Publications by authors named "Kotaro Ishida"

4 Publications

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Endoplasmic reticulum-associated degradation controls virus protein homeostasis that is required for the flavivirus propagation.

J Virol 2021 May 12. Epub 2021 May 12.

Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan

Many positive-stranded RNA viruses encode polyproteins and viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though their required amounts are different. In this study, we found that the extra membrane-anchored non-structural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 predominantly localized in the convoluted membrane (CM) of viral replication organelle, and viral NS proteins degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses.The results of this study reveal that the cellular ERAD system controls the amount of each viral protein in virus-infected cells; this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/Sel1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.
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http://dx.doi.org/10.1128/JVI.02234-20DOI Listing
May 2021

Triplet induction chemotherapy followed by less invasive surgery without reconstruction for human papillomavirus-associated oropharyngeal cancers: Why is it successful or unsuccessful?

Int J Clin Oncol 2021 Jun 8;26(6):1039-1048. Epub 2021 Mar 8.

Division of Radiation Oncology, Shizuoka Cancer Center, Shizuoka, Japan.

Background: De-escalating treatments have been focused on for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). We assessed the efficacy of a triplet induction chemotherapy (ICT) followed by surgery with or without neck dissection (ND) for locally advanced OPSCC, aiming at less invasive surgery without free-flap reconstruction and avoiding postoperative irradiation.

Methods: This was a retrospective study of 41 patients with advanced resectable HPV-positive OPSCC who underwent ICT followed by surgery of primary resection with or without ND. Patients underwent triplet ICT, including docetaxel, cisplatin, and 5-fluorouracil, or carboplatin, paclitaxel, and cetuximab.

Results: Twenty-nine patients had tonsillar cancer, 15 patients were current smokers, and 18 and 12 patients had T2N1M0 and T1N1M0 status (UICC 8th), respectively. After ICT, a surgical procedure without free-flap reconstruction and tracheostomy was possible in 90.2%. Pathological complete response at both the primary site and lymph nodes was achieved in 73.2%. Of the patients who underwent surgery, no adjuvant radiotherapy was required in 85.0%. Two patients (4.9%) experienced recurrence at regional lymph nodes, but were cured by salvage ND followed by adjuvant radiotherapy.

Conclusions: Upfront ICT using highly responsive triplet chemotherapeutic regimens may enable us to perform less invasive surgery without free-flap reconstruction and to avoid postoperative irradiation to the locoregional field through excellent postoperative pathological features.
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http://dx.doi.org/10.1007/s10147-021-01894-zDOI Listing
June 2021

Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus.

Bio Protoc 2020 May 5;10(9):e3606. Epub 2020 May 5.

Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki-shi, Aomori, Japan.

Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such analyses. We developed a method for viral particle analysis that integrates a high-sensitivity split luciferase system and density gradient centrifugation. This protocol enables high-resolution profiling of particles produced by cells expressing Japanese encephalitis virus factors.
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http://dx.doi.org/10.21769/BioProtoc.3606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842715PMC
May 2020

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

J Virol 2019 10 12;93(19). Epub 2019 Sep 12.

Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles. This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.
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http://dx.doi.org/10.1128/JVI.00612-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744241PMC
October 2019