D型肝炎抗原調控轉錄因子TAp63之基因表現

Abstract

D 型肝炎病毒（hepatitis delta virus, HDV）是一種小型球狀RNA 病毒，內部由單股RNA 基因體以及兩種delta 抗原所構成，外套膜則由B 型肝炎表面抗原（hepatitis B virus surface antigen, HBsAg）及脂雙層（lipid bilayer）形成。雖然已知HDV 在HBsAg 存在下具有感染力，但是目前對於HDV 造成肝炎發生的機制目前仍未完全瞭解。 HDV 內部的兩種delta 抗原是其基因體轉譯產生的唯一蛋白質，分別為對於病毒顆粒包裹所必須的大型delta（HDAg-L）以及複製所必須的小型delta（HDAg-S）抗原。先前的研究利用報導基因表現分析（reporter gene assay）發現大型delta 抗原可以活化血清反應因子（serum response factor, SRF）調控的基因轉錄以及轉形生長因子β（transforming growth factor beta, TGF-β）訊息傳遞路徑。為了進一步瞭解delta 抗原在基因表現調控所扮演的角色，我們首先利用染色質免疫沉澱技術（chromatin immunoprecipitation, ChIP）證實大型及小型delta 抗原皆會坐落在宿主細胞血漿纖溶酶原活化劑抑制物-1（plasminogen activator inhibitor 1, PAI-1）基因啟動子上。接著以免疫沉澱（immunoprecipitation, IP）及質譜分析，進一步探討delta 抗原與宿主細胞中參與轉錄因子之結合發現，可能與delta 抗原有結合的蛋白質中有5 個與轉錄調控有關。以免疫共沉澱證實，與細胞生長分化相關的TAp63γ確實和delta 抗原有專一性結合。另外，我們同時也發現在穩定表現大型delta 抗原的HepG2 細胞中，TAp63γ 的mRNA 及蛋白質表現量皆有增加的情形。到底delta抗原與p63 的結合和轉錄調控對宿主細胞的影響亦進行討論。

Hepatitis delta virus (HDV) is a spherical enveloped virus. The viral genome consists of a single-stranded, negative sense, circular RNA of 1.7 kb that encodes the only known HDV protein, hepatitis delta antigen (HDAg). There are two forms of delta antigen, a small delta antigen (HDAg-S, 195 amino acids, 24 kDa) and a large delta antigen (HDAg-L, 214 amino acids, 27 kDa). HDAg-S is essential for the viral genome replication, whereas HDAg-L is required for the viral assembly. HDV is a satellite virus that requires hepatitis B virus as its helper virus to provide surface antigen (HBsAg) for natural transmission. Although it is known that HDV infection increases liver disease severity, the underlying mechanisms are poorly understood. Previous studies have demonstrated that HDAg-L could activate serum response factor-associated transcription and TGF-β signaling pathway. To further understand the role of HDAg in the expression of host genes, chromatin immunoprecipitation assay was conducted in this study. The results emonstrated that both HDAg-L and HDAg-S bound to the promoter region of plasminogen activator inhibitor (PAI-1) gene. In addition, immunoprecipitation followed by LC-MS/MS analysis were performed to identify other cellular factors that interact with HDAg. Results showed that p63, a member of p53 family, known to be involved in the transcription regulation of cell growth and differentiation was identified to be associated with HDAg. Further studies demonstrated that TAp63γ, an isoform of p63, interacted with both HDAg-L and HDAg-S. Moreover, the expression levels of TAp63γ mRNA and protein were remarkedly increased in cells stably expressing HDAg-L. How HDAg regulates expression of host genes through p63 remains to be elucidated.