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Characterization of intracellular interactions between dengue virus and host proteins.

机译：登革热病毒和宿主蛋白之间的细胞内相互作用的表征。

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摘要

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of world population live in areas where dengue is prevalent, leading to high levels of morbidity and mortality in many areas. Currently there are no vaccines or effective treatments. The virus is transmitted from one person to another by the yellow fever mosquito, Aedes aegypti. The genome of dengue virus encodes only ten proteins implying that the virus needs to interact with and utilize several host proteins for replication. In this project, I used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. I detected 46 dengue-human and 102 dengue-mosquito protein interactions, including some that had been discovered previously and many novel interactions. I further confirmed 38 out of 136 testable interactions using co-affinity purification assays from cultured cells. I tested each host protein against the proteins from all four serotypes of dengue virus and found that 57 out of 102 (56.9%) dengue-mosquito PPI and 34 out of 46 (73.9%) dengue-human PPI interacted with corresponding dengue proteins from all four serotypes.;To further analyze biological significance of these protein interactions, I selected to study capsid-NAP1 interaction. I employed the domain mapping of capsid using yeast two-hybrid and co-affinity purification. I also over-expressed or silenced NAP1L1 in HepG2 cells stably expressing capsid. I found that NAP1L1 might bind the bipartite sequence of capsid blocking importin binding and sequestering capsid in the cytoplasm.;I also showed that the mosquito cells, AAG2, were capable of uptaking double stranded RNA without a transfection vehicle. Thus, a large-scale RNA interference study in AAG2 as previously published is feasible.;Finally, I showed that using two 2A sequences to generate three separate peptides form a single mRNA was possible in the insect cells. This construct may be applied to design a non-infectious dengue replicon, which may be a safer substitute of the live dengue virus.;The dengue-host interaction maps and the new tools that I generated should be useful for understanding how dengue interacts with its hosts and may provide candidates for drug targets and vector control strategies.
机译：登革热病毒是登革热，登革出血热和登革热休克综合症的病原体。大约五分之二的世界人口生活在登革热流行的地区，导致许多地区的发病率和死亡率很高。当前没有疫苗或有效的治疗方法。该病毒是由黄热伊蚊埃及伊蚊（Aedes aegypti）从一个人传播到另一个人的。登革热病毒的基因组仅编码十种蛋白质，这意味着该病毒需要与多种宿主蛋白​​质相互作用并利用其进行复制。在该项目中，我使用了高通量酵母双杂交筛选技术来鉴定与登革热蛋白发生物理相互作用的蚊子和人类蛋白。我检测到46种登革热-人和102种登革热-蚊子蛋白相互作用，包括以前发现的相互作用和许多新颖的相互作用。我使用培养细胞的共亲和纯化试验进一步证实了136种可测相互作用中的38种。我针对每种四种登革热病毒血清型的蛋白质测试了每种宿主蛋白​​质，发现102种登革热蚊子PPI中有57种（56.9％）和46种登革热人PPI中有34种（73.9％）与所有登革热中的相应登革热蛋白相互作用四种血清型。为了进一步分析这些蛋白质相互作用的生物学意义，我选择研究衣壳-NAP1相互作用。我采用了酵母双杂交和共亲和纯化方法对衣壳进行结构域定位。我还在稳定表达衣壳的HepG2细胞中过表达或沉默了NAP1L1。我发现NAP1L1可能结合了衣壳的二分体序列，从而阻断了importin的结合并隔离了衣壳中的衣壳。我还表明，蚊子细胞AAG2能够摄取双链RNA而无需转染载体。因此，如先前发表的在AAG2中进行大规模RNA干扰研究是可行的。最后，我证明了在昆虫细胞中使用两个2A序列生成三个分离的肽形成单个mRNA是可行的。该构建体可用于设计非感染性登革热复制子，可以更安全地替代活登革热病毒。登革热与宿主的相互作用图和我生成的新工具应有助于理解登革热如何与其登革热相互作用。宿主，并可能提供药物靶点和媒介控制策略的候选对象。

著录项

  • 作者

    Mairiang, Dumrong.;

  • 作者单位

    Wayne State University.;

  • 授予单位 Wayne State University.;
  • 学科 Biology Microbiology.;Biology Entomology.;Biology Genetics.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 394 p.
  • 总页数 394
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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