Intracellular localization of influenza virus A/PR/8/34 NS1A protein in live cells
碩士 === 中興大學 === 生物科技學研究所 === 98 === The influenza A viruses are the prototype of the family Orthomyxoviridae. They contain eight single-stranded, negative-sense, segmented viral RNA (vRNA), which encode 11 known proteins. The vRNA segment eight encodes two nonstructural proteins, NS1A and NEP. Th...
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| Format: | Others |
| Language: | en_US |
| Online Access: | http://ndltd.ncl.edu.tw/handle/18613562676648046750 |
| Summary: | 碩士 === 中興大學 === 生物科技學研究所 === 98 === The influenza A viruses are the prototype of the family Orthomyxoviridae. They contain eight single-stranded, negative-sense, segmented viral RNA (vRNA), which encode 11 known proteins. The vRNA segment eight encodes two nonstructural proteins, NS1A and NEP. The NS1 protein is a multifunctional protein that counteracts host cell antiviral responses and inhibits host cell pre-mRNA processing. In order to study the dynamics of localization of NS1 protein during virus infection in live cells, tetracysteine-tag (TC-tag) which can bind the membrane permeable bis-arsenical fluorescein FlAsH with fluorescent was fused with NS1 at the linker region between RNA-binding domain and effector domain and the loop region inside the effector domain. The construct containing TC-tag at the linker region of NS1A is cotransfected with all other plasmids of reverse genetic system into 293 cells to generate the recombinant virus. The mutant virus shows similar the infectivity with that of wild type. In vitro labeling the mutant NS1-tc-linker protein with FlAsH was successful and specific. The specific localization of NS1 in infected cells can be visualized after FlAsH labeling by confocal microscopy. NS1 was found to localize in mitochondria at very early time point of infection (1.5 hpi); and at 4 hpi and 8hpi, it was localized predominantly in nucleus at 4 hpi and 8 hpi and formed a granular pattern. These results suggested that the biarsenical labeling technique is successfully established and it can be an important strategy to visualize the location of target proteins dynamically in live cell.
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