臺灣博碩士論文加值系統

登革熱是目前世界上非常嚴重的蟲媒傳染疾病，目前尚無疫苗與有效的治療方式。在先前文獻中已經證實登革熱的核心蛋白(core)擁有三段入核訊號，可以在細胞質、細胞核、甚至是在核仁中被發現。目前在病毒的複製或是致病機轉中，核心蛋白在細胞核以及核仁中的功能為何至今仍不清楚。先前本實驗室透過了酵母菌雙雜合系統、共免疫沉澱法等方式已經證實了登革熱的核心蛋白與宿主的轉錄因子 TAF7 之間會有交互作用，並且得知兩者交互作用的功能區域。令人驚訝的是，我們發現了當共同表達兩者時， TAF7 會從細胞質中轉移到細胞核中，而 TAF7 本身是沒有入核訊號的。因此，我提出一個假設，認為登革熱的核心蛋白會帶著 TAF7 一同進到細胞核中。為了證實這個假設，我在 HeLa 細胞中同時表達全長的 TAF7 蛋白以及完整或是具缺失不同片段的核心蛋白，透過免疫染色以螢光顯微鏡觀察並計算TAF7入核比例。從結果中可以發現:相較於同時表達全長的 TAF7 和全長的核心蛋白時，若同時表達全長的 TAF7 和無交互作用功能區域的核心蛋白時，TAF7 在細胞質的比例有明顯上升的趨勢。另外，為了得知核心蛋白將 TAF7 帶進細胞核中是否會影響病毒的複製，我透過慢病毒系統在 HEK293T 以及 HuH 7 細胞弱化降低 TAF7 的表現量，並透過西方墨點法以及即時定量核酸聚合酶連鎖反應去確認表現量確實降低。之後透過病毒斑點試驗以檢測 TAF7 的表現量是否影響病毒的複製能力。結果中顯示在降低 TAF7 的表現量後，病毒的複製能力在兩種不同的細胞株以及兩種不同來源的病毒株中都可以提高病毒的效價會。這結果或許對於將來在登革熱的致病機轉上能夠有更進一步的了解，也有助於未來在登革熱的藥物的開發。

Dengue fever is one of the most important arthropod-borne diseases. Previous study demonstrates that the core protein has three nuclear location signals (NLS), and can be detected not only in the cytoplasm, but also in the nucleus, or even in the nucleolus. The functions of core protein in the nucleus/nucleolus and the roles in virus production/pathogenesis still remain unclear. From previous results of our laboratory, we identified human host factor TAF7 as the interacting protein of core from yeast two-hybrid screening, confirmed the interaction and mapped the interaction regions of core protein and TAF7. Surprisingly, we observed the distribution of TAF7 between cytoplasm and nucleus was changed in the presence of the full-length core protein. Since the core protein has NLS signals but TAF7 does not, we hypothesized that core protein might facilitate TAF7 to be translocated into the nucleus. In this study, I co-expressed both full-length, the N- or the C- terminus truncated core and full-length TAF7 in HeLa cells, and evaluated the distribution of TAF7 by confocal microscope. The results showed the distribution of TAF7 were changed when co-expressed both bull-length, the N- or C- terminus truncated core and full-length TAF7. In addition, in order to understand whether the TAF7 is important for virus production, the lenti-virus system has been used for knockdown TAF7 in HEK293T and Huh7 cells through Western blot and qRT-PCR test. DENV-2 strains PL046 and 16681 were used to infect the stable knocked-down HEK293T cells and transient HuH7 cells, and the viral titer after infection by plaque assay was used to evaluate the effect of TAF7 on virus production. The virus production was increased in TAF7 knockdown cells if challenged with different strain DENV. These results may be helpful in understanding the molecular mechanism of viral pathogenesis and in developing the drugs for dengue diseases in the future.

Abstract (Chinese version) I
Abstract II
Index IV
Introduction 1
1. Dengue Diseases 1
1.1 Dengue virus 1
1.2 Dengue virus core protein 5
2. Human TBP-associated factors (hTAFs) 7
2.1 hTAFs 7
2.2 TAF7s 8
1. Materials 10
1.1 Bacterial 10
1.2 Cell lines 10
1.3 Virus 11
1.4 Medium 12
1.5 Primers 12
1.6 Plasmids 12
1.7 Chemicals and Reagents 13
1.8 Antibodies 13
2. Methods 15
2.1 DNA preparation 15
2.2 E.coli transformation 17
2.3 Cell culture 17
2.4 Transfection 18
2.5 Immunofluorescence assay 18
2.6 SDS-PAGE 19
2.7 Western blot 20
2.8 RNA extraction 21
2.9 qRT-PCR 21
2.10 Lenti-virus production 22
2.11 Lenti-virus infection 23
2.12 Virus infection 23
2.13 Plaque assay 24
2.14 TCID50 25
2.15 Virus amplification 25
Result 27
1. Interaction between TAF7 and core protein of dengue virus can affect the distribution of TAF7 27
2. Lenti-virus knockdown system was established 28
3. TAF7 knockdown increased the DENV production 28
4. Overexpression of the full-length TAF7 and central region-deleted TAF7 (Δ180-230) do not affect the viral production 30
Discussion 31
1. The nuclear location signal of DENV core protein 31
2. The amount of TAF7 was limited in host cell 32
3. The relationship of TAF7 and core interaction how to affect the dengue virus infection 32
4. Virion plaque size harvested from Huh7 cells is smaller than that harvested from HEK293T cells 34
References 35
Tables and Figures 42
Appendix 58

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