人類核仁磷酸化蛋白質140及小型D型肝炎抗原共同調控核醣體RNA合成及病毒複製

Abstract

D型肝炎病毒(hepatitis delta virus, HDV)為一具有外套膜之球狀病毒顆粒。其病毒顆粒內部是一負向、單股、環狀，全長約為1.7 kb之RNA基因體所組成。D型肝炎病毒是一種缺陷型的病毒(defective virus)，必須獲得B型肝炎病毒的外套膜才具有感染力，因此被歸類為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，分別是小型delta抗原(small delta antigen, HDAg-S；195個胺基酸，約24 kDa)及大型delta抗原(large delta antigen, HDAg-L；214個胺基酸，約27 kDa)。小型delta抗原參與病毒基因體之複製，而大型delta抗原則與病毒顆粒之組裝有關。 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抗原分佈於核仁及D型肝炎病毒複製是重要的，並且證實小型delta抗原會與RNA polymerase I之最大次單元RPA194有交互作用。再綜合其他學者的研究，許多的證據皆指向RNA polymerase I可能參與D型肝炎病毒antigenomic RNA的合成，或小型delta抗原可能透過與RNA polymerase I之結合而調控rRNA之合成。 先前的研究發現，人類核仁磷酸化蛋白質140 (human nucleolar phosphoprotein 140, hNopp140；699 個胺基酸，約140 kDa)與RPA194相結合，此hNopp140最小結合區域已被鑑定在其胺基酸204至382之序列中。hNopp140被認為是組成核仁結構之最基礎單元，且與協助rRNA 合成有關。由序列比對發現小型delta抗原與hNopp140的第204至382個胺基酸片段有一定程度的相似性。 本研究利用GST pull-down assay證明了RPA194之region a分別會和小型delta抗原及hNopp140結合。且初步的研究結果顯示，小型delta抗原的第88至164個胺基酸與RPA194之region a有交互作用。此外我們利用蔗糖濃度梯度離心實驗觀察到，RPA194分別與hNopp140及小型delta抗原皆有共沈降之現象，而RPA194與小型delta抗原的分佈具有高度重疊性。因此我們認為小型delta抗原之存在可能會影響hNopp140與RNA polymerase I 之交互作用，進而抑制宿主細胞中rRNA的合成。此外利用間接免疫螢光染色發現，利用低劑量的actinomycin D選擇性的抑制RNA polymerase I或利用過度表現hNopp140的方式使得rRNA的合成無法進行時，此時觀察到小型delta抗原會離開核仁而轉移至核質，因此小型delta抗原在核仁中的分佈，可能與active rDNA transcription相關。更進一步利用RT real-time PCR分析發現，當小型delta抗原離開核仁時，D型肝炎病毒之複製會增加；另一方面利用干擾性核醣核酸(RNA interference; RNAi)降低RNA polymerase I 之RPA194的表現量，意外地發現D型肝炎病毒antigenomic RNA的合成有增加之現象。因此，RNA polymerase I對於D型肝炎病毒antigenomic RNA之複製是否扮演重要的角色，需要更進一步的釐清。

Hepatitis delta virus (HDV) is a spherical virus particle with envelope. It contains a 1.7 kb single-stranded circular RNA genome of negative polarity. HDV is considered to be a satellite virus of hepatitis B virus (HBV). It requires HBV surface envelope protein to form infectious virus particles. HDV encodes two forms of delta antigen (HDAg). The small form of HDAg (HDAg-S, 195 amino acids, 24 kDa) is required for the HDV RNA replication, while the large form of delta antigen (HDAg-L, 214 amino acids, 27 kDa) is required for the viral assembly. Earlier studies from our laboratory have identified an interaction between HDAg-S and nucleolin that is critical for the nucleolar targeting of HDAg-S and HDV replication. In addition, HDAg-S interacts with the largest subunit of RNA polymerase I, RPA194. Furthermore, HDAg-S inhibits the de novo synthesis of rRNA. These results indicate that HDAg-S may regulate the synthesis of HDV antigenomic RNA and rRNA through interacting with RNA polymerase I. Previous studies have demonstrated an interaction of RPA194 with the middle domain of human nucleolar phosphoprotein 140 (hNopp140) from amino acid residues 204 to 382 of hNopp140. hNopp140 is thought to be the basic unit of nucleolus structure and involves in the synthesis of ribosomal RNA. Interestingly, HDAg-S shares conserved sequences with the middle domain of hNopp140. In this study, an association among the conserved region a of RPA194, HDAg-S, and hNopp140 was demonstrated by GST pull-down assay. HADg-S interacts with RPA194 through its amino acid residues 88 to 164. In addition, RPA194 co-sedimented with both HDAg-S and hNopp140 in sucrose gradient centrifugation. The presence of HDAg-S may therefore influence the interaction between hNopp140 and RNA polymerase I, resulting in an inhibition of the de novo synthesis of rRNA. At low dose actinomycin D which selectively inhibits RNA polymerase I activity, HDAg-S translocated from the nucleolus to the nucleoplasm of transfected cells. The phenomenon was also observed when hNopp140 known to inhibit synthesis of rRNA was overexpressed. Unexpectedly, HDV total RNA as well as antigenomic RNA increased in RPA194 knockdown cells. In summary, hNopp140 and HDAg-S coordinately regulate the synthesis of rRNA and HDV RNA.