Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis. / Land and Food Systems, Faculty of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22810号 / 生博第444号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...

摘 要 本研究主要是探討利用新式生物反應器以固定化細胞培養技術生產日本腦炎病毒(Japanese encephalitis virus；JEV)之研究，首先根據所培養細胞的生長特性與原有生物反應器之缺點，利用已改良設計之新式生物反應器，評估此新式生物反應器適用性、效能，以及所培養細胞之生長代謝情形與病毒力價。整個實驗過程大致分為幾個階段，第一個階段探討細胞固定化培養之最適化培養條件與生長代謝情形，第二個階段找出細胞固定化培養於此新式生物反應器中最佳生長狀態，最後一個階段為病毒的培養。實驗後發現Vero細胞經固定化貼附於FIBRA-CEL®載體上，可擴大培養於新式生物反應器，Vero細胞最佳生長量達到6.6×106cells/mL。希望藉由此改良之新式生物反應器提供細胞與病毒一個良好之生長培養環境，獲得高產量、品質穩定一致之細胞生物製品，以提供ㄧ設備簡單與製程操作容易、低成本、低能源消耗之細胞製品生產基座。 / Abstract In this study, we investigated the production of Japanese encephalitis virus (JEV) by the immobilized cell technology in a novel bioreactor. According to the disadvantages of original bioreactor and growth characteristics of cell culture, we evaluated the suitability and efficiency of a design-improved novel bioreactor as well as the growth and metabolic situation of cultured cells and titers of JEV. All studies including three major stages: (1) investigation of the optimal conditions and metabolic situation for the growth of immobilized cells, (2) finding the optimal conditions for the growth of immobilized cells in this novel bioreactor, and (3) growth of JEV using immobilized cells in this novel bioreactor. Our results showed that after immobilization on the FIBRA-CEL® carries, Vero cells can grow on the novel bioreactor up to the density of 6.6 × 106 cells/mL. Hopefully, the improvement of the novel bioreactor will provide an optimal growth condition for both the cells and viruses. Furthermore, it will also provide the basis for the production of cell products with advantages of simple-equipped, easy-to-operate, low cost, and low energy consumption. / 目 錄 誌謝------------------------------------------------- i 中文摘要 -------------------------------------------- ii 英文摘要 -------------------------------------------- iii 目錄 -------------------------------------------- iv 表目錄 -------------------------------------------- v 圖目錄 -------------------------------------------- vi 第一章 緒論---------------------------------------- 1 第二章 文獻探討------------------------------------ 3 第一節 日本腦炎病毒疫苗---------------------------- 3 第二節 動物細胞的培養------------------------------ 4 第三節 載體上動物細胞的培養------------------------ 5 第四節 動物細胞培養於生物反應器-------------------- 7 第三章 材料與方法---------------------------------- 10 一 細胞株的培養-------------------------------- 10 二 細胞冷凍保存與解凍培養---------------------- 10 三 細胞滾瓶培養-------------------------------- 11 四 病毒株的培養-------------------------------- 12 五 固定化載體材料製備-------------------------- 12 六 載體上細胞數的測定-------------------------- 12 七 細胞貼壁率的計算---------------------------- 13 八 生物反應器結構特性與固定化細胞培養---------- 13 九 日本腦炎病毒力價測定------------------------ 19 十 葡萄糖的測定-------------------------------- 19 第四章 結果與討論---------------------------------- 20 一 固定化載體材料比例對Vero細胞生長的影響------ 20 二 細胞貼附固定化時間對Vero細胞生長的影響------ 23 三 細胞接種量對Vero細胞生長的影響-------------- 24 四 生物反應器培養系統對Vero細胞生長的影響------ 25 五 新鮮培養基更換對Vero細胞生長的影響---------- 27 六 最適化細胞生長條件培養日本腦炎病毒---------- 29 第五章 結論與建議---------------------------------- 30 參考文獻 -------------------------------------------- 31 附錄一 PBS配製方法--------------------------------- 62 附錄二 Medium 199配製方法-------------------------- 62 附錄三 MEM medium配製方法-------------------------- 62