探討核酸內切酶 FEN1 之抑制劑 PTPD 於抑制肝病毒複製之機轉

Abstract

B 型肝炎病毒隸屬於肝病毒科（Hepadnaviridae），目前全球仍有超過2.5億人口慢性感染此疾病，而最終可能導致肝硬化（liver cirrhosis）或是肝癌（hepatocellular carcinoma）的產生。目前臨床上用於治療 B 型肝炎之抗病毒藥物為核苷酸類似物以及干擾素α（interferon-α），它們雖然能抑制病毒複製，卻無法清除病毒，導因於病毒感染後會在細胞核中形成一種穩定的共價閉合環狀DNA (covalently closed circular DNA, cccDNA)，降解的速率相當緩慢，此外，其可作為病毒複製模版轉錄出四種主要的 mRNA，大小分別為3.5, 2.4, 2.1及0.6 kb，除了可轉譯出各種病毒蛋白外，其中3.5 kb之 mRNA更可透過自身所轉譯出的反轉錄酶來合成新的病毒 DNA，總括來說，欲解決B型肝炎的慢性感染問題勢必需找出清除cccDNA之方法，否則殘存在肝細胞當中之 cccDNA，可能會使停藥的病人症狀復發。由於cccDNA是 B 肝病患能否治癒的關鍵角色，又目前對於 B 型肝炎的 cccDNA 形成的機轉，以及有何宿主因子參與其中都並不明朗，所以我們對此十分的感興趣。B型肝炎病毒經由反轉錄酶合成之rcDNA (relaxed circular DNA)為一部分雙股的DNA，其負股之5'端連接著反轉錄酶，正股則有一段缺口，在B型肝炎病毒感染人類肝細胞後 rcDNA 會進入到細胞核內形成cccDNA。因為從rcDNA形成cccDNA的過程與DNA 修補的機轉類似，所以我們想找出是否有DNA修補相關的宿主因子參與在其中，在比較後我們發現在BER (base excision repair)這種DNA修補機轉中的蛋白質FEN1 (flap endonuclease 1)，由於其可切除5'端的flap結構以及RNA的primer這都是在形成cccDNA時必須經過的步驟，因此我們推測其可能參與在cccDNA形成之過程當中，而在前人的研究中指出在加入FEN1之抑制劑 PTPD (3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidin-e-2,4(1H,3H)-dione)後rcDNA跟cccDNA之合成會受到抑制。因此我接著利用新挑選出來，可調控鴨子Ｂ型肝炎病毒（DHBV）表達的細胞株，在背景比較乾淨的狀況下觀察PTPD抑制DHBV DNA形成的現象，並研究其是否是透過抑制FEN1來影響DHBV DNA的合成，因此我們也使用了另一種 FEN1的抑制劑 aurintricarboxylic acid (ATA) 來看是否同樣會抑制 rcDNA 以及 cccDNA 的形成，但效果並不明顯。此外，在2013年的 B 型肝炎國際會議中，參與在修復雙股斷裂 DNA 的蛋白 Mre11被報導可能也會參與在 cccDNA 的形成當中，因此我們也利用了我們所建立的細胞株 iDBHG-a 去看其抑制劑 Mirin 是否能抑制 cccDNA之形成，初步看來確實能影響cccDNA的產生，但進一步的機制仍須釐清。

Hepatitis B virus belongs to the Hepadnaviridae family. There are over 250 million people being chronically infected by HBV, leading to liver cirrhosis and hepatocellular carcinoma. When HBV infects host hepatocytes, it will form a covalently closed circular DNA (cccDNA) with a long half-life. Because currently approved antiviral drugs cannot directly target cccDNA, relapse often occurs when the treatment stops. However, little is known about the mechanisms regulating cccDNA formation, so we decided to investigate the host factors involved in this process. The relaxed circular DNA (rcDNA) is a partially double-stranded DNA which is the precursor of cccDNA. Upon entry, the HBV genome is translocated into the nucleus and forms cccDNA. The processes from rcDNA to cccDNA are likely to utilize host DNA repair mechanisms. We hypothesized that flap endonuclease 1 (FEN1), a critical nuclease in the BER (base excision repair) pathway, might involve HBV cccDNA formation, because it functions in removal of the 5' flap structure and the RNA primer that are analogous to the rcDNA structure. In the previous study we found that the FEN1 inhibitor PTPD (3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidin -e-2,4(1H,3H)-dione) could suppress both rcDNA and cccDNA formation in 293T cells. To confirm this observation, I generated a new HepG2-derived duck hepatitis B virus (DHBV)-inducible cell line named iDBHG-a and utilized it to test the effect of PTPD. The cell line expresses the DHBV pregenomic RNA (pgRNA) by induction with doxycycline. Compared to the previous transfection system with the DHBV-expression vector, it has a cleaner background and is easier to manipulate. To characterize this cell line, I examined the DHBV RNA and replicative DNA intermediates expression driven by the tet-on system and confirmed the identity of the replicative DNA intermediates by restriction enzyme digestion. By using this inducible cell line, I examined the effect of PTPD, ATA (another FEN1 inhititor), and mirin (Mre11 inhibitor) treatment on cccDNA formation. We found that only the mirin could suppress the cccDNA formation in a dose-dependent manner, but the detailed mechanisms need to further investigating. To summarize, we established a new DHBV inducible system in HepG2, and demonstrated its utility in studying the mechanisms and novel inhibitors for the hepadnavirus cccDNA formation.