小型Delta抗原參與D型肝炎病毒反基因股RNA合成之分子機制

Molecular Mechanisms of the Small Delta Antigen Involved in the Antigenomic RNA Synthesis of Hepatitis Delta Virus

王品堯(Pin-Yao Wang)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03400

摘要

D型肝炎病毒(hepatitis delta virus, HDV)為一種經由B型肝炎表面抗原包裹形成的病毒顆粒，其基因體為一全長約1.7 kb的單股負向環狀RNA。D型肝炎病毒需要獲得B型肝炎病毒的外套膜才具有感染力，因此也被歸類為B型肝炎病毒的衛星病毒。D型肝炎病毒可轉譯出兩種抗原：小型delta抗原(small delta antigen, HDAg-S；195個胺基酸，約24 kDa)參與病毒基因體之複製，而大型delta抗原(large delta antigen, HDAg-L；214個胺基酸，約27 kDa)則與病毒顆粒之組裝有關。由於D型肝炎病毒沒有自己的RNA聚合酶，因此需要利用宿主的RNA聚合酶來進行基因體的複製。許多研究指出，D型肝炎病毒基因股RNA之生成是利用RNA聚合酶 II來執行，而反基因股RNA之生成則需要RNA聚合酶 I或具有α-amanitin resistant的聚合酶來執行。先前本實驗室發現小型delta抗原與核仁素(nucleolin)的結合對於小型delta型抗原分佈於核仁及D型肝炎病毒之複製是重要的，並且小型delta抗原中間區域第89-163個胺基酸的片段與RNA聚合酶 I最大次單元RPA194之間有交互作用，而此中間區域與人類核仁磷酸化蛋白質140 (human nucleolar phosphoprotein 140; hNopp140)的RNA聚合酶 I之結合區域部分呈現保守性，經過序列交叉比對後建構了KA106AD以及EEE125AAA兩種突變型小型delta抗原的表現質體。在D型肝炎病毒RNA複製的實驗中，發現這兩種突變型小型delta抗原皆無法支持病毒total RNA之複製以及反基因股RNA之生成。初步實驗結果顯示，這兩種突變皆影響到小型delta抗原與RPA194之交互作用。
為了探究106AD以及125AAA兩種突變型小型delta抗原是透過何種分子機制影響D型肝炎病毒RNA之複製，本研究首先利用免疫螢光染色法觀察小型delta抗原在細胞中的分布。結果顯示野生型及125AAA突變型小型delta抗原和RPA194共同位於核仁，而106AD突變型小型delta抗原則分布在核仁與核質。此外，利用共同免疫沉澱法發現106AD突變型小型delta抗原和RPA194交互作用之能力有減弱的趨勢。進一步於MBP pull-down assay中發現小型delta抗原第89-163個胺基酸的片段能與RPA194 N端409個胺基酸的片段直接交互作用，而106AD突變型小型delta抗原第89-163個胺基酸的片段與RPA194直接交互作用之能力減弱了50 %。另一方面，在RT real-time PCR的分析中發現，106AD以及125AAA兩種突變型小型delta抗原皆不影響以反基因股RNA為模板之D型肝炎病毒total RNA複製以及基因股RNA之生成。透過濾紙結合試驗得知，不管是106AD或125AAA突變型小型delta抗原皆失去了與病毒RNA結合之能力。綜合以上結果推論，K106、A107及E125-127胺基酸對於小型delta抗原與RNA結合之能力是重要的。而K106、A107胺基酸對於小型delta抗原在細胞中的分布以及和RNA聚合酶 I直接交互作用之能力扮演相當重要的角色，並且與反基因股RNA之生成有關。

關鍵字

D型肝炎病毒 ； 小型Delta抗原

並列摘要

Hepatitis delta virus (HDV) is a viral particle enveloped by hepatitis B surface antigens (HBsAg). The RNA genome of HDV is a 1.7 kb, single stranded, negative-polarity circular RNA. Because of the requirement of HBsAg for transmission and propagation, HDV is also considered as a satellite virus of hepatitis B virus. The HDV antigenome encodes two forms of hepatitis delta antigen (HDAg). The small delta antigen (HDAg-S, 195 amino acids, 24 kDa) is essential for HDV RNA replication, while the large delta antigen (HDAg-L, 214 amino acids, 27 kDa) is involved in the viral assembly. HDV doesn’t have its own RNA polymerase, host RNA polymerase is used for the viral RNA replication. Accumulated evidences have indicated that host RNA polymerase II mediates the synthesis of HDV genomic RNA, whereas the RNA polymerase I or an α-amanitin resistant RNA polymerase is necessary for the synthesis of HDV antigenomic RNA. Previous studies in our laboratory have demonstrated that the interaction between HDAg-S and nucleolin is critical for the nucleolus targeting of HDAg-S and HDV replication. In addition, the middle domain (a.a. 89-163) of HDAg-S, which is partially conserved with the RNA polymerase I binding region of human nucleolar phosphoprotein 140 (hNopp140), could interact with the largest subunit of RNA polymerase I, RPA194. According to sequence alignment analysis, expression plasmids encoding HDAg-S mutants, HDAg-S106AD (KA106AD) and HDAg-S125AAA (EEE125AAA), were generated. Neither of the mutants could support HDV RNA replication and antigenomic RNA synthesis. Both mutations affected the interaction of HDAg-S with RPA194 in vitro.
To study the molecular mechanisms that HDAg-S106AD and HDAg-S125AAA affect the HDV RNA replication, immunofluorescence assay was first performed to examine the subcellular localizations of HDAg-S. Results showed that the wild type HDAg-S and HDAg-S125AAA predominantly co-localized with RPA194 in nucleolus, whereas the HDAg-S106AD localized throughout the nucleoplasm and nucleolus. In addition, the interaction of HDAg-S106AD with RPA194 was reduced as compared to the wild type HDAg-S. In MBP pull-down assay, a direct interaction between the middle domain (a.a. 89-163) of HDAg-S and the N terminus (a.a. 1-409) of RPA194 was detected, but the interaction was reduced to 50 % with the HDAg-S106AD mutant. Furthermore, neither 106AD nor 125AAA substitutions affected HDAg-S on the HDV total RNA replication and genomic RNA synthesis. On the other hand, both HDAg-S106AD and HDAg-S125AAA failed to interact with HDV RNA in filter binding assay. These results indicate that the K106、A107 and E125-127 amino acids are critical for the binding between HDAg-S and HDV RNA. Nevertheless, K106 and A107 amino acids play important roles in the subcellular localization of HDAg-S, and in the direct interaction of HDAg-S with RPA194 that is involved in the antigenomic RNA synthesis.

並列關鍵字

Hepatitis Delta Virus ； Small Delta Antigen

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