人類B型肝炎病毒之殼體蛋白及顆粒來回穿梭於細胞核質之間的探討

Abstract

是什麼因素決定B型肝炎病毒（HBV）之核心蛋白（HBc）和顆粒在肝細胞內的位置，至今仍然不清楚。為了探索此一基礎科學問題，本論文用免疫螢光染色以共軛聚焦顯微鏡觀察和細胞核與細胞質分離法結合西方墨點分析，確定了在HBc上富含精氨酸的蛋白質結構模組（ARD）中有四個不同的HBc位置決定訊號。ARD主要是由四個緊密群集的富含精氨酸的次功能區域所組成。第一個和第三個ARD次功能區域是兩個互相依賴的細胞核輸入訊號（NLS），而第二個和第四個ARD次功能區域，功能上是兩個獨立的細胞核運出信號（NES）。此結論是由五個獨立的實驗證據所支持： 一）在肝癌細胞中，使用B型肝炎病毒複製系統並且以雙盲的方式分析，結果在15種ARD次功能區域之HBc突變株中，很有趣地，只有突變第二個和第四個ARD次功能區域的HBc可以很明顯地位於細胞核中。 二）上述這些實驗結果也能夠在猿病毒腫瘤抗原（SV40 large T antigen）結合突變或正常HBc穿梭訊號的異源性嵌合蛋白質報告系統中研究得到證實。 三）由異核或同核融合分析方法， SV40 large T antigen結合HBc穿梭信號的嵌合蛋白質的確能從轉染細胞的細胞核穿梭到另一個接受體細胞的細胞核中，這也表示HBc的ARD存在有新穎的NES，而此NES是對於leptomycin B具有抵抗性。 四）由免疫共沉澱的方法，很驚訝地發現HBc的ARD可以和TAP/NXF1細胞因子（Tip結合蛋白/細胞核運出因子-1）在物理上有交互結合作用；TAP已知對於mRNA和蛋白質的細胞核運出非常重要。在肝癌細胞中，使用B型肝炎病毒複製系統並且轉染專一性的TAP小干擾RNA (siRNA）可將HBc顯著地從細胞質轉移到細胞核，並且導致降低病毒的複製近7倍，以及B型肝炎病毒表面抗原的分泌減少近10倍。 五）在老鼠實驗模式中，以尾端靜脈流體動力注射法，同樣地也支持突變第二個和第四個ARD次功能區域的HBc能夠明顯累積在細胞核中。 在本篇論文中，除了重新修訂HBc的NLS位置，更重要的是我們發現HBc的NES能夠透過新穎的TAP細胞因子，可以快速來回穿梭於細胞核和細胞質之間。

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signal (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.