41 results on '"Ujike M"'
Search Results
2. Optical measurement of change of nonuniform electric field vector distribution in liquid insulator.
- Author
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Ihori, H., Ujike, M., Hiromoto, K., Fujii, M., and Arii, K.
- Published
- 2001
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3. Monitoring and characterization of oseltamivir-resistant pandemic (H1N1) 2009 virus, Japan, 2009-2010.
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Ujike M, Ejima M, Anraku A, Shimabukuro K, Obuchi M, Kishida N, Hong X, Takashita E, Fujisaki S, Yamashita K, Horikawa H, Kato Y, Oguchi A, Fujita N, Tashiro M, Odagiri T, and the Influenza Virus Surveillance Group of Japan, Ujike, Makoto, Ejima, Miho, and Anraku, Akane
- Abstract
To monitor and characterize oseltamivir-resistant (OR) pandemic (H1N1) 2009 virus with the H275Y mutation, we analyzed 4,307 clinical specimens from Japan by neuraminidase (NA) sequencing or inhibition assay; 61 OR pandemic (H1N1) 2009 viruses were detected. NA inhibition assay and M2 sequencing indicated that OR pandemic (H1N1) 2009 virus was resistant to M2 inhibitors, but sensitive to zanamivir. Full-genome sequencing showed OR and oseltamivir-sensitive (OS) viruses had high sequence similarity, indicating that domestic OR virus was derived from OS pandemic (H1N1) 2009 virus. Hemagglutination inhibition test demonstrated that OR and OS pandemic (H1N1) 2009 viruses were antigenically similar to the A/California/7/2009 vaccine strain. Of 61 case-patients with OR viruses, 45 received oseltamivir as treatment, and 10 received it as prophylaxis, which suggests that most cases emerged sporadically from OS pandemic (H1N1) 2009, due to selective pressure. No evidence of sustained spread of OR pandemic (H1N1) 2009 was found in Japan; however, 2 suspected incidents of human-to-human transmission were reported. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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4. Oseltamivir-resistant influenza viruses A (H1N1) during 2007-2009 influenza seasons, Japan.
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Ujike M, Shimabukuro K, Mochizuki K, Obuchi M, Kageyama T, Shirakura M, Kishida N, Yamashita K, Horikawa H, Kato Y, Fujita N, Tashiro M, Odagiri T, Working Group for Influenza Virus Surveillance in Japan, Ujike, Makoto, Shimabukuro, Kozue, Mochizuki, Kiku, Obuchi, Masatsugu, Kageyama, Tsutomu, and Shirakura, Masayuki
- Abstract
To monitor oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y in neuraminidase (NA) in Japan during 2 influenza seasons, we analyzed 3,216 clinical samples by NA sequencing and/or NA inhibition assay. The total frequency of ORVs was 2.6% (45/1,734) during the 2007-08 season and 99.7% (1,477/1,482) during the 2008-09 season, indicating a marked increase in ORVs in Japan during 1 influenza season. The NA gene of ORVs in the 2007-08 season fell into 2 distinct lineages by D354G substitution, whereas that of ORVs in the 2008-09 season fell into 1 lineage. NA inhibition assay and M2 sequencing showed that almost all the ORVs were sensitive to zanamivir and amantadine. The hemagglutination inhibition test showed that ORVs were antigenetically similar to the 2008-09 vaccine strain A/Brisbane/59/2007. Our data indicate that the current vaccine or zanamivir and amantadine are effective against recent ORVs, but continuous surveillance remains necessary. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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5. Characterization of sugi (Cryptomeria japonica) bark extracts
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Tatsuya Ashitani, Ujike, M., Nagahama, S., Ueno, T., and Sakai, K.
6. Kanzo
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Kosaka, K., primary, Shimada, Y., additional, Ohta, Y., additional, Ujike, M., additional, Kihara, K., additional, and Amioka, T., additional
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- 1960
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7. Nonuniform electric field vector distribution in a dielectric liquid using optical measurement system with parallel processing
- Author
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Ihori, H., primary, Ujike, M., additional, Fujii, M., additional, and Arii, K., additional
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8. Optical measurement of change of nonuniform electric field vector distribution in liquid insulator
- Author
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Ihori, H., primary, Ujike, M., additional, Hiromoto, K., additional, Fujii, M., additional, and Arii, K., additional
- Full Text
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9. A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir
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Itagaki Tsutomu, Takashita Emi, Sugawara Kanetsu, Sanjoh Kanako, Abiko Chieko, Suto Asuka, Aoki Yoko, Mizuta Katsumi, Matsuzaki Yoko, Katsushima Yuriko, Ujike Makoto, Obuchi Masatsugu, Odagiri Takato, and Tashiro Masato
- Subjects
Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. Results Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). Conclusion Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.
- Published
- 2010
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10. Progress of research on coronaviruses and toroviruses in large domestic animals using reverse genetics systems.
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Ujike M and Suzuki T
- Subjects
- Animals, Swine, Cattle, Torovirus Infections veterinary, Torovirus Infections virology, Coronavirus Infections veterinary, Coronavirus Infections virology, Coronavirus Infections epidemiology, Swine Diseases virology, Cattle Diseases virology, Animals, Domestic virology, Reverse Genetics methods, Torovirus genetics, Coronavirus genetics
- Abstract
The generation of genetically engineered recombinant viruses from modified DNA/RNA is commonly referred to as reverse genetics, which allows the introduction of desired mutations into the viral genome. Reverse genetics systems (RGSs) are powerful tools for studying fundamental viral processes, mechanisms of infection, pathogenesis and vaccine development. However, establishing RGS for coronaviruses (CoVs) and toroviruses (ToVs), which have the largest genomes among vertebrate RNA viruses, is laborious and hampered by technical constraints. Hence, little research has focused on animal CoVs and ToVs using RGSs, especially in large domestic animals such as pigs and cattle. In the last decade, however, studies of porcine CoVs and bovine ToVs using RGSs have been reported. In addition, the coronavirus disease-2019 pandemic has prompted the development of new and simple CoV RGSs, which will accelerate RGS-based research on animal CoVs and ToVs. In this review, we summarise the general characteristics of CoVs and ToVs, the RGSs available for CoVs and ToVs and the progress made in the last decade in RGS-based research on porcine CoVs and bovine ToVs., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2024
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11. Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.
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Kawase M, Suwa R, Sugimoto S, Kakizaki M, Kume Y, Chishiki M, Ono T, Okabe H, Norito S, Ujike M, Hosoya M, Hashimoto K, and Shirato K
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- Humans, Child, Real-Time Polymerase Chain Reaction, Respiratory Tract Infections diagnosis, Viruses genetics, Virus Diseases diagnosis, Pneumonia
- Abstract
Importance: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses., Competing Interests: The authors declare no conflict of interest.
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- 2024
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12. Trends of Mismatches in Real-Time RT-PCR Assays Developed by the National Institute of Infectious Diseases, Japan for Omicron Variant of Severe Acute Respiratory Syndrome Coronavirus 2.
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Shirato K, Ujike M, and Kawase M
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- Animals, SARS-CoV-2 genetics, Japan epidemiology, Reverse Transcriptase Polymerase Chain Reaction, COVID-19 Testing, COVID-19 diagnosis, COVID-19 epidemiology, Communicable Diseases
- Abstract
The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2021 and gradually overtook the Delta variant, which was the predominant variant at that time. The Omicron variant has been consecutively replaced by related sublineages. The real-time RT-PCR assays developed by the National Institute of Infectious Diseases (NIID), Japan (i.e., the NIID-N2 and NIID-S2 assays) are the reference assays that have been used in Japan since the outbreak of SARS-CoV-2. To evaluate the applicability of the NIID assays for the Omicron variants, trends in the prevalence of nucleotide mismatches in the primer/probe sequences were traced using sequences registered in the Global Initiative on Sharing Avian Influenza Data database. Approximately 99% of the deposited Omicron variant sequences did not have any mismatches in the NIID assay primer/probes from January to August 2022. This indicates that the NIID assays have been able to detect the changing SARS-CoV-2 Omicron variants.
- Published
- 2023
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13. Single Amino Acid Substitution in the Receptor Binding Domain of Spike Protein Is Sufficient To Convert the Neutralization Profile between Ethiopian and Middle Eastern Isolates of Middle East Respiratory Coronavirus.
- Author
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Sugimoto S, Kakizaki M, Kawase M, Kawachi K, Ujike M, Kamitani W, Sentsui H, and Shirato K
- Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes MERS, which is endemic in the Middle East. The absence of human cases in Africa despite the presence of MERS-CoV suggests virological differences between MERS-CoVs in Africa and the Middle East. In fact, in the laboratory, recombinant MERS-CoV carrying the spike (S) protein of Ethiopian isolates exhibits attenuated properties, being more easily neutralized and replicating slower than viruses carrying the S protein of Middle Eastern isolate, EMC. In this study, to identify the amino acids that define the different virological features between Ethiopian and Middle Eastern MERS-CoVs, neutralization titers and viral replication were evaluated using recombinant MERS-CoVs carrying amino acid substitution(s) in the S protein. A single amino acid difference introduced into the receptor binding domain was sufficient to reverse the difference in the neutralizing properties of the S protein between Ethiopian and Middle Eastern MERS-CoVs. Furthermore, amino acid mutations in the S1 and S2 regions of S protein were collectively involved in slow viral replication. Since even a single amino acid difference in S protein can reverse the viral properties of MERS-CoV, it should be noted that multiple mutations may induce a significant change. Careful monitoring of genetic alterations in MERS-CoVs in Africa is therefore required to detect the emergence of virulent strains generated by a few genetic differences. IMPORTANCE There have been no reported cases of human Middle East respiratory syndrome (MERS) in Africa, despite the presence of MERS coronavirus (MERS-CoV). Previous studies have shown that recombinant MERS-CoV carrying the S protein of an Ethiopian isolate replicated slower and was more easily neutralized relative to MERS-CoV carrying the S protein of a Middle Eastern isolate. In this study, we investigated the amino acid(s) in S protein associated with the different viral characteristics between Ethiopian and Middle Eastern MERS-CoVs. The results revealed that a single amino acid difference in the receptor binding domain was sufficient to reverse the neutralization profile. This implies that slight genetic changes can alter the predominant population of MERS-CoV, similar to the transition of variants of severe acute respiratory syndrome coronavirus-2. Careful genetic monitoring of isolates is important to detect the spread of possible virulent MERS-CoVs generated by mutation(s).
- Published
- 2023
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14. Identification of trypsin-degrading commensals in the large intestine.
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Li Y, Watanabe E, Kawashima Y, Plichta DR, Wang Z, Ujike M, Ang QY, Wu R, Furuichi M, Takeshita K, Yoshida K, Nishiyama K, Kearney SM, Suda W, Hattori M, Sasajima S, Matsunaga T, Zhang X, Watanabe K, Fujishiro J, Norman JM, Olle B, Matsuyama S, Namkoong H, Uwamino Y, Ishii M, Fukunaga K, Hasegawa N, Ohara O, Xavier RJ, Atarashi K, and Honda K
- Subjects
- Administration, Oral, Animals, Bacterial Secretion Systems, Bacterial Vaccines administration & dosage, Bacterial Vaccines immunology, Bacteroidetes isolation & purification, Bacteroidetes metabolism, COVID-19 complications, Citrobacter rodentium immunology, Diarrhea complications, Feces microbiology, Humans, Immunoglobulin A metabolism, Mice, Murine hepatitis virus metabolism, Murine hepatitis virus pathogenicity, Proteolysis, SARS-CoV-2 pathogenicity, Virus Internalization, Gastrointestinal Microbiome genetics, Intestine, Large metabolism, Intestine, Large microbiology, Symbiosis, Trypsin metabolism
- Abstract
Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions
1-3 . However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5 . Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection., (© 2022. The Author(s).)- Published
- 2022
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15. Reduction of Cell Fusion by Deletion in the Hypervariable Region of the Spike Protein of Mouse Hepatitis Virus.
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Tennakoon N, Ryu J, Ujike M, Taguchi F, and Shin HJ
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- Animals, Cell Line, Mice, Murine hepatitis virus genetics, Murine hepatitis virus physiology, Sequence Deletion, Spike Glycoprotein, Coronavirus genetics, Cell Fusion, Murine hepatitis virus pathogenicity, Spike Glycoprotein, Coronavirus physiology
- Abstract
Deletions in the spike gene of mouse hepatitis virus (MHV) produce several variants with diverse biological characteristics, highlighting the significance of the spike gene in viral pathogenesis. In this study, we characterized the JHM-X strain, which has a deletion in the hypervariable region (HVR) of the spike gene, compared with the cl-2 strain, which has a full spike gene. Cytopathic effects (CPEs) induced by the two strains revealed that the size of the CPE produced by cl-2 is much greater than that produced by JHM-X in delayed brain tumor (DBT) cells. Thus, this finding explains the greater fusion activity of cl-2 than JHM-X in cultured cells, and we speculate that the deletion region of the spike protein is involved in the fusion activity differences. In contrast with the fusion activity, a comparison of the virus growth kinetics revealed that the titer of JHM-X was approximately 100 times higher than that of cl-2. We found that the deletion region of the spike protein was involved in fusion activity differences, whereas cl-2 produced significantly higher luciferase activity than JHM-X upon similar expression levels of the spike protein. However, the reason behind the growth difference is still unknown. Overall, we discovered that deletion in the HVR of the spike gene could be involved in the fusion activity differences between the two strains.
- Published
- 2022
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16. Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.
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Ujike M, Etoh Y, Urushiyama N, Taguchi F, Asanuma H, Enjuanes L, and Kamitani W
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- Animals, Cattle, Cattle Diseases virology, Cell Line, Cells, Cultured, Chromosomes, Artificial, Bacterial, Cloning, Molecular, Genes, Reporter, Hemagglutinins, Viral genetics, Hemagglutinins, Viral metabolism, Mutation, Plasmids genetics, Torovirus isolation & purification, Torovirus Infections, Transfection, DNA, Complementary, Genome, Viral, Reverse Genetics, Torovirus genetics
- Abstract
Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.
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- 2022
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17. Recent Progress in Torovirus Molecular Biology.
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Ujike M and Taguchi F
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- Animals, Coronavirus genetics, Coronavirus physiology, Coronavirus Infections virology, Humans, Torovirus genetics, Torovirus physiology, Torovirus Infections virology
- Abstract
Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.
- Published
- 2021
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18. Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.
- Author
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Ujike M, Kawachi Y, Matsunaga Y, Etho Y, Asanuma H, Kamitani W, and Taguchi F
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- Amino Acid Sequence, Animals, Cell Line, Cell Nucleolus metabolism, Cytoplasm metabolism, Humans, Mutation, Nuclear Export Signals, Nuclear Localization Signals, Nucleocapsid Proteins genetics, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Torovirus growth & development, Torovirus metabolism, Virus Replication genetics, Cell Nucleus metabolism, Nucleocapsid Proteins chemistry, Nucleocapsid Proteins metabolism, Torovirus physiology
- Abstract
Torovirus (ToV) has recently been classified into the new family Tobaniviridae , although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth. IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses., (Copyright © 2021 American Society for Microbiology.)
- Published
- 2021
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19. The Inhaled Steroid Ciclesonide Blocks SARS-CoV-2 RNA Replication by Targeting the Viral Replication-Transcription Complex in Cultured Cells.
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Matsuyama S, Kawase M, Nao N, Shirato K, Ujike M, Kamitani W, Shimojima M, and Fukushi S
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- Animals, Chlorocebus aethiops, Dogs, HeLa Cells, Humans, Madin Darby Canine Kidney Cells, Vero Cells, COVID-19 Drug Treatment, COVID-19 metabolism, Pregnenediones pharmacology, RNA, Double-Stranded biosynthesis, RNA, Viral biosynthesis, SARS-CoV-2 physiology, Virus Replication drug effects
- Abstract
Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC
90 ) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 μM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS and COVID-19., (Copyright © 2020 American Society for Microbiology.)- Published
- 2020
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20. A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes.
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Imai R, Nagai M, Oba M, Sakaguchi S, Ujike M, Kimura R, Kida M, Masuda T, Kuroda M, Wen R, Li K, Katayama Y, Naoi Y, Tsuchiaka S, Omatsu T, Yamazato H, Makino S, and Mizutani T
- Subjects
- Animals, Apoptosis Regulatory Proteins genetics, Cysteine Proteases metabolism, Enterovirus Infections epidemiology, Enterovirus Infections virology, Feces virology, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Viral, Phylogeny, Swine, Swine Diseases epidemiology, Swine Diseases virology, Apoptosis Regulatory Proteins metabolism, Cysteine Proteases genetics, Enterovirus Infections veterinary, Enteroviruses, Porcine genetics, Viral Structural Proteins genetics
- Abstract
Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G., (Copyright © 2019. Published by Elsevier B.V.)
- Published
- 2019
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21. Identification of tumor-initiating cells derived from two canine rhabdomyosarcoma cell lines.
- Author
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Kishimoto TE, Yashima S, Nakahira R, Onozawa E, Azakami D, Ujike M, Ochiai K, Ishiwata T, Takahashi K, and Michishita M
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- Animals, Antineoplastic Agents pharmacology, Cell Line, Tumor, Dogs, Doxorubicin pharmacology, Drug Resistance, Neoplasm, Female, Mice, Mice, Inbred BALB C, Mitoxantrone pharmacology, Muscle Neoplasms drug therapy, Muscle Neoplasms pathology, Neoplasm Transplantation, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells pathology, Rhabdomyosarcoma drug therapy, Rhabdomyosarcoma pathology, Vincristine pharmacology, Dog Diseases pathology, Muscle Neoplasms veterinary, Neoplastic Stem Cells cytology, Rhabdomyosarcoma veterinary
- Abstract
Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 10
3 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis.- Published
- 2017
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22. Phylogenetic and antigenic characterization of newly isolated porcine epidemic diarrhea viruses in Japan.
- Author
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Islam MT, Kubota T, Ujike M, Yahara Y, and Taguchi F
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Antigens, Viral chemistry, Antigens, Viral immunology, Japan, Neutralization Tests, Phylogeny, Porcine epidemic diarrhea virus classification, Porcine epidemic diarrhea virus isolation & purification, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus immunology, Swine, Swine Diseases epidemiology, Coronavirus Infections veterinary, Porcine epidemic diarrhea virus genetics, Porcine epidemic diarrhea virus immunology, Swine Diseases immunology, Swine Diseases virology
- Abstract
To evaluate the mechanism by which a large outbreak of porcine epidemic diarrhea (PED) occurred in Japan, where the majority of sows are vaccinated, we isolated two new strains of PED virus (PEDV) from the intestines of piglets and found that they showed greater similarity to US isolates (group II PEDV) than to the Japanese vaccine strain (group I PEDV). We compared the antigenicity of the vaccine type strain and newly isolated strains by means of a neutralization test using sera from a number of pigs from various farms; the results revealed that they are antigenically similar. This is the first report of the similarity of group I and II viruses using sera from individual pigs vaccinated with group I virus. These data suggest that the large outbreak of PED in Japan cannot be attributed to inefficient vaccination but may be due to the extremely high virulence of the newly appearing viruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
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23. The contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of S proteins and incorporation of S protein into virus-like particles.
- Author
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Ujike M, Huang C, Shirato K, Makino S, and Taguchi F
- Subjects
- Animals, Cell Line, Coronavirus M Proteins, Golgi Apparatus chemistry, Humans, Mutant Proteins genetics, Mutant Proteins metabolism, Protein Sorting Signals, Protein Transport, Spike Glycoprotein, Coronavirus genetics, Viral Matrix Proteins metabolism, Severe acute respiratory syndrome-related coronavirus physiology, Spike Glycoprotein, Coronavirus metabolism, Virosomes metabolism, Virus Assembly
- Abstract
The cytoplasmic tails of some coronavirus (CoV) spike (S) proteins contain an endoplasmic reticulum retrieval signal (ERRS) that can retrieve S proteins from the Golgi to the endoplasmic reticulum (ER); this process is thought to accumulate S proteins at the CoV budding site, the ER-Golgi intermediate compartment (ERGIC), and to facilitate S protein incorporation into virions. However, we showed previously that porcine epidemic diarrhoea CoV S proteins lacking the ERRS were efficiently incorporated into virions, similar to the original virus. Thus, the precise role of the ERRS in virus assembly remains unclear. Here, the roles of the S protein ERRS in severe acute respiratory syndrome CoV (SARS-CoV) intracellular trafficking and S incorporation into virus-like particles (VLPs) are described. Intracellular trafficking and indirect immunofluorescence analysis suggested that when M protein was present, wild-type S protein (wtS) could be retained in the pre- and post-medial Golgi compartments intracellularly and co-localized with M protein in the Golgi. In contrast, mutant S protein lacking the ERRS was distributed throughout the ER and only partially co-localized with M protein. Moreover, the intracellular accumulation of mutant S protein, particularly at the post-medial Golgi compartment, was significantly reduced compared with wtS. A VLP assay suggested that wtS that reached the post-medial compartment could be returned to the ERGIC for subsequent incorporation into VLPs, while mutant S protein could not. These results suggest that the ERRS of SARS-CoV contributes to intracellular S protein accumulation specifically in the post-medial Golgi compartment and to S protein incorporation into VLPs.
- Published
- 2016
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24. Identification of CCL2, RARRES2 and EFNB2 as host cell factors that influence the multistep replication of respiratory syncytial virus.
- Author
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Shirato K, Ujike M, Kawase M, and Matsuyama S
- Subjects
- Animals, Cell Line, Gene Expression Profiling, Humans, Microarray Analysis, Chemokine CCL2 metabolism, Chemokines metabolism, Ephrin-B2 metabolism, Host-Pathogen Interactions, Intercellular Signaling Peptides and Proteins metabolism, Respiratory Syncytial Virus, Human physiology, Virus Replication
- Abstract
Human respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infections in children worldwide. Preterm children or those with underlying cardiopulmonary disorders are at particularly high risk of developing severe and lethal RSV respiratory tract infections; however, there are currently no effective vaccines or anti-viral drugs. To identify targets for the development of drugs to treat RSV infections, we investigated host cell factors involved in the replication of RSV. To this end, MDCK cells with low susceptibility to RSV were transfected with cDNA libraries derived from RSV-susceptible human lung or HeLa cells. A microarray analysis was subsequently performed on parental MDCK cells and MDCK cells that were converted to an RSV-susceptible form. Among the genes identified, chemokine (C-C motif) ligand 2 (CCL2), retinoic acid receptor responder protein 2 (RARRES2) and ephrin-B2 (EFNB2) had a positive effect on RSV replication. Expression of these genes in MDCK cells resulted in a 10- to 100-fold increase in RSV replication. CCL2 expression also disrupted the distribution of claudin-1, a tight junction protein, suggesting that CCL2 plays a role in claudin-based tight junction formation during RSV replication. The knockdown of EFNB2 and RARRES2 by siRNA in RSV-susceptible cell lines (HEp-2 and A549) resulted in reduced RSV replication, suggesting that EFNB2 and RARRES2 participate in RSV replication. Together, our findings suggest that CCL2, RARRES2 and EFNB2 are host cell factors involved in RSV replication., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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25. Host Adaptation and the Alteration of Viral Properties of the First Influenza A/H1N1pdm09 Virus Isolated in Japan.
- Author
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Ainai A, Hasegawa H, Obuchi M, Odagiri T, Ujike M, Shirakura M, Nobusawa E, Tashiro M, and Asanuma H
- Subjects
- Amino Acid Substitution, Animals, Chickens, Dogs, Female, Ferrets virology, Influenza A Virus, H1N1 Subtype genetics, Influenza A Virus, H1N1 Subtype immunology, Japan, Kinetics, Madin Darby Canine Kidney Cells, Mice, Ovum virology, Adaptation, Physiological, Host-Pathogen Interactions, Influenza A Virus, H1N1 Subtype physiology
- Abstract
A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions.
- Published
- 2015
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26. Incorporation of spike and membrane glycoproteins into coronavirus virions.
- Author
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Ujike M and Taguchi F
- Subjects
- Protein Binding, Protein Multimerization, Protein Transport, Coronavirus physiology, Membrane Glycoproteins metabolism, Viral Envelope Proteins metabolism, Virion metabolism, Virus Assembly
- Abstract
The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein-protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein-protein interactions.
- Published
- 2015
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27. Hemagglutination mediated by the spike protein of cell-adapted bovine torovirus.
- Author
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Shimabukuro K, Ujike M, Ito T, Tsunemitsu H, Oshitani H, and Taguchi F
- Subjects
- Animals, Erythrocytes virology, Spike Glycoprotein, Coronavirus, Virus Attachment, Hemagglutination, Membrane Glycoproteins metabolism, Torovirus pathogenicity, Viral Envelope Proteins metabolism, Virulence Factors metabolism
- Abstract
Bovine torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity.
- Published
- 2013
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28. Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M-S co-localization.
- Author
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Ujike M, Huang C, Shirato K, Matsuyama S, Makino S, and Taguchi F
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Coronavirus M Proteins, Cysteine metabolism, Mice, Palmitic Acid metabolism, Severe acute respiratory syndrome-related coronavirus physiology, Spike Glycoprotein, Coronavirus, Virion metabolism, Virion physiology, Virus Assembly physiology, Membrane Glycoproteins metabolism, Severe acute respiratory syndrome-related coronavirus metabolism, Severe Acute Respiratory Syndrome virology, Viral Envelope Proteins metabolism, Viral Matrix Proteins metabolism
- Abstract
The endodomain of several coronavirus (CoV) spike (S) proteins contains palmitylated cysteine residues and enables co-localization and interaction with the CoV membrane (M) protein. Depalmitylation of mouse hepatitis virus S proteins abolished this interaction, resulting in the failure of S incorporation into virions. In contrast, an immunofluorescence assay (IFA) showed that depalmitylated severe acute respiratory syndrome coronavirus (SCoV) S proteins still co-localized with the M protein in the budding site. Here, we determined the ability of depalmitylated SCoV S mutants to incorporate S into virus-like particles (VLPs). IFA confirmed that all SCoV S mutants co-localized with the M protein intracellularly. However, the mutants lacking two cysteine residues (C(1234/1235)) failed to incorporate S into VLPs. This indicated that these palmitylated cysteines are essential for S incorporation, but are not involved in S co-localization mediated by the M protein. Our findings suggest that M-S co-localization and S incorporation occur independently of one another in SCoV virion assembly.
- Published
- 2012
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29. Increased replication of respiratory syncytial virus in the presence of cytokeratin 8 and 18.
- Author
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Shirato K, Ujike M, Kawase M, and Matsuyama S
- Subjects
- Cell Line, Humans, Keratin-18 genetics, Keratin-8 genetics, RNA, Viral biosynthesis, Respiratory Syncytial Virus Infections genetics, Respiratory Syncytial Virus Infections metabolism, Respiratory Syncytial Viruses genetics, Respiratory Syncytial Viruses metabolism, Keratin-18 metabolism, Keratin-8 metabolism, Respiratory Syncytial Virus Infections virology, Respiratory Syncytial Viruses physiology, Virus Replication
- Abstract
Previously, it was reported that productive viral infection, viral protein synthesis, and viral RNA replication of respiratory syncytial virus (RSV) operated efficiently in two human epithelial cell lines (HEp-2 and A549), but not in a human mast-cell line, HMC-1. Based on these observations, it was hypothesized that HMC-1 cells lack the machinery required for RSV replication. To identify the host factors required for RSV replication, cDNA subtraction using A549, HEp-2, and HMC-1 cells was performed, and cytokeratin 18 (C18) was identified as a candidate host factor. Because C18 is generally expressed in simple epithelia with cytokeratin 8 (C8), HMC-1 cells that constitutively express C18 and C8 (HMC-1-C8/18) were established to evaluate the role of C8/18 in RSV replication. In HMC-1-C8/18 cells, RSV RNA replication was increased, and the amount of infective virus produced was also increased in the cellular fraction after RSV spinoculation, whereas RSV production was decreased in A549 cells in which C18 expression was knocked down. These data suggest that the replication of RSV increases in the presence of C8/18., (Copyright © 2011 Wiley Periodicals, Inc.)
- Published
- 2012
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30. Mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (S) protein is responsible for enhanced fusion activity.
- Author
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Shirato K, Maejima M, Matsuyama S, Ujike M, Miyazaki A, Takeyama N, Ikeda H, and Taguchi F
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Coronavirus Infections virology, Cytoplasm virology, Membrane Glycoproteins chemistry, Molecular Sequence Data, Porcine epidemic diarrhea virus physiology, Protein Transport, Spike Glycoprotein, Coronavirus, Swine, Viral Envelope Proteins chemistry, Coronavirus Infections veterinary, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Mutation, Missense, Porcine epidemic diarrhea virus genetics, Protein Sorting Signals, Swine Diseases virology, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Virus Internalization
- Abstract
Murine-adapted porcine epidemic diarrhea virus (PEDV), MK-p10, shows high neurovirulence and increased fusion activity compared with a non-adapted MK strain. MK-p10 S protein had four mutations relative to the original virus S, and one of these (H→R at position 1381, H1381R) in the cytoplasmic tail (CT) was suggested to be responsible for the increased fusion activity. To explore this, we examined fusion activity using recombinant S proteins. We expressed and compared the fusion activity of MK-p10 S, S with the H1381R mutation, S with the three other mutations that were not thought to be involved in high fusion activity, and the original S protein. The MK-p10 and MK-H1381R S proteins induced larger cell fusions than others. We also examined the distribution of these S proteins; the MK-p10 and MK-H1381R S proteins were transported onto the cell surface more efficiently than others. These findings suggest that the H1381R mutation is responsible for enhanced fusion activity, which may be attributed to the efficient transfer of S onto the cell surface. H1381 is a component of the KxHxx motif in the CT region, which is a retrieval signal of the S protein for the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Loss of this motif could allow for the efficient transfer of S proteins from ERGIC onto the cell surface and subsequent increased fusion activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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31. Role of proteases in the release of porcine epidemic diarrhea virus from infected cells.
- Author
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Shirato K, Matsuyama S, Ujike M, and Taguchi F
- Subjects
- Animals, Base Sequence, Blotting, Western, Cell Fusion, Chlorocebus aethiops, DNA Primers, Microscopy, Electron, Polymerase Chain Reaction, Vero Cells, Virus Replication, Peptide Hydrolases metabolism, Porcine epidemic diarrhea virus physiology
- Abstract
Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhea, requires a protease(s) for multicycle replication in cultured cells. However, the potential role of proteases in the infection process remains unclear. In order to explore this, we used two different approaches: we infected either Vero cells in the presence of trypsin or Vero cells that constitutively express the membrane-associated protease TMPRSS2 (Vero/TMPRSS2 cells). We found that PEDV infection was enhanced, and viruses were efficiently released into the culture fluid, from Vero cells infected in the presence of protease, while in cells without protease, the virus grew, but its release into the culture fluid was strongly hampered. Cell-to-cell fusion of PEDV-infected cells and cleavage of the spike (S) protein were observed in cells with protease. When infected Vero cells were cultured for 3 days in the absence of trypsin but were then treated transiently with trypsin, infectious viruses were immediately released from infected cells. In addition, treatment of infected Vero/TMPRSS2 cells with the protease inhibitor leupeptin strongly blocked the release of virus into the culture fluid. Under electron microscopy, PEDV-infected Vero cells, as well as PEDV-infected Vero/TMPRSS2 cells treated with leupeptin, retained huge clusters of virions on their surfaces, while such clusters were rarely seen in the presence of trypsin and the absence of leupeptin in Vero and Vero/TMPRSS2 cells, respectively. Thus, the present study indicates that proteases play an important role in the release of PEDV virions clustered on cells after replication occurs. This unique observation in coronavirus infection suggests that the actions of proteases are reminiscent of that of the influenza virus neuraminidase protein.
- Published
- 2011
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32. Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay.
- Author
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Nakauchi M, Ujike M, Obuchi M, Takashita E, Takayama I, Ejima M, Oba K, Konomi N, Odagiri T, Tashiro M, and Kageyama T
- Subjects
- Amino Acid Substitution, Animals, Antiviral Agents pharmacology, Cell Line, Dogs, Drug Resistance, Viral drug effects, Drug Resistance, Viral genetics, Genetic Testing methods, Genotype, Hemagglutination Inhibition Tests, Humans, Influenza A Virus, H1N1 Subtype drug effects, Influenza, Human drug therapy, Influenza, Human epidemiology, Influenza, Human virology, Japan, Mutation, Missense, Pandemics, Sequence Analysis, DNA, Zanamivir pharmacology, Influenza A Virus, H1N1 Subtype genetics, Influenza, Human genetics, Molecular Typing methods, Neuraminidase genetics, Oseltamivir pharmacology, RNA, Viral analysis, Reverse Transcriptase Polymerase Chain Reaction methods, Viral Proteins genetics
- Abstract
Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity., (Copyright © 2011 Wiley-Liss, Inc.)
- Published
- 2011
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33. Molecular evolutionary analysis of the influenza A(H1N1)pdm, May-September, 2009: temporal and spatial spreading profile of the viruses in Japan.
- Author
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Shiino T, Okabe N, Yasui Y, Sunagawa T, Ujike M, Obuchi M, Kishida N, Xu H, Takashita E, Anraku A, Ito R, Doi T, Ejima M, Sugawara H, Horikawa H, Yamazaki S, Kato Y, Oguchi A, Fujita N, Odagiri T, Tashiro M, and Watanabe H
- Subjects
- Bayes Theorem, Cluster Analysis, Humans, Influenza, Human virology, Japan epidemiology, Evolution, Molecular, Influenza A Virus, H1N1 Subtype isolation & purification, Influenza, Human epidemiology
- Abstract
Background: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009., Methods/principal Findings: We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer., Conclusions: Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan.
- Published
- 2010
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34. A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir.
- Author
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Matsuzaki Y, Mizuta K, Aoki Y, Suto A, Abiko C, Sanjoh K, Sugawara K, Takashita E, Itagaki T, Katsushima Y, Ujike M, Obuchi M, Odagiri T, and Tashiro M
- Subjects
- Adolescent, Amino Acid Substitution genetics, Animals, Antiviral Agents pharmacology, Cell Line, Child, Preschool, Dogs, Humans, Infant, Influenza A Virus, H1N1 Subtype classification, Influenza A Virus, H1N1 Subtype growth & development, Influenza A Virus, H1N1 Subtype isolation & purification, Japan, Molecular Epidemiology, Molecular Sequence Data, Mutation, Missense, Neuraminidase genetics, Oseltamivir pharmacology, Phylogeny, Sequence Analysis, DNA, Treatment Outcome, Viral Proteins genetics, Virulence, Antiviral Agents therapeutic use, Drug Resistance, Viral, Influenza A Virus, H1N1 Subtype drug effects, Influenza, Human drug therapy, Influenza, Human virology, Oseltamivir therapeutic use, Zanamivir therapeutic use
- Abstract
Background: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir., Results: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356)., Conclusion: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.
- Published
- 2010
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35. Isolation of oseltamivir-resistant influenza A/H1N1 virus of different origins in Yokohama City, Japan, during the 2007-2008 influenza season.
- Author
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Kawakami C, Obuchi M, Saikusa M, Noguchi Y, Ujike M, Odagiri T, Iwata M, Toyozawa T, and Tashiro M
- Subjects
- Adult, Antiviral Agents administration & dosage, Child, Child, Preschool, Disease Outbreaks, Drug Resistance, Viral genetics, Female, Humans, Influenza A Virus, H1N1 Subtype drug effects, Influenza A Virus, H1N1 Subtype genetics, Influenza, Human drug therapy, Influenza, Human virology, Japan, Male, Middle Aged, Seasons, Antiviral Agents pharmacology, Influenza A Virus, H1N1 Subtype isolation & purification, Influenza, Human epidemiology, Oseltamivir pharmacology
- Published
- 2009
36. Heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway.
- Author
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Ujike M, Nishikawa H, Otaka A, Yamamoto N, Yamamoto N, Matsuoka M, Kodama E, Fujii N, and Taguchi F
- Subjects
- Animals, Chlorocebus aethiops, Endosomes virology, Peptide Hydrolases physiology, Vero Cells, Antiviral Agents pharmacology, Peptides pharmacology, Severe acute respiratory syndrome-related coronavirus physiology, Viral Proteins pharmacology, Virus Internalization drug effects
- Abstract
The peptides derived from the heptad repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two heptad repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface.
- Published
- 2008
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37. A point mutation at the C terminus of the cytoplasmic domain of influenza B virus haemagglutinin inhibits syncytium formation.
- Author
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Ujike M, Nakajima K, and Nobusawa E
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Chlorocebus aethiops, Flow Cytometry, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Hydrophobic and Hydrophilic Interactions, Influenza B virus physiology, Molecular Sequence Data, Mutagenesis, Site-Directed, Giant Cells physiology, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Hemagglutinin Glycoproteins, Influenza Virus genetics, Influenza B virus pathogenicity, Membrane Fusion, Point Mutation
- Abstract
The C-terminal sequence of the cytoplasmic tail (CT) of influenza B haemagglutinin (BHA) consists of strictly conserved, hydrophobic amino acids, and the endmost C-terminal amino acid of the CT is Leu. To elucidate the role of this amino acid in the fusion activity of BHA (B/Kanagawa/73), site-specific mutant HAs were created by replacing Leu at this position with Arg, Lys, Ser, Try, Val or Ile or by the deletion of Leu altogether. All mutants were expressed at the cell surface, bound to red blood cells, were cleaved properly into two subunits and could be acylated like the wild-type (wt) HA. The membrane-fusion ability of these mutants was examined with a lipid (R18) and aqueous (calcein) dye-transfer assay and quantified with a syncytium-formation assay. All mutant HAs showed no measurable effect on lipid mixing or fusion-pore formation. However, mutant HAs with a hydrophobic value of the C-terminal amino acid lower than that of Leu had a reduced ability to form syncytia, whereas mutants with a more hydrophobic amino acid (Val or Ile) promoted fusion to the extent of the wt HA. On the other hand, the mutant HA with the deletion of Leu supported full fusion. These results demonstrate that Leu at the endmost portion of the C terminus of the BHA-CT is not essential for BHA-mediated fusion, but that the hydrophobicity of the single amino acid at this position plays an important role in syncytium formation.
- Published
- 2006
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38. Enhancement of SARS-CoV infection by proteases.
- Author
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Matsuyama S, Ujike M, Ishii K, Fukushi S, Morikawa S, Tashiro M, and Taguchi F
- Subjects
- Animals, Cell Membrane metabolism, Chlorocebus aethiops, Endosomes virology, Intestines virology, Lung virology, Macrolides pharmacology, Peptide Hydrolases metabolism, Reverse Transcriptase Polymerase Chain Reaction, Vero Cells, Peptide Hydrolases chemistry, Severe acute respiratory syndrome-related coronavirus metabolism
- Published
- 2006
- Full Text
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39. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection.
- Author
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Matsuyama S, Ujike M, Morikawa S, Tashiro M, and Taguchi F
- Subjects
- Animals, Chlorocebus aethiops, Lung enzymology, Lung virology, Pancreatic Elastase pharmacology, Severe acute respiratory syndrome-related coronavirus drug effects, Severe acute respiratory syndrome-related coronavirus genetics, Severe acute respiratory syndrome-related coronavirus physiology, Severe Acute Respiratory Syndrome enzymology, Severe Acute Respiratory Syndrome virology, Thermolysin pharmacology, Trypsin pharmacology, Vero Cells, Virulence drug effects, Virulence physiology, Virus Replication drug effects, Peptide Hydrolases metabolism, Severe acute respiratory syndrome-related coronavirus pathogenicity, Severe Acute Respiratory Syndrome etiology
- Abstract
A unique coronavirus severe acute respiratory syndrome-coronavirus (SARS-CoV) was revealed to be a causative agent of a life-threatening SARS. Although this virus grows in a variety of tissues that express its receptor, the mechanism of the severe respiratory illness caused by this virus is not well understood. Here, we report a possible mechanism for the extensive damage seen in the major target organs for this disease. A recent study of the cell entry mechanism of SARS-CoV reveals that it takes an endosomal pathway. We found that proteases such as trypsin and thermolysin enabled SARS-CoV adsorbed onto the cell surface to enter cells directly from that site. This finding shows that SARS-CoV has the potential to take two distinct pathways for cell entry, depending on the presence of proteases in the environment. Moreover, the protease-mediated entry facilitated a 100- to 1,000-fold higher efficient infection than did the endosomal pathway used in the absence of proteases. These results suggest that the proteases produced in the lungs by inflammatory cells are responsible for high multiplication of SARS-CoV, which results in severe lung tissue damage. Likewise, elastase, a major protease produced in the lungs during inflammation, also enhanced SARS-CoV infection in cultured cells.
- Published
- 2005
- Full Text
- View/download PDF
40. Influence of additional acylation site(s) of influenza B virus hemagglutinin on syncytium formation.
- Author
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Ujike M, Nakajima K, and Nobusawa E
- Subjects
- Acylation, Animals, COS Cells, Chlorocebus aethiops, Hemagglutinin Glycoproteins, Influenza Virus genetics, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Hydrophobic and Hydrophilic Interactions, Influenza B virus metabolism, Influenza B virus physiology, Mutation, Giant Cells physiology, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Influenza B virus chemistry, Membrane Fusion
- Abstract
We studied the effects of an increase in the hydrophobicity of the transmembrane domain (TM) and cytoplasmic tail (CT) of influenza B virus hemagglutinin (BHA) on fusion activities. For this purpose, we created mutant HAs with novel acylation site(s) in the TM and/or CT. All mutants were able to induce hemifusion and to form fusion pores as well as could wild type (wt) BHA. However, the ability of these mutants to form syncytia was impaired, indicating that the increase in the hydrophobicity of these domains (especially the CT) affected fusion pore dilation.
- Published
- 2005
- Full Text
- View/download PDF
41. Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation.
- Author
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Ujike M, Nakajima K, and Nobusawa E
- Subjects
- Acylation, Amino Acid Sequence, Animals, COS Cells, Dogs, Hemagglutinin Glycoproteins, Influenza Virus physiology, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Influenza B virus physiology, Membrane Fusion
- Abstract
The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.
- Published
- 2004
- Full Text
- View/download PDF
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