調節rRNA合成及D型肝炎病毒複製的重要小型D型抗原保守胺基酸序列

Abstract

D型肝炎病毒(hepatitis delta virus, HDV)為一具有外套膜之負向單股環狀RNA病毒顆粒，其基因體全長約為1.7 kb。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，小型D型抗原(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D型抗原(large delta antigen, HDAg-L；214個胺基酸，約27 kDa)則與病毒顆粒之組裝有關。許多研究指出D型肝炎病毒的genomic及antigenomic RNA複製可能是透過不同的宿主聚合酶來達成。目前已知genomic RNA複製是利用宿主第二型RNA聚合酶(RNA polymerase II)，小型D型抗原的羧基端(C-terminus)會與第二型RNA聚合酶的clamp domain有交互作用，並且可能參與調控genomic RNA複製的精確性。然而，antigenomic RNA的合成需要何種聚合酶則尚未定論。研究發現，antigenomic RNA的複製不受高濃度α-amanitin影響。先前本實驗室發現小型D型抗原與核仁素(nucleolin)的結合對於小型D型抗原分佈於核仁及D型肝炎病毒複製是重要的。小型D型抗原的中間片段(胺基酸序列87-165)會與第一型RNA聚合酶之最大次單元RPA194有交互作用，而且RPA194與D型肝炎病毒之genomic RNA可以被共同沈澱下來。再綜合其他學者的研究，許多的證據皆指向第一型RNA聚合酶可能參與D型肝炎病毒antigenomic RNA的合成。除此之外，大量表達小型D型肝炎會抑制宿主rRNA新生成。其他研究發現，人類核仁磷酸化蛋白140 (human nucleolar phosphoprotein 140; hNopp140)的中間片段(胺基酸序列204-382)會與RPA194結合，hNopp140為組成核仁的最小單元並且與rRNA合成相關。根據序列比對結果發現，小型D型抗原及hNopp140的RPA194結合區域，其胺基酸序列具有相當程度的相似性，推測可能是小型D型抗原會利用相似序列競爭hNopp140與RPA194或rDNA promoter之交互作用。 本研究首先利用同源性模擬法(homology modeling)預測RPA194 clamp domain可能包含胺基酸序列1-409 (pol IA)。利用pull-down assay證明此片段會和小型D型抗原結合。進一步，分析小型D型抗原的保守胺基酸序列並交叉比對hNopp140的演化保守胺基酸序列，將小型D型抗原保守性胺基酸Lys106及Ala107分別突變成Ala及Asp (HDAg-S-106AD)，125EEE127則皆突變成Ala (HDAg-S-125AAA)。由pull-down assay的結果發現HDAg-S-106AD與pol IA的結合能力較野生型小型D型抗原弱，而HDAg-S-125AAA與pol IA的結合卻會增強。RT real-time PCR的結果發現HDAg-S-125AAA幾乎不會抑制45S rRNA的新生成，而HDAg-S-106AD抑制45S rRNA新生成的情況則與野生型小型D型抗原差異不大。此外，RT real-time PCR結果也顯示此兩種突變型小型D型抗原皆不支持D型肝炎病毒total RNA及antigenomic RNA之複製。以上結果顯示保守性胺基酸Lys106、Ala107及Glu125-127對於病毒RNA的複製及抑制宿主rRNA合成扮演不同的重要角色，然而pol I是否參與病毒RNA複製及小型D型抗原抑制rRNA新生成的詳細機制，則需要更進一步的釐清。

Hepatitis delta virus (HDV) is a spherical enveloped virus which contains a single-stranded, negative sense circular RNA genome of 1.7 kb in length. Specifically, HDV was considered as a satellite virus with the requirement of its helper virus, hepatitis B virus (HBV), to provide the envelope proteins in forming infectious virus particles. HDV encodes two forms of delta antigen (HDAg). Firstly, the small delta antigen (HDAg-S, 195 amino acids, 24 kDa) is needed for HDV RNA replication. Secondly, the large delta antigen (HDAg-L, 214 amino acids, 27 kDa) is required for viral assembly. According to accumulated evidences, two independent host transcriptional machineries may be necessary for the replication of the viral genomic and antigenomic RNAs. RNA polymerase II is essential for HDV genomic RNA synthesis. The C-terminus of HDAg-S can interact with pol II clamp domain and may affect transcriptional fidelity. However, the synthesis of antigenomic RNA was resistant to α-amanitin. Our laboratory members have previously identified an interaction between HDAg-S and nucleolin, as well as demonstrated that the interaction is critical for the nucleolar targeting of HDAg-S and HDV replication. In addition, the central domain (a.a. 87-165) of HDAg-S interacted with the largest subunit of RNA polymerase I, RPA194, and the RPA194 could be co-precipitated with HDV genomic DNA. Thus, the results indicated that RNA polymerase I may participate in the synthesis of HDV antigenomic RNA. Furthermore, overexpression of HDAg-S inhibited the de novo synthesis of rRNA. Other studies also demonstrated an interaction of RPA194 with the middle domain of human nucleolar phosphoprotein 140 (hNopp140) from amino acid residues 204 to 382. Besides, hNopp140 was regarded as the basic unit of nucleolar structure and was demonstrated to be involved in the synthesis of rRNA. Interestingly, the RPA194-interacting domains of HDAg-S and hNopp140 share certain similarities in sequences, suggesting that HDAg-S may compete with the association among hNopp140, RPA194 and rDNA promoter. In this study, the interaction between HDAg-S and RPA194 clamp domain (a.a. 1-409, pol IA, predicted by homology modeling) was first demonstrated by pull-down assay. Evolutionarily conserved residues of the similar sequences between HDAg-S and hNopp140 were analyzed. The conserved residues Lys106 and Ala107 of HDAg-S were mutated to Ala and Asp respectively (HDAg-S-106AD), and 125EEE127 were all mutated to Ala (HDAg-S-125AAA). Results from pull-down assay showed that the interaction between HDAg-S-106AD and pol IA was reduced, while HDAg-S-125AAA had a stronger interaction with pol IA. In addition, HDAg-S-106AD had an inhibitory effect on the de novo synthesis of rRNA similar to that of the wild type HDAg-S. Unexpectedly, HDAg-S-125AAA no longer inhibited the de novo synthesis of rRNA. Results from RT real-time PCR indicated that both HDAg-S-106AD and HDAg-S-125AAA did not surpport the viral and antigenomic RNA synthesis. In summary, the conserved residues Lys106, Ala107 and Glu125-127 play differential roles in HDV RNA replication and the inhibitory effect on host rRNA synthesis. However, molecular mechanisms of pol I involved in HDV antigenomic RNA synthesis and the inhibition of rRNA synthesis need to be further studied.