摘要:C 型肝炎病毒感染全球約 1 億 7 千萬人口,在台灣約有 42 萬名帶原者。C 型肝炎的慢性帶原者通常沒有症狀,但長期感染下經常造成肝硬化與肝癌等疾病。雖然市面上已經有許多有效的C 肝病毒藥物,但一個療程需花費約250 萬台幣,故大多數病人仍依賴傳統療效較差、副作用多的α 干擾素與Ribavirin 治療。再加上目前尚未有C 型肝炎病毒疫苗,因此開發具有競爭性的替代藥物仍是學界與業界積極發展的目標。藉由蛋白質質譜儀、分子病毒學及免疫學等方法,本實驗室成功證明C 肝病毒非結構性蛋白質 NS5A 中絲氨酸 235 (S235)的磷酸化在病毒複製過程中扮演舉足輕重的角色。希望透過抑制S235 的磷酸化,達到阻止C 肝病毒的複製,與避免C 肝病毒引起的癌化狀況。然而,對於S235 磷酸化的機制與功能我們尚未有太多的了解,另外,S235 磷酸化是否參與肝癌的進程也是我們想探討的問題。本計畫擬一、探討NS5A S235 的磷酸化是否藉促進NS5A 形成dimer、增加其RNA 結合的能力、刺激NS5B RNA 聚合酶的活性或促進複製體的形成來增加C 肝病毒的複製能力。本計畫擬二、運用點突變的方法系統性地探討NS5A 上六個磷酸化位點(S222、S225、S229、S232、S235、S238)在C 肝病毒生活史中的功能,並運用我們現有且專一性極高的抗體釐清各個磷酸化位點間是否擁有關聯性。我們也將鑑定出NS5A 的磷酸化激酶並探討NS3 絲氨酸蛋白質水解酶參與NS5A 磷酸化的機轉。本計畫擬三、探討NS5A S235 的磷酸化是否藉改變細胞「微環境」或「表觀遺傳」的變異導致或惡化肝癌的機轉。自1994 年發現NS5A磷酸化以來,我們團隊首次能針對NS5A 個別的磷酸化位點做系統性的探討,其中,NS5A S235的磷酸化無疑地是C 肝病毒的活性指標,可以成為開發C 肝病毒的新標靶與C 肝病毒治療的指標。另外,如果我們能夠了解NS5A S235 的磷酸化參與肝癌的機轉,我們可以從抑制C 肝病毒的複製與抑制肝癌的進程,雙管齊下,造福近一億七千萬的C肝病毒帶原者。
Abstract: HCV infects approximately 170 million people worldwide including 420,000 people in Taiwan. Chronic HCV carriers are asymptomatic until development of severe liver diseases including fibrosis, cirrhosis and cancer, leaving HCV infection the major cause of liver transplant. Already, there are many approved highly efficient HCV antivirals; however, their costs as high as NT$2,500,000 per treatment course greatly prohibit their accessibility. Most patients still rely on less effective drugs interferon α and ribavirin that have serious side effects. Research and development for effective alternatives are desirable. Using tools of protein mass spectrometry, molecular virology and immunology, my lab has demonstrated that phosphorylation of serine 235 (S235) of the HCV non-structural protein NS5A plays a critical role in HCV viral replication. Thus, NS5A S235 phosphorylation is a superb marker for active HCV infection and a target for treatment; however, the molecular mechanisms by which NS5A S235 phosphorylation participates in viral replication remain largely unknown. Whether NS5A S235 phosphorylation participates in liver cancer is also unknown. Here, I proposed the following aims to investigate three questions. (1) How does NS5A S235 participate in viral replication, viz. whether S235 promotes NS5A dimerization, RNA binding ability, NS5B RNA polymerase activity or replication complex formation to enhance viral replication? (2) How is NS5A phosphorylated? NS5A has 6 functional serine phosphorylation sites (S222, S225, S229, S232, S235 and S238). Each is phosphorylated with different levels and functions differently. We will systematically investigate functions of each site and study whether they are interdependent using phosphorylation-specific antibodies that we have already generated. Moreover, we will identify the kinases responsible for their phosphorylation and investigate how the NS3 protease activity may be involved in NS5A phosphorylation. (3) Whether and how does NS5A S235 phosphorylation participate in liver cancer, viz. whether NS5A S235 phosphorylation alters the microenvironment or epigenetic modifications of the liver cells to enhance cancer cell growth, migration or invasion? Since the discovery of NS5A phosphorylation in 1994, my lab now has the best opportunity to systematically study functions of different NS5A phosphorylation sites using phosphorylation-specific antibodies. In particular, understanding the functions and regulations of NS5A S235 phosphorylation will help to eliminate HCV infection. In addition, understanding how NS5A S235 phosphorylation may cause liver cancer will benefit a large number of chronic HCV carriers. This revised grant now fits in the new NHRI research aims in the category of “Cancer development, progression and metastasis”. It investigates tumorigenic potentials of NS5A via S235 phosphorylation-mediated alterations in the cancer cell microenvironment and epigenetics. Successful implementation of this grant will lead to development of active HCV diagnosis markers and novel therapeutics.