Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system
碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 99 === Dengue virus (DENV) which belongs to the Flaviviridae family is the causative agent of dengue fever and dengue hemorrhagic fever. DENV contain a 10.7 kb single-stranded positive sense RNA genome. After cell entry, the viral genome is translated into a polyp...
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ndltd-TW-099YM0051050032015-10-13T20:37:07Z http://ndltd.ncl.edu.tw/handle/14127823787969208946 Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system 利用以膜為基礎的分裂泛素酵母菌雙雜交系統探討登革病毒非結構蛋白的交互作用 Jan-Ing Chang 張峻瑛 碩士 國立陽明大學 生命科學暨基因體科學研究所 99 Dengue virus (DENV) which belongs to the Flaviviridae family is the causative agent of dengue fever and dengue hemorrhagic fever. DENV contain a 10.7 kb single-stranded positive sense RNA genome. After cell entry, the viral genome is translated into a polyprotein and processed into three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The structural proteins are the components of virus particles, while the rest of the NS proteins are involved in viral genome replication. NS1 is a secreted protein; NS2A, NS2B, NS4A, and NS4B are membrane proteins, NS3 and NS5 are multifunctional cytosolic proteins. According to previous studies, NS3 can interact with NS2B, NS4B and NS5, while the interaction among membrane associated NS proteins is still unknown. In this study, I used the Split-Ubiquitin Membrane based Yeast Two-Hybrid System (MbYTH) to analyze the possible interaction between DENV membrane associated NS proteins and cytosolic NS proteins in order to predict the role of membrane associated NS proteins in virus replication. We found that NS2A, NS2B, NS4A, and NS2k4B (NS4B with its upstream 2 kDa signal peptide) not only interacting among themselves, but also having self-interaction. NS2B and NS4B were interacting with NS3 cytosolic protein. To gain further understanding of the possible interacting domains of NS4B (the biggest membrane associated NS proteins) with other NS proteins, we used the truncated NS2k4B to study the possible interacting domains. We found that NS4B with 191~248 aa deletion abolished the interaction with NS3. The interactions of NS4B with 93~248 aa deletion with NS2A, NS2B, NS4A proteins were abolished. NS4B can interact with itself with only the N terminal 93 aa. By introducing NS4B P200L mutation that has found to abolish the viral replication in our preliminary study, we found that NS4B P200L will disrupt the interaction between NS2k4B and NS3. All these results suggested that NS4B was having specific domain to influence the interaction. Huey-Nan Wu 吳惠南 2011 學位論文 ; thesis 71 zh-TW |
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碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 99 === Dengue virus (DENV) which belongs to the Flaviviridae family is the causative agent of dengue fever and dengue hemorrhagic fever. DENV contain a 10.7 kb single-stranded positive sense RNA genome. After cell entry, the viral genome is translated into a polyprotein and processed into three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The structural proteins are the components of virus particles, while the rest of the NS proteins are involved in viral genome replication. NS1 is a secreted protein; NS2A, NS2B, NS4A, and NS4B are membrane proteins, NS3 and NS5 are multifunctional cytosolic proteins. According to previous studies, NS3 can interact with NS2B, NS4B and NS5, while the interaction among membrane associated NS proteins is still unknown. In this study, I used the Split-Ubiquitin Membrane based Yeast Two-Hybrid System (MbYTH) to analyze the possible interaction between DENV membrane associated NS proteins and cytosolic NS proteins in order to predict the role of membrane associated NS proteins in virus replication. We found that NS2A, NS2B, NS4A, and NS2k4B (NS4B with its upstream 2 kDa signal peptide) not only interacting among themselves, but also having self-interaction. NS2B and NS4B were interacting with NS3 cytosolic protein. To gain further understanding of the possible interacting domains of NS4B (the biggest membrane associated NS proteins) with other NS proteins, we used the truncated NS2k4B to study the possible interacting domains. We found that NS4B with 191~248 aa deletion abolished the interaction with NS3. The interactions of NS4B with 93~248 aa deletion with NS2A, NS2B, NS4A proteins were abolished. NS4B can interact with itself with only the N terminal 93 aa. By introducing NS4B P200L mutation that has found to abolish the viral replication in our preliminary study, we found that NS4B P200L will disrupt the interaction between NS2k4B and NS3. All these results suggested that NS4B was having specific domain to influence the interaction.
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author2 |
Huey-Nan Wu |
author_facet |
Huey-Nan Wu Jan-Ing Chang 張峻瑛 |
author |
Jan-Ing Chang 張峻瑛 |
spellingShingle |
Jan-Ing Chang 張峻瑛 Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
author_sort |
Jan-Ing Chang |
title |
Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
title_short |
Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
title_full |
Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
title_fullStr |
Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
title_full_unstemmed |
Studies of the Dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
title_sort |
studies of the dengue virus nonstructural proteins interaction by the split-ubiquitin membrane-based yeast two-hybrid system |
publishDate |
2011 |
url |
http://ndltd.ncl.edu.tw/handle/14127823787969208946 |
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