探討不同基因型C型肝炎病毒之F蛋白質對干擾素表現及反應之調控

Genotypic Effects of HCV Frame-Shift (F) Protein in Interferon Induction and Response Pathways

賴羿廷(Yi-Ting Lai)

指導教授 ： 劉旻禕

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10335

摘要

第一型干擾素是抵抗病毒感染的第一道防線，細胞在受到病毒感染後會刺激第一型干擾素的產生，活化其訊息傳遞路徑並刺激下游抗病毒基因表現來抵禦病毒的入侵，然而C型肝炎病毒有多種機制能逃脫先天性免疫反應的攻擊，進而造成宿主慢性感染，甚至可能演變成肝癌。C型肝炎病毒的F蛋白質與Core蛋白質的基因序列重疊，是Core基因序列發生框移的產物。F蛋白質在感染C型肝炎病毒的患者體內確實有表現，且最常在感染基因型第一型病毒的患者體內發現。也有研究顯示在慢性感染患者體內抗F抗體含量顯著較高。先前研究發現C型肝炎病毒Core蛋白質能調控干擾素訊息傳遞路徑，但並未排除該現象是由F蛋白質調控的可能性。因此本篇論文欲研究不同基因型C型肝炎病毒之F蛋白質在干擾素表現及反應訊息傳遞路徑之調控。
首先我們建構不同質體，包含四種基因型〈1a、2a、3a及4a〉C型肝炎病毒的Core序列，且將序列突變使該序列只能表現F蛋白質或是Core蛋白質。接著比較不同基因型胺基酸序列之差異，四種基因型的Core胺基酸序列長度一樣且組成相似，但F蛋白質的胺基酸序列長度及組成差異皆大，因此我們推測不同基因型F蛋白質對於第一型干擾素訊息傳遞路徑作用不同，可能影響先天性免疫反應以及干擾素相關治療。
接著我們利用 dual luciferase reporter assay 觀察F蛋白質對於第一型干擾素表現訊息傳遞路徑的影響。在表現1a型F〈F1a〉蛋白質的Huh7細胞內IFNβ的啟動子活性較控制組來得高，相反的在2a型F〈F2a〉蛋白質表現的細胞內IFNβ啟動子活性則較低。IFNβ上游的IRF3啟動子活性與IFNβ啟動子活性結果一樣，IRF7啟動子活性只有在表現2a型F蛋白質的細胞內較低，NFκB的啟動子活性則都無差異，在MAVS knock-down的Huh7細胞內，表現1a型F蛋白質的細胞IFNβ啟動子活性也較高，而表現2a型F蛋白質的細胞則較低，F蛋白質可能作用於MAVS到IRF3的傳遞路徑之間。以BX795抑制TBK1後觀察IFNβ啟動子活性，也得到相同的結果，表現1a型F蛋白質的細胞IFNβ啟動子活性也較高，而2a型較低。因此我們推測F蛋白質可能直接作用在IRF3上。另外我們也觀察了F蛋白質對IFNβ反應訊息傳遞路徑的影響，在表現1a型F蛋白質的細胞內IFN-stimulated response element(ISRE)的啟動子活性較控制組來得高，相反的在2a型F蛋白質表現的細胞內ISRE啟動子活性則較低，我們也利用real-time PCR觀察不同ISGs mRNA表現的差異來再次驗證前面實驗的結果。我們也使用了另一個細胞株PH5CH8重複上述實驗，一樣得到相同的趨勢。
我們的研究結果顯示，F蛋白質確實能調控第一型干擾素訊息傳遞路徑，1a型F蛋白質會加強此路徑的訊息傳遞，而2a型則會抑制。在第一型干擾素訊息傳遞路徑上游的調控應是作用於IRF3，下游的調控位置還需要進一步研究。而不同基因型F蛋白質調控此路徑進一步的分子機制尚待研究。

關鍵字

C 型肝炎病毒 ； F 蛋白質 ； 第一型干擾素訊息傳遞路徑

並列摘要

Type I interferons (IFN) are the first line of defense against virus infection, production of IFN might induce hundreds of IFN stimulated genes (ISGs) to establish an antiviral state inside the cells. HCV has evolved mechanisms to evade innate immunity, resulting in chronic infections. It has been reported that HCV Core protein can interfere with the IFN signaling pathway, but these studies did not rule out the contributions of F protein, which is a frameshift product of Core coding sequence. The F protein is produced during natural HCV infection, and is found most commonly in genotype 1 HCV, it had also been reported that the titers of anti-F antibody were significantly higher in the group of chronically infected patients. Our research is to investigate whether the F protein plays a role in interferon induction and signaling pathway.
We engineered F expression constructs from Core coding sequences of 4 genotypes (1a, 2a, 3a and 4a) of HCV as well as the sequences which would only be able to produce Core protein. The amino acid sequences of Core and F proteins were aligned and compared. The peptide lengths and amino acid sequences of Core are conserved, but those of F are highly variable. Therefore, we hypothesized that the F protein from different genotypes might control the type I IFN production and response differently to evade innate immunity and IFN-based therapy.
To investigate the function of F protein in type I IFN induction and response pathway, we performed luciferase reporter assay of IFNβ and found that the IFNβ promoter activity is higher in genotype 1a F protein expressing cells. Conversely, the IFNβ promoter activity is minor in genotype 2a F protein expressing cells. And the same phenotype on IRF3 promoter activity. There is no difference among different genotypes of F protein on NFκB promoter activity. Genotype 2a also reduce the IRF7 promoter activity. And then we used MAVS k/d cells to perform luciferase reporter assay of IFNβ, and observed the same phenotype. The regulation of F protein might happened between MAVS and IRF3. Treating the cells with TBK1 inhibitor did not change the phenotype, therefore we predicted that the F protein might affect directly on IRF3. We also looked at the ISRE promoter activity, the promoter activity is enhanced in genotype 1a F protein expression cells and reduced in genotype 2a F protein expressing cells. Different ISRE mRNA expressing levels were confirmed by real-time PCR. We used PH5CH8 cells to make sure the influence is not Huh7-specific.
Our results showed that the F protein is involved in both type I IFN induction and response signaling pathways. Genotype 1a F protein enhances the signaling, genotype 2a F protein inhibits the signaling. In the type I IFN induction pathway, F protein might affect directly on IRF3, and this effect may be dependent on amino acid 40-57.

並列關鍵字

Hepatitis C virus ； F protein ； Type I IFN signaling pathway

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