Takeshi Morikawa

Bio: Takeshi Morikawa is an academic researcher from Kyoto University. The author has contributed to research in topics: Virology & Nucleolus. The author has an hindex of 3, co-authored 4 publications receiving 16 citations.

Syntheses of some polymers containing cyclopropane rings in the main chain

Abstract: Polyesters, polyamides, and polyurethanes containing cyclopropane rings in the main chains were prepared, using 1,2-dimethylol-cyclopropanedicarboxylic acid or 1,2-dimethylol-cyclopropane as one component. 1,2-Cyclopropane dicarboxylic acid and its homologs were synthesized according to the method of McCoy. The separation of the cis and trans isomers of these acids was performed by treatment with thionyl chloride, where the trans isomers were converted into acid chlorides, while the cis isomers into acid anhydrides. 1,2-Dimethylol-cyclopropane was prepared by the reduction of 1,2-cyclopropane dicarboxylic chloride with LiAlH4. The polymerization was carried out by the interfacial technique, solution polycondensation or the ester interchange method. Some polymers and their films showed good technological properties. Polyester, Polyamide und Polyurethane, welche Cyclopropanringe in der Hauptkette enthalten, wurden unter Verwendung von 1,2-Cyclopropan-dicarbonsaure oder 1,2-Dimethylol-cyclopropan als eine Ausgangskomponente dargestellt. Die 1,2-Cyclopropan-dicarbonsaure und deren Homologe wurden nach der Methode von McCoy hergestellt. Zur Trennung der dabei entstandenen cis- und trans-Isomeren wurde das Gemisch mit Thionylchlorid behandelt, dabei das trans-Isomere in das Saurechlorid, das cis-Isomere in das Dicarbonsaureanhydrid umgewandelt. Diese zwei Verbindungen konnten leicht durch fraktionierte Destillation getrennt werden. Das 1,2-Dimethylol-cyclopropan wurde durch die Reduktion von 1,2-Cyclopropan-dicarbonsaurechlorid mit LiAlH4 erhalten. Es wurden die Grenzflachenpolykondensation, die Polykondensation in Losung und die Esteraustausch-Reaktion angewendet. Einige erhaltene Polymere zeigten ausgezeichnete Filmeigenschaften.

Contribution of RNA-RNA Interactions Mediated by the Genome Packaging Signals for the Selective Genome Packaging of Influenza A Virus

Abstract: While numerous viral genomes comprise a single genome segment, the influenza A virus possesses eight segmented genomes. Influenza A virus can benefit from having a segmented genome because the segments can reassort with other strains of the influenza virus to create new genetically distinct strains. ABSTRACT The influenza A virus genome is composed of eight single-stranded negative-sense viral RNA segments (vRNAs). The eight vRNAs are selectively packaged into each progeny virion. This process likely involves specific interactions between the vRNAs via segment-specific packaging signals located in both the 3′- and 5′-terminal regions of the respective vRNAs. To assess the importance of vRNA-vRNA interactions via packaging signals for selective genome packaging, we generated mutant viruses possessing silent mutations in the packaging signal region of the hemagglutinin (HA) vRNA. A mutant virus possessing silent mutations in nucleotides (nt) 1664 to 1676 resulted in defects in HA vRNA incorporation and showed a reduction in viral growth. After serial passage, the mutant virus acquired additional mutations in the 5′-terminal packaging signal regions of both the HA and polymerase basic 2 (PB2) vRNAs. These mutations contributed to the recovery of viral growth and HA vRNA packaging efficiency. In addition, an RNA-RNA interaction between the 5′ ends of HA and PB2 vRNAs was confirmed in vitro, and this interaction was disrupted following the introduction of silent mutations in the HA vRNA. Thus, our results demonstrated that RNA-RNA interactions between the packaging signal regions of HA vRNA and PB2 vRNA are important for selective genome packaging. IMPORTANCE While numerous viral genomes comprise a single genome segment, the influenza A virus possesses eight segmented genomes. Influenza A virus can benefit from having a segmented genome because the segments can reassort with other strains of the influenza virus to create new genetically distinct strains. The influenza A virus efficiently incorporates one copy of each of its eight genomic segments per viral particle. However, the mechanism by which each segment is specifically selected is poorly understood. The genome segments contain RNA signals that facilitate the incorporation of segments into virus particles. These regions may facilitate specific interactions between the genome segments, creating an eight-segment complex, which can then be packaged into individual particles. In this study, we provide evidence that RNA signals contribute to specific interactions between two of the influenza virus genome segments.

vRNA-vRNA interactions in influenza A virus HA vRNA packaging

Abstract: The genome of the influenza A virus is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight different vRNAs are selectively packaged into progeny virions. This process likely involves specific interactions among vRNAs via segment-specific packaging signals located in the 3’ and 5’ terminal coding regions of vRNAs. To identify vRNA(s) that interact with hemagglutinin (HA) vRNA during genome packaging, we generated a mutant virus, HA 5m2, which possessed five silent mutations in the 5’ packaging signal region of HA vRNA. The HA 5m2 virus had a specific defect in HA vRNA incorporation, which reduced the viral replication efficiency. After serial passaging in cells, the virus acquired additional mutations in the 5’ terminal packaging signal regions of both HA and PB2 vRNAs. These mutations contributed to recovery of viral growth and packaging efficiency of HA vRNA. A direct RNA-RNA interaction between the 5’ ends of HA and PB2 vRNAs was confirmed in vitro. Our results indicate that direct interactions of HA vRNA with PB2 vRNA via their packaging signal regions are important for selective genome packaging and enhance our knowledge on the emergence of pandemic influenza viruses through genetic reassortment.

Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

Abstract: Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process. ABSTRACT Influenza A virus double-helical ribonucleoprotein complex (RNP) performs transcription and replication of viral genomic RNA (vRNA). Although RNP formation occurs in the nuclei of virus-infected cells, the nuclear domains involved in this process remain unclear. Here, we show that the nucleolus is an essential site for functional RNP formation. Viral nucleoprotein (NP), a major RNP component, temporarily localized to the nucleoli of virus-infected cells. Mutations in a nucleolar localization signal (NoLS) on NP abolished double-helical RNP formation, resulting in a loss of viral RNA synthesis ability, whereas ectopic fusion of the NoLS enabled the NP mutant to form functional double-helical RNPs. Furthermore, nucleolar disruption of virus-infected cells inhibited NP assembly into double-helical RNPs, resulting in decreased viral RNA synthesis. Collectively, our findings demonstrate that NP migration into the nucleolus is a critical step for functional RNP formation, showing the importance of the nucleolus in the influenza virus life cycle. IMPORTANCE Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process. Here, we reveal by using several microscopic techniques that its major component, viral nucleoprotein (NP), temporally stays in the nucleolus, the assembly site of ribosomal RNAs/proteins, and that the formation is dependent on a nucleolar localization signal in NP. We also show that nucleolar disruption causes abortive RNP formation, resulting in a significant reduction in virus replication. Our findings demonstrate the importance of the nucleolus as the site of RNP formation and for virus replication.

Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm.

Abstract: Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

The influence of spatial orientation of free orbitals in pmr spectroscopy influence of vicinal orbitals on 3J and 2J

Abstract: In accordance with recent literature data, it is proposed that an increment of + 2.3 cps and + 1.8 cps is to be added to the vicinal (3J) and gem (2J) coupling values respectively, each time an α-oxygen (or α-nitrogen) atom has one of its free electron pair p-orbitals parallel with the carbon hydrogen bond of one of the protons involved in the coupling under consideration. An influence on 3J coupling values in three membered rings (epoxides) is however not to be considered. Molecular deformations which disturb the parallelity (i.e. in the rigid half chair form of pentacyclic compounds or in the twist form of hexacyclic compounds) nullify this effect. The Karplus rule and the electronegativity rule have regained a great amount of their reliability with this supplementary effect.

Synthesis and synthetic applications of 1-metallo-1-selenocyclopropanes and -cyclobutanes and related 1-metallo-1-silylcyclopropanes

Abstract: Among the α-heterosubstituted cyclopropylmetals α-selenocyclopropyllithiums represent some of the most valuable synthetic intermediates. They are quantitatively prepared from selenoacetals of cyclopropanones and butyllithiums, are thermally stable at ∼−78° for several hours and are particularly nucleophilic especially towards carbonyl compounds. The cyclopropyl derivatives containing a selenenyl moiety have been transformed to selenium free derivatives such as alkylidene cyclopropanes, vinyl cyclopropanes, allylidene cyclopropanes, cyclobutanones and α-silyl cyclopropyllithiums. The latter compounds have been used as starting material for the synthesis of alkylidene cyclopropanes and cyclopentenyl derivatives. α-Seleno cyclobutyllithiums, which are available in two steps from cyclobutanones, also permit the synthesis of various selenium free homologues such as alkylidene cyclobutanes, vinyl cyclobutanes, oxaspirohexanes and cyclopentanones.