neurovirulence test for oral live poliovaccines using poliovirus-sensitive transgenic mice.

The transgenic mice, ICR-PVRTg21, carrying the human poliovirus receptor gene, were intraspinally inoculated with oral poliovirus vaccine viruses and the viruses that had been derived from a vaccine preparation by passagings at 38 degrees. Dose-dependent incidence of paralysis was observed in the transgenic mice inoculated with any of the viruses used. All transgenic mice showing histopathological lesions in the central nervous system appeared to display clinical signs that resembled those observed in human poliomyelitis. These suggest that relative neurovirulence levels of individual virus preparations can be estimated by values of 50% paralysis dose. Indeed, the parameter correlated well with monkey lesion scores of viruses used. Histopathological changes such as degeneration of neurons resemble those displayed by monkeys and were observed in the spinal cord, medulla oblongata, and pons. Thus, the transgenic mice, ICR-PVRTg21, may become a new animal model in place of monkeys for safety testing of oral poliovirus vaccine preparations.

Animals↗
PubMed1995

A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.

Animals↗
PubMed1995

Mechanism of translation initiation on hepatitis C virus RNA.

In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site [IRES]) within the 5' noncoding region (5'NCR). Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests. A protein p57 (polypyrimidine tract binding protein [PTB]), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES. To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E. coli. Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells. In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs. As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells. Recombinant HCV IRESs were constructed and tested for their function in the in vitro system. Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher. This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level.

Animals↗
PubMed1994

Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor.

A set of genes (SWI1, SWI2/SNF2, SWI3, SNF5 and SNF6) in Saccharomyces cerevisiae are required for transcription of a variety of yeast genes. It was recently reported that the mammalian glucocorticoid receptor failed to activate transcription when transiently expressed in swi1-, swi2- or swi3- yeast strains. We report here that two highly related human cDNAs, hSNF2 alpha and -beta, encode amino acid sequences homologous to both the yeast SWI2/SNF2 and the Drosophila brahma. Similar to their yeast and Drosophila counterparts, both human cDNAs contain helicase motifs, a bromodomain, a highly charged C-terminal sequence and an N-terminal sequence rich in proline, glutamine and glycine. Tissue distribution of the mRNAs varied slightly. Transcriptional activation by the estrogen receptor and the retinoic acid receptor was enhanced by co-expression of either hSNF2 cDNA. No enhancement was observed for promoters which do not respond to nuclear receptors. We suggest that global transcriptional coactivators equivalent to the yeast SWI/SNF complex exist in mammalian cells.

Adenosine Triphosphatases↗
PubMed1994

Amino acid residues on human poliovirus receptor involved in interaction with poliovirus.

We have previously demonstrated that the N-terminal immunoglobulin-like domain (domain 1; 115 amino acids) of human poliovirus receptor (hPVR) is essential for poliovirus binding and infection to cells. To identify amino acids involved in the interaction with poliovirus, we constructed a number of cDNAs encoding mutant hPVRs whose domain 1 was partially derived from mouse PVR (mPVR) homolog, which does not serve as a binding site for poliovirus. Poliovirus binding and infection assays were performed on mouse L cells that express these chimera cDNAs. Anti-hPVR monoclonal antibodies were employed to confirm the presence of mutant PVRs on the surface of mouse cells and to know conformational alteration of these PVRs. A significant decrease in efficiency of both poliovirus binding and infection to the cells was observed when one or a few amino acids of hPVR at Gly73, Ser74, Gln82, Leu99-Glu102, or Gln130-Ser132 were substituted by the corresponding amino acids of mPVR. Similar results were obtained when a 2-amino acid insertion of mPVR, which was missing in hPVR, was introduced at the corresponding site (between Arg98 and Leu99) of hPVR. These amino acids were highly conserved in functional PVRs of primates but not in unfunctional PVRs of rodents. These results indicate that the amino acids identified may have important roles in interaction of PVR with poliovirus that leads to the establishment of the virus infection. In the three-dimensional model of the domain 1 of hPVR, these amino acids are located on one side of the molecule. This suggests that the interaction with poliovirus occurs on this side of the domain 1.

Amino Acid Sequence↗
PubMed1994

Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene--influence of the transgene expression on pathogenesis.

Three transgenic mouse lines, ICR-PVRTg1, ICR-PVRTg5, and ICR-PVRTg21, which are susceptible to poliovirus, have been established by introducing the human gene for poliovirus receptor (PVR) into the genome of mouse strain ICR. Genetic characterizations of the PVR gene were carried out on these mouse lines to define the approximate copy number, insertion site, and expression of the transgene in the central nervous system (CNS). The transgene was integrated in the chromosome 4, 12, and 13 of ICR-PVRTg1, ICR-PVRTg5 and ICR-PVRTg21 mice, respectively, and was stably transmitted to progeny mice. ICR-PVRTg1 appeared to have the most abundant copy numbers of the transgene and showed the highest level of PVR mRNA and membrane associated PVR protein in the CNS among the three mouse lines. Those in ICR-PVRTg21 and ICR-PVRTg5 were at intermediate and lowest levels, respectively. In the CNS, PVR mRNA was detected at high levels only in neurons of the spinal cord and brain stem where poliovirus can replicate, suggesting that the PVR mRNA expression confers cell specificity to poliovirus in the CNS. ICR-PVRTg1 and ICR-PVRTg5 showed the highest and the lowest sensitivity to poliovirus, respectively, whereas ICR-PVRTg21 was in-between. These results may suggest that poliovirus sensitivity of the mice is attributed to relative levels of PVR expression.

Animals↗
PubMed1994

Tissue tropism and species specificity of poliovirus infection.

Paralytic poliomyelitis is caused by the destruction of neurons in the central nervous system during the lytic replication of poliovirus. The human gene for the poliovirus receptor has been cloned recently, and transgenic mice generated that are susceptible to this virus. This has allowed the critical amino acids that determine the specificity of poliovirus to be identified.

Amino Acid Sequence↗
PubMed1993

Molecular biology and cell-free synthesis of poliovirus.

Poliovirus is a small icosahedral particle consisting of only five species of macromolecules: 60 copies each of the capsid protein VP1-4; and one copy of single-stranded RNA, approximately 7500 nt long. The genome, linked at the 5' end to a small protein VPg and 3' polyadenylylated, is of plus strand polarity. After receptor-mediated uptake of the virus and release of the RNA into the cytoplasm, the genome serves as mRNA, encoding only a single polypeptide, the polyprotein. The polyprotein is cleaved co-translationally into numerous polypeptides by its own, internal proteinases 2Apro, 3Cpro and 3CDpro. Initiation of translation is mediated by a novel genetic element, called internal ribosomal entry site (IRES). IRES elements, which are 400 nt long RNA segments located within the 5' non-translated region of the viral genome, are common to all picornaviruses. Their function renders translation of picornavirus mRNAs cap- and 5'-independent, an observation that has upset the dogma of cap-dependent translation in eukaryotic cells. IRES elements have also been used to genetically dissect the viral genome and to construct novel expression vectors. Genome replication is not fully understood, the major conundrum being the initiation of RNA synthesis by the primer-dependent viral RNA polymerase 3Dpol, a process leading to VPg-linked RNA products. Nearly all non-structural proteins appear to be involved in initiation, the proteinases 2Apro and 3CDpro included. A HeLa cell-free system has been developed that, on programming with plasmid-transcribed viral RNA, will perform viral translation, protein processing, RNA replication, and assembly of capsid protein and newly made genomic RNA. The final yield is infectious poliovirus.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Antigenicities of Group I and II hepatitis C virus polypeptides--molecular basis of diagnosis.

Comparative nucleotide sequence studies on the putative NS3 and NS4 regions of the genomes of hepatitis C viruses (HCV) have revealed that there are at least two groups of HCV, group I and group II. The cDNA clone E, corresponding to a boundary between the NS3 and NS4 (NS3-4) region of the group II HCV genome, encodes antigens that react to antibodies specific to group II HCV (Tsukiyama-Kohara et al. (1991) Virus Genes 5, 243-254). To understand the molecular basis of the group-specific antigenicity of HCV peptides, the predicted amino acid sequences around the NS3-4 region of our group II HCV cDNAs were compared with those of other HCV isolates. The analysis revealed the presence of group-specific amino acids in this peptide region. Evolutionary analysis of nucleotide sequences within this region of these HCV isolates also led to the same classification. A similar result was obtained by sequence analysis of cloned cDNAs corresponding to the core region. A cDNA of the group II HCV core region was prepared by polymerase chain reaction from the cDNA synthesized with group II-specific primer complementary to the NS3-4 region. The products directed by the cDNA of the core region did not have group-specific antigenicity. The NS3 peptide region also appeared not to carry group-specific antigens. Our results indicate that most HCV isolates can be classified into either group I or II, and that the existence of two groups of HCV does not disturb HCV diagnosis as long as core and/or NS3 peptides are used to detect HCV antibodies.

Amino Acid Sequence↗
PubMed1993

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.

Adsorption↗
PubMed1993

Poliovirus-sensitive transgenic mice as a new animal model.

Transgenic mice susceptible to poliovirus infection were produced by introducing a human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of receptor mRNAs in tissues of the transgenic mice was analysed using Northern blot hybridization. The results indicate that the human gene is expressed in many tissues of the transgenic mice just as in human tissues, and that the amount of the receptor mRNAs varies from tissue to tissue. The transgenic mice inoculated with poliovirus by any of the routes tested in this study show clinical symptoms similar to those in humans and monkeys, although the sensitivity of the mice depended on the inoculation route. In any route, the virulent Mahoney strain of type 1 poliovirus is much more virulent than the attenuated Sabin 1 strain in the transgenic mice. These observations suggest that the transgenic mice become an excellent new animal model for studying molecular mechanisms of pathogenesis of poliovirus and for assessing oral poliovirus vaccines.

Administration, Oral↗