C型肝炎病毒非結構性蛋白質NS5A對於趨化素CCL3基因之調控

Regulation of CCL3 Gene by the Non-structural Protein 5A of Hepatitis C Virus

王浩宇(Hao-Yu Wang)

指導教授 ： 張鑫

國立臺灣大學/醫學院/微生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10447

摘要

感染C型肝炎病毒(hepatitis C virus, HCV)的患者有高比例的機會發展為慢性持續感染，進而造成肝纖維化、肝硬化甚至是肝細胞癌。而HCV的非結構性蛋白質5A (non-structural protein 5A, NS5A)已知會和宿主蛋白質進行交互作用，進而影響宿主細胞的訊息傳遞及基因表現，因此被認為可能是造成慢性感染的原因之一。在先前實驗室的研究中，將帶有肝臟專一性啟動子的NS5A蛋白質表現質體送入小鼠體內，挑選成功表現NS5A蛋白質的肝臟細胞，利用cDNA微陣列系統分析細胞內的基因表現，發現趨化素CCL3的基因表現量有顯著的下降。另外，在HCV複製子及穩定表現NS5A蛋白質的細胞株中也偵測到相同現象。因此，本研究進一步探討NS5A蛋白質如何調控趨化素CCL3的基因表現。
首先，相較於沒有表現NS5A蛋白質的細胞，在穩定表現NS5A蛋白質的Huh7細胞株以及轉染NS5A蛋白質表現質體的293T細胞中，均偵測到CCL3 mRNA表現量有明顯降低的情形，因此接著探討NS5A蛋白質是否會在轉錄階段調控CCL3的基因表現。透過建構一系列含有不同長度CCL3啟動子的螢火蟲冷光酶基因報導質體，分析CCL3啟動子的轉錄活性，發現在NS5A蛋白質的表現下，相較於CCL3(-996/+59)，CCL3(-653/+59)、CCL3(-272/+59)以及CCL3(-173/+59)的啟動子活性均有下降的現象，其中又以CCL3(-173/+59)啟動子活性降低幅度最為顯著，因此認為NS5A蛋白質主要藉由啟動子核苷酸-173到+59區間負調控CCL3基因表現。
進一步對於此區間內的轉錄因子NF-κB及AP-1結合位分別進行定點突變，結果顯示在不表現NS5A蛋白質時，定點突變組的CCL3啟動子的活性相對於野生型組均明顯下降，因此NF-κB及AP-1對於調控CCL3啟動子的基礎活性是重要的。在NS5A蛋白質的參與下，NF-κB及AP-1結合位突變對於NS5A蛋白質負調控CCL3啟動子的能力只有些微影響，顯示NS5A蛋白質可能不是透過NF-κB及AP-1結合位，影響CCL3(-173/+59)啟動子活性。利用西方墨點法偵測NS5A蛋白質是否會影響轉錄因子在細胞內的分布及表現量，結果顯示NS5A蛋白質會促進細胞質中磷酸化的NF-κB subunit p65 (p-p65)表現，但細胞核內p-p65並未增加。另一方面，NS5A蛋白質對於p53及AP-1的表現量均無明顯影響。NS5A蛋白質是否會透過與轉錄因子結合而調控CCL3啟動子活性，以及是否會影響p-p65進入細胞核中則有待進一步證實。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質5A ； 趨化素CCL3 ； 基因調控

並列摘要

Patients who were infected with hepatitis C virus (HCV) had a high opportunity to turn to persistent infection, and might progress to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. The non-structural protein 5A (NS5A) of HCV was known to interact with cellular proteins and influence signaling pathways and gene expression in host cells. Therefore, it is convinced that NS5A is one of the cause of persistent infection of HCV. In our previous studies, mice were introduced with NS5A-expressing plasmids harboring a liver-specific promoter. cDNA microarray was performed to analyze the gene expression profile in the hepatocytes which successfully expressed NS5A. The results demonstrated a decreased level of chemokine CCL3 mRNA under the expression of NS5A. The effect of NS5A was also observed in HCV replicon cells and in Huh7 cell lines stably expressing NS5A. The current study aims to further investigate how NS5A regulates the gene expression of CCL3.
The down-regulation of CCL3 mRNA was first analyzed and confirmed in Huh7 cell lines stably expressing NS5A and in 293T cells transfected with NS5A-expressing plasmids. To examine whether NS5A regulates CCL3 gene expression at transcriptional level, a series of luciferase reporter plasmids carrying various lengths of CCL3 promoter were constructed and analyzed for the promoter activities. In the presence of NS5A, the promoter activities of CCL3(-653/+59), CCL3(-272/+59) and CCL3(-173/+59) were reduced as compared to the CCL3(-996/+59). Among them, the CCL3(-173/+59) showed a most significant decrease. Therefore, the negative regulation of NS5A on the CCL3 promoter activity mainly involved in the CCL3 promoter region from -173 to +59. Next, the binding site of NF-κB and AP-1 in this region was mutated individually and subjected to luciferase assay. The results demonstrated significant decreases on the luciferase activity, indicating the involvement of the NF-κB(-80/-71) and AP-1(-155/-149) binding sites on the basal promoter activity of CCL3. On the other hand, the inhibition ability of NS5A on CCL3 promoter activity was not significantly affected with the binding site being mutated. To examine whether NS5A would affect the expression and distribution of transcriptional factors, cellular proteins were fractionationed and analyzed by Western blotting. The results showed that NS5A induced the expression of phosphorylated NF-κB subunit p65 (p-p65) localized in the cytoplasm but not in nuclei. In addition, NS5A had no significant effects on the expression level of p53 and AP-1. Whether NS5A regulates CCL3 promoter activity through interacting with transcriptional factors, and whether NS5A influences the translocation of p-p65 from cytoplasm to nuclei remain to be examined.

並列關鍵字

Hepatitis C virus ； NS5A ； CCL3 ； gene regulation

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