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Koki Taniguchi - One of the best experts on this subject based on the ideXlab platform.

  • Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.
    Journal of virology, 2018
    Co-Authors: Kumiko Ishikawa-sasaki, Koki Taniguchi, Shigeo Nagashima, Jun Sasaki
    Abstract:

    Positive-strand RNA Viruses, including picornaViruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi Virus (AiV), a picornaVirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaViruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA Viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi Virus (AiV), a picornaVirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

  • A Complex Comprising Phosphatidylinositol 4-Kinase IIIβ, ACBD3, and Aichi Virus Proteins Enhances Phosphatidylinositol 4-Phosphate Synthesis and Is Critical for Formation of the Viral Replication Complex
    Journal of virology, 2014
    Co-Authors: Kumiko Ishikawa-sasaki, Jun Sasaki, Koki Taniguchi
    Abstract:

    ABSTRACT Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornaVirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi Virus (AiV), a picornaVirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754–766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. IMPORTANCE The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornaVirus genomes. Aichi Virus, a picornaVirus belonging to the genus KobuVirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that Virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by Viruses to support the replication of their genomes.

  • acbd3 mediated recruitment of pi4kb to picornaVirus rna replication sites
    The EMBO Journal, 2012
    Co-Authors: Jun Sasaki, Kumiko Ishikawa, Minetaro Arita, Koki Taniguchi
    Abstract:

    Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for genome RNA replication of enteroViruses, small non-enveloped Viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornaVirus, Aichi Virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroViruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enteroVirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication.

  • 3cd but not 3c cleaves the vp1 2a site efficiently during Aichi Virus polyprotein processing through interaction with 2a
    Virus Research, 2012
    Co-Authors: Jun Sasaki, Kumiko Ishikawa, Koki Taniguchi
    Abstract:

    PicornaVirus genomes are translated into a single large polyprotein, which is processed by Virus-encoded proteases into individual functional proteins. 3C of all picornaViruses is a protease, and the leader (L) and 2A proteins of some picornaViruses are also involved in polyprotein processing. Aichi Virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus KobuVirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site.

  • Overall linkage map of the nonstructural proteins of Aichi Virus.
    Virus Research, 2009
    Co-Authors: Kumiko Ishikawa, Jun Sasaki, Koki Taniguchi
    Abstract:

    Abstract Aichi Virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus KobuVirus of the family Picornaviridae . PicornaVirus genome replication occurs in replication complexes that include viral nonstructural proteins, host proteins and viral RNA. In polioVirus, all nonstructural proteins are found in the replication complexes, suggesting the ability of the viral nonstructural proteins to interact with each other. In this study, we examined the interactions between the AiV nonstructural proteins using a mammalian two-hybrid system. The results showed that all of the tested proteins could interact with more than one protein. We observed homodimerization of five proteins, bidirectional heterodimerization of six protein pairs, and unidirectional heterodimerization of eighteen protein pairs. Among the interactions detected in this study, the 2A–2BC, 2A–2BC, 2A–2C, 2BC–3CD, 2BC–3C, 2C–3C, 2C–3CD and 3AB–3C interactions have not been observed in the previous two-hybrid studies with other picornaViruses. The strongest interaction was observed between 2A and 3CD. AiV 2A has already been shown to be involved in genome replication. Domain mapping of the 2A and 3CD interaction in mammalian two-hybrid analysis revealed that the C-terminal quarter of 2A is not required for the interaction with 3CD.

Jun Sasaki - One of the best experts on this subject based on the ideXlab platform.

  • Bacterial Stabilization of a Panel of PicornaViruses
    American Society for Microbiology, 2019
    Co-Authors: Elizabeth R. Aguilera, Jun Sasaki, Y Nguyen, Julie K. Pfeiffer
    Abstract:

    Recent studies have shown that bacteria promote infection and stabilization of polioVirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the Viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornaVirus stability.Several Viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaViruses are not completely understood. Previous work determined that polioVirus (PV), an enteric Virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for interhost transmission, since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaViruses from three different genera: EnteroVirus (PV and coxsackieVirus B3 [CVB3]), KobuVirus (Aichi Virus), and CardioVirus (mengoVirus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while mengoVirus and Aichi Virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi Virus, and mengoVirus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria, since Viruses bound to bacteria in a pulldown assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaViruses

  • Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.
    Journal of virology, 2018
    Co-Authors: Kumiko Ishikawa-sasaki, Koki Taniguchi, Shigeo Nagashima, Jun Sasaki
    Abstract:

    Positive-strand RNA Viruses, including picornaViruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi Virus (AiV), a picornaVirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaViruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA Viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi Virus (AiV), a picornaVirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

  • A Complex Comprising Phosphatidylinositol 4-Kinase IIIβ, ACBD3, and Aichi Virus Proteins Enhances Phosphatidylinositol 4-Phosphate Synthesis and Is Critical for Formation of the Viral Replication Complex
    Journal of virology, 2014
    Co-Authors: Kumiko Ishikawa-sasaki, Jun Sasaki, Koki Taniguchi
    Abstract:

    ABSTRACT Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornaVirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi Virus (AiV), a picornaVirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754–766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. IMPORTANCE The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornaVirus genomes. Aichi Virus, a picornaVirus belonging to the genus KobuVirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that Virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by Viruses to support the replication of their genomes.

  • acbd3 mediated recruitment of pi4kb to picornaVirus rna replication sites
    The EMBO Journal, 2012
    Co-Authors: Jun Sasaki, Kumiko Ishikawa, Minetaro Arita, Koki Taniguchi
    Abstract:

    Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for genome RNA replication of enteroViruses, small non-enveloped Viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornaVirus, Aichi Virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroViruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enteroVirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication.

  • 3cd but not 3c cleaves the vp1 2a site efficiently during Aichi Virus polyprotein processing through interaction with 2a
    Virus Research, 2012
    Co-Authors: Jun Sasaki, Kumiko Ishikawa, Koki Taniguchi
    Abstract:

    PicornaVirus genomes are translated into a single large polyprotein, which is processed by Virus-encoded proteases into individual functional proteins. 3C of all picornaViruses is a protease, and the leader (L) and 2A proteins of some picornaViruses are also involved in polyprotein processing. Aichi Virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus KobuVirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site.

Teruo Yamashita - One of the best experts on this subject based on the ideXlab platform.

  • Structure of human Aichi Virus and implications for receptor binding.
    Nature microbiology, 2016
    Co-Authors: Ling Zhu, Teruo Yamashita, Xiangxi Wang, Jingshan Ren, Abhay Kotecha, Thomas S. Walter, Shuai Yuan, Tobias J. Tuthill, Elizabeth E. Fry, Zihe Rao
    Abstract:

    Aichi Virus (AiV), an unusual and poorly characterized picornaVirus, classified in the genus KobuVirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries1,2. The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life3,4. There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 A. This first high-resolution structure for a kobuVirus is intermediate between those of the enteroViruses and cardioViruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroViruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaViruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaViruses. A peptide corresponding to this polyproline motif somewhat attenuates Virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

  • molecular detection and nucleotide sequence analysis of a new Aichi Virus closely related to canine kobuVirus in sewage samples
    Journal of Medical Microbiology, 2014
    Co-Authors: Teruo Yamashita, Shinichi Kobayashi, Miyabi Ito, Hirokazu Adachi, Emi Hirose, Noriko Nakamura, Yoshihiro Yasui, Hiroko Minagawa
    Abstract:

    Between 2001 and 2005, 207 raw sewage samples were collected at the inflow of a sewage treatment plant in Aichi Prefecture, Japan. Of the 207 sewage samples, 137 (66.2 %) were found to be positive for amplification of Aichi Virus (AiV) nucleotide using reverse transcription (RT)-PCR with 10 forward and 10 reverse primers in the 3D region corresponding to the nucleotide sequence of all kobuViruses. AiV genotype A sequences were detected in all 137 samples. New sequences of AiV were detected in nine samples, exhibiting 83 % similarity with AiV A846/88, but 95 % similarity with canine kobuVirus (CKV) US-PC0082 in this region. The nucleotide sequences from the VP3 region to the 3′ untranslated region (UTR) of sewage sample Y12/2004 were determined. The number of nucleotides in each region was the same as that of CKV. The similarity of the nucleotide (amino acid) identity of a complete VP1 region was 90.5 % (94.8 %) between Y12/2004 and CKV US-PC0082. The phylogenic analyses based on the nucleotide and the deduced amino acid sequences of VP1 and 3D showed that Y12/2004 was independent from AiV, but closely related to CKV. These results suggested that CKV is present in Aichi Prefecture, Japan.

  • isolation and characterization of a new species of kobuVirus associated with cattle
    Journal of General Virology, 2003
    Co-Authors: Teruo Yamashita, Hideaki Tsuzuki, Miyabi Ito, Yuka Kabashima, Akira Fujiura, Kenji Sakae
    Abstract:

    A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi Virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaViruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi Virus. The Virus sequence had 33 and 75 % amino acid identity with the Aichi Virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other Picornaviridae. The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus KobuVirus but is distinct from Aichi Virus. Of 72 cattle sera, 43 (59·7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16·7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus KobuVirus, now termed bovine kobuVirus.

  • vi 3 molecular biology and epidemiology of Aichi Virus and other diarrhoeogenic enteroViruses
    Perspectives in Medical Virology, 2003
    Co-Authors: Teruo Yamashita, Kenji Sakae
    Abstract:

    The virion of the Aichi Virus contains a single-stranded RNA molecule as the genome. The homology of Aichi Virus structural proteins (VP0, VP3, and VP1) with corresponding polypeptides of other picornaViruses varies between 19% and 32%. The epidemiology of the Aichi Virus as a medically important pathogen has not been well defined. Stool samples from adult patients in six oyster-associated gastroenteritis outbreaks were examined for variation, based on their reactivity with a monoclonal antibody raised against the standard strain (A486/88) and on reverse transcription-polymerase chain reactions (RT-PCR) of three genomic regions. Antibody to the Aichi Virus could be detected using a neutralization test and an enzyme-linked immunosorbent assay (ELISA). These methods were used for the identification of Aichi Virus infection in paired serum samples. The chapter concludes with a discussion on other diarrheagenic enteroViruses.

  • identification of Aichi Virus infection by measurement of immunoglobulin responses in an enzyme linked immunosorbent assay
    Journal of Clinical Microbiology, 2001
    Co-Authors: Teruo Yamashita, Hideaki Tsuzuki, Miyabi Ito, Kenji Sakae
    Abstract:

    Using inhibitory enzyme-linked immunosorbent assay, seroconversions to Aichi Virus were detected in 24 (42.9%) of 56 patients with gastroenteritis in six outbreaks. Virus-specific immunoglobulin M (IgM) was detected in convalescent-phase sera from 7 of 24 patients. Of the other 17 patients, 12 developed a significant increase in both IgA and IgG levels and 5 developed a significant increase in IgG alone.

Shigeo Nagashima - One of the best experts on this subject based on the ideXlab platform.

  • Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.
    Journal of virology, 2018
    Co-Authors: Kumiko Ishikawa-sasaki, Koki Taniguchi, Shigeo Nagashima, Jun Sasaki
    Abstract:

    Positive-strand RNA Viruses, including picornaViruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi Virus (AiV), a picornaVirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaViruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA Viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi Virus (AiV), a picornaVirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

  • interaction between polypeptide 3abc and the 5 terminal structural elements of the genome of Aichi Virus implication for negative strand rna synthesis
    Journal of Virology, 2008
    Co-Authors: Shigeo Nagashima, Jun Sasaki, Koki Taniguchi
    Abstract:

    Secondary structural elements at the 5′ end of picornaVirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaViruses. In polioVirus, ribonucleoprotein complex formation at the 5′ end of the genome is required for negative-strand synthesis. We have previously shown that the 5′-end 115 nucleotides of the Aichi Virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5′-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5′-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5′-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5′-terminal region did not correlate with the RNA replication ability.

  • the 5 terminal region of the Aichi Virus genome encodes cis acting replication elements required for positive and negative strand rna synthesis
    Journal of Virology, 2005
    Co-Authors: Shigeo Nagashima, Jun Sasaki, Koki Taniguchi
    Abstract:

    Aichi Virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi Virus genome required for RNA replication using a series of Aichi Virus-encephalomyocarditis Virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi Virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.

  • Aichi Virus leader protein is involved in viral rna replication and encapsidation
    Journal of Virology, 2003
    Co-Authors: Jun Sasaki, Shigeo Nagashima, Koki Taniguchi
    Abstract:

    Aichi Virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi Virus L protein exhibits no significant sequence homology to those of other picornaViruses. In this study, we investigated the function of the Aichi Virus L protein in Virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Δ114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the Virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Δ114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi Virus L protein is involved in both viral RNA replication and encapsidation.

  • functional analysis of the stem loop structures at the 5 end of the Aichi Virus genome
    Virology, 2003
    Co-Authors: Shigeo Nagashima, Jun Sasaki, Koki Taniguchi
    Abstract:

    Aichi Virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5' end) within the 5'-end 120 nucleotides of the genome. We have already shown that the most 5'-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable Viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during Virus infection.

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  • applicability of crassphage pepper mild mottle Virus and tobacco mosaic Virus as indicators of reduction of enteric Viruses during wastewater treatment
    Scientific Reports, 2020
    Co-Authors: Sarmila Tandukar, Samendra P. Sherchan, Eiji Haramoto
    Abstract:

    This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle Virus (PMMoV), and tobacco mosaic Virus (TMV) as indicators of the reduction of human enteric Viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator Viruses, eight human enteric Viruses [human adenoViruses, JC and BK polyomaViruses, Aichi Virus 1 (AiV-1), enteroViruses, and noroViruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator Viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested Viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric Viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric Viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested Viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.

  • temporal variations in genotype distribution of human sapoViruses and Aichi Virus 1 in wastewater in southern arizona united states
    Journal of Applied Microbiology, 2018
    Co-Authors: Masaaki Kitajima, Eiji Haramoto, Andri Taruna Rachmadi, Brandon C Iker, Charles P Gerba
    Abstract:

    AIMS To investigate the molecular epidemiology, especially temporal variations in genotype distribution, of sapoViruses and Aichi Virus 1 (AiV-1) in Arizona, United States, by examining wastewater. METHODS AND RESULTS A total of 26 wastewater samples (13 influent and 13 effluent) were collected monthly from a wastewater treatment plant and viral strains were identified through nested reverse transcription-PCR followed by cloning and sequencing analysis. Identified sapoVirus strains were classified into seven genotypes belonging to three genogroups (GI, GII, and GV): GI.1, GI.2, GI.3, GII.1, GII.2, GII.8 and GV.1, with a clear temporal shift. The majority of AiV-1 strains identified from the wastewater samples were classified into genotype B, and genotype A strains were identified in only two samples. CONCLUSIONS We identified a number of sapoVirus and AiV-1 strains belonging to multiple genotypes in wastewater samples collected over a 13-month period. Our results suggested a temporal shift in prevalent genotypes in the community. SIGNIFICANCE AND IMPACT OF THE STUDY This is the first study elucidating the genotype distribution of human sapoViruses and AiV-1 in wastewater in the United States. Wastewater surveillance is especially useful for understanding molecular epidemiology of Viruses that are less commonly tested in clinical diagnosis, including sapoViruses and AiV-1.

  • Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment
    2018
    Co-Authors: Bradley W. Schmitz, Samendra P. Sherchan, Eiji Haramoto, Masaaki Kitajima, Charles P Gerba, Hitoha Moriyama, Ian L. Pepper
    Abstract:

    Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)­cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)­cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)­cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and Viruses (pepper mild mottle Virus, Aichi Virus 1, adenoVirus, and polyomaViruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters

  • quantification and genotyping of Aichi Virus 1 in water samples in the kathmandu valley nepal
    Food and Environmental Virology, 2017
    Co-Authors: Eiji Haramoto, Masaaki Kitajima
    Abstract:

    Aichi Virus 1 genomes were detected by quantitative PCR in groundwater from shallow dug (10/22) and tube wells (1/15), river water (14/14), and sewage (1/1), with the maximum concentration of 4.0 × 109 copies/l. Nucleotide sequencing analysis demonstrated the prevalence of genotype B in the Virus positive samples.