Evaluation on the Feasibility of Using GFPTax and Mutants for Investigation on Tax-Mediated Effects

• Main Authors: Chia-Ming Yang, 楊嘉銘
• Other Authors: Shin-Lian Doong
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/26496513870966188719

碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === HTLV-1 is the etiological agent of adult T cell leukemia (ATL), the neurological disease (HTLV-1-assoicated myelopathy/tropical spastic paraparesis, HAM/TSP) and other clinical disorders. The HTLV-1 encoded protein, Tax, affects a variety of cellular functions, prompting it to be considered as the pivotal factor in the process leading to these diseases. Tax modulates the expression of many viral and cellular genes through the CREB/ATF-, SRF-, and NF-κB -associated pathways. In this study, we would like to evaluate the potential of using GFPTax to examine the Tax-mediated cellular growth. Tax was fused with green fluorescence protein (GFP) for better visualization under fluorescence microscope and detection. The three Tax mutants utilized in this study were TaxH43Q (defect in NF-κB activating ability), TaxS274A (defect in both NF-κB and CREB/ATF activating ability), and TaxL320G (defect in CREB/ATF activating ability). In the first part of the study, we investigated if fusion with GFP would change the cellular distribution and trans-activating function of Tax. Fused GFPTax and GFPTax mutant genes ( GFPTaxH43Q , GFPTaxS274A, and GFPTaxL320G) were cloned into doxycyclin -inducible expression vector respectively. Northern blot and Western blot analysis confirmed the expression of these fusion products as expected. Reporter assay using pNF-κB-Luc (for detection of NF-κB activating ability) and pTxRE-Luc (containing 5 tandem repeats of Tax-responsive element from HTLV-1 LTR, for detection of CREB-activating ability) was performed to evaluate the trans-activating ability of these fusion products. Surprisingly, while GFPTax retained the ability to activate the NF-κB pathway as Tax, the fusion of GFP diminished the Tax-mediated CREB- activating ability. TaxH43Q was shown to be defective in the NF-κB pathway, however, GFPTaxH43Q resumed its NF-κB activating ability. Confocal microscope was utilized to examine the cellular distribution of these fusion products as compared to their authentic proteins. Both TaxS274A and GFPTaxS274A were evenly and predominantly distributed in the cytoplasm. GFPTax, GFPTaxH43Q and GFPTaxL320G were found to be localized near the nuclear membrane in punctuated form. Tax, TaxH43Q and TaxL320G were mainly distributed in the nucleus in punctuated form. The fusion with GFP did affect the cellular distribution and trans-activating function of Tax. Meanwhile, lentivirus-base expression system was employed to transduce the fused GFPTax and mutant genes into the hepatoblastoma cell line – Hep G2 and T-cell leukemia cell line – Jurkat to examine their effects on population doubling time and colony formation ability. The transduction efficiency of EGFP and GFPTax could reach 80%, however, transduction efficiency of GFPTax mutants could only up to 50%. GFPTax increased the population doubling time in Hep G2 and Jurkat cells. Colony formation ability decreased in Hep G2 cells with expression of GFPTax. No obvious cellular growth changes were observed in Hep G2 cells with expression of the three GFPTax mutants. Taken together, our results raised an uncertainty in utilizing GFPTax for investigation on Tax-mediated effects. Problems and solutions will be discussed in the text.