小型delta抗原與宿主第一型RNA聚合酶共同結合至 D型肝炎病毒基因體特定末端環狀區段之研究

Abstract

D型肝炎病毒(hepatitis delta virus, HDV)為目前已知最小的動物病毒，含一單股負向環狀RNA，其基因體全長約1.7 kb，並且被由B型肝炎表面抗原所形成的外套膜所包覆。由於需要倚賴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)主要參與病毒的複製，而大型delta抗原(large delta antigen, HDAg-L; 214個胺基酸，約27 kDa)則參與病毒顆粒的包裹與組裝。D型肝炎病毒並不具有自己的聚合酶，因此其RNA的複製必須仰賴宿主的聚合酶及相關宿主因子。儘管D型肝炎病毒的複製機制目前仍有許多尚未清楚之處，最近的研究指出，D型肝炎病毒基因股及反基因股的合成，可能是由不同形式的宿主聚合酶及細胞因子所負責。有文獻指出，宿主的第二型RNA聚合酶參與病毒基因股的合成，而α-amanitin resistant 的RNA聚合酶I可能是負責反基因股的合成。然而宿主的RNA聚合酶為DNA-dependent RNA polymerase，皆是以DNA作為模板之RNA聚合酶，而為何在病毒的複製中獲得以RNA作為模板的能力則是值得進一步探討的。本實驗室先前的研究發現，小型delta抗原的中間第89至163個胺基酸片段與人類核仁磷酸化蛋白質140 (human nucleolar phophoprotein 140; hNopp140)之RNA聚合酶I結合區域部分呈現保守性，並且同樣能與RNA聚合酶I最大次單元RPA194之間有交互作用。而此中間區段也足以支持病毒反基因股的複製。這些證據也加強了第一型RNA聚合酶參與病毒反基因股複製的可能性。本研究希望進一步探討第一型RNA聚合酶參與病毒反基因股合成的機制。首先利用能表現帶有TAT-domain的小型delta抗原中間區段之質體，轉入C43大腸桿菌中表現融合蛋白質，再以親和性管柱層析的方式純化出此融合蛋白質，TAT-HDAg-S-m與其突變型TAT-HDAg-S-m106AD，將此融合蛋白質在加入Huh7細胞培養液後30分鐘，可以共軛焦顯微鏡觀察到能轉導進入細胞內並且進入細胞核。而real-time PCR的實驗結果可以看到，相較於突變型的對照組，小型delta抗原確實能夠增加D型肝炎病毒反基因股之複製。而為了進一步瞭解是否有未知的宿主因子參與D型肝炎病毒與小型delta抗原所形成的複製複合體，本實驗利用可還原之cross-linker在in vivo的狀況下將細胞內複合體固定並以親和性管柱層析將此複合體分離出來，再以LC-MS/MS分析方法進行蛋白質鑑定。而鑑定的結果找到符合先前研究報導過之D型肝炎病毒複製相關蛋白質及一些未知角色之蛋白質，未來需做進一步的探討其在病毒複製所扮演之角色。另外，為了探討小型delta抗原及第一型RNA聚合酶與病毒基因股RNA是否有交互作用，本研究合成病毒基因股及反基因股末端區段，並與純化之小型delta抗原及第一型RNA聚合酶進行免疫沈澱實驗。在in vitro條件下的實驗結果發現，小型delta抗原與RNA聚合酶I專一性協同結合至D型肝炎病毒基因股末端的環狀區段，而反基因股則否。綜合以上結果推論，D型肝炎病毒在宿主細胞中的複製，很可能還有本研究所鑑定之宿主因子參與其中，而D型肝炎病毒基因體RNA末端桿狀結構之一環狀區段可能存在著病毒反基因體合成之啟動區域及起始位置。

Hepatitis delta virus (HDV) is the smallest known animal virus, with a single-stranded, negative-polarity circular RNA of 1.7 kb, enveloped by hepatitis B surface antigen (HBsAg). HDV is thus considered as a satellite virus of hepatitis B virus (HBV). HDV contains only one known gene which encodes two forms of hepatitis delta antigen (HDAg). The small delta antigen (HDAg-S, 195 amino acid, 24 kDa) is involved in HDV RNA replication, and the large delta antigen (HDAg-L, 214 amino acids, 27 kDa) mainly participates in the viral assembly. HDV doesn’t have its own RNA polymerase. Relevant evidences suggested that α-aminatin resistant RNA polymerase I is necessary for the synthesis of HDV antigenomic RNA, while RNA polymerase II mediates the synthesis of HDV genomic RNA. However, detailed mechanisms of HDV replication are still unknown. Our laboratory has previously demonstrated that the middle domain of HDAg-S, HDAg-S-m (a.a. 89-163), shares partial sequence identity with the RNA polymerase I binding region of human nucleolar phosphoprotein 140 (hNopp140). HDAg-S-m interacted with the largest subunit of RNA polymerase I, RPA194, and was able to support the synthesis of HDV antigenomic RNA. In this study, whether the DNA-dependent RNA polymerase I is directly involved in the HDV antigenomic RNA synthesis was further investigated. TAT-HDAg-S-m representing TAT fusion protein of HDAg-S-m was purified by affinity chromatography. Following incubating with Huh7 cells for 30 mins, the partially purified TAT-HDAg-S-m was detected by confocal microscopy in the cell nucleus. TAT-HDAg-S-m increased the level of HDV antigenomic RNA. In addition, host factors associated with HDV RNA-HDAg-S-m replication complex were cross-linked, pulled down, and further identified by LC-MS/MS analysis. Functional analysis of the host factors will be further elucidated to understand the detailed mechanisms involved in the antigenomic RNA synthesis of HDV. On the other hand, to determine RNA regions bound by HDAg-S-m and Pol I, subdomains of the HDV genomic RNA were synthesized by in vitro transcription and subjected to RNA co-immunoprecipitation assay. The results indicated that HDAg-S-m and Pol I cooperatively interacted with a specific terminal region of HDV genomic RNA. In conclusion, this study identified several host factors that may involve in the replication of HDV antigenomic RNA and determined that the specific terminal stem-loop region of HDV genomic RNA may contain the promotor region and replication start site for the antigenomic RNA synthesis.