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Author Mairiang, Dumrong
Title Characterization of intracellular interactions between dengue virus and host proteins [electronic resource] / by Dumrong Mairiang.
Publication Info. 2012
Location Call No. Status Notes
 Libraries Electronic Books  Electronic Resource - WSU ETD    AVAIL. ONLINE
Description 395 p. : ill.
Note Advisor: Russell L. Finley.
Thesis Thesis (Ph.D.) -- Wayne State University, 2012.
Summary 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.
Subject Microbiology
Molecular biology
Added Title Wayne State University thesis (Ph.D.) : Molecular Biology and Genetics
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