D 型肝炎病毒抗原的核仁位置訊息區域：可能乙醯化的影響

中文摘要 D 型肝炎病毒的基因體 RNA為單股、環狀，長度約為1700個核苷酸。在其生活史中，HDV可以透過轉譯同一開放閱讀架構而表現兩種 D 型肝炎病毒抗原，分別是 195 個胺基酸的小型delta抗原(small delta antigen，S-HDAg，24kDa)和 214 個胺基酸的大型delta抗原(large delta antigen，L-HDAg，27 kDa)。其中，S-HDAg 是 HDV 複製時所必需的，而 L-HDAg 會與 HBsAg 作用而為形成病毒顆粒所必需。根據之前的文獻報導指出，當只把會表現S-HDAg 的DNA 質體轉染於細胞內時，以免疫螢光染色可以觀察到S-HDAg大部分是位於核仁的位置，但是對於S-HDAg位於核仁和其幫助HDV 複製的能力之間的關聯性目前尚不清楚。我們實驗室在之前的研究發現S-HDAg的賴胺酸 72 ( lysine 72 )可以被乙醯化，當把賴胺酸 72 ( lysine 72 ) 突變成精胺酸 72 ( arginine 72 ) ，S-HDAg 在細胞內的分佈位置從細胞核變成主要位在細胞質內。此外，當有去乙醯化(deacetylation)功能的酵素SIRT大量表現存在時，或是於S-HDAg的N端產生賴胺酸的單一點突變，皆觀察到S-HDAg在細胞內分佈形態的改變。基於以上的發現，我們假設S-HDAg可能藉由乙醯化這種轉譯後修飾作用調控其在細胞內的分佈位置。關於HDAg位於細胞核仁內的位置的研究，有研究報告顯示，HDAg能夠與細胞核蛋白nucleolin結合，而且他們推測HDAg的第51-65的胺基酸殘基可能和HDAg分佈於細胞核仁內有關。在經過S-HDAg的胺基酸序列組成分析後，我們發現在S-HDAg蛋白質 N 端第35到第65胺基酸殘基(amino acid residues)中，有3個 (R/K) (R/K)X(R/K) motif (在被報導具有nucleolar localization sequence的蛋白質中是highly conserved) 的存在及兩個 (K/R)XKK motif (被報導是一個highly conserved且能夠被乙醯化的motif) 的存在。因此，我們假設S-HDAg N 端第35-50胺基酸殘基(amino acid residues)可能是一段使其位於細胞核仁的nucleolar retention sequence，且可能藉由賴胺酸(lysine)的乙醯化當作retention signal，而第51-65胺基酸殘基則可能是一段使其位於細胞核仁的nucleolar translocation sequence，我們假設這兩段胺基酸殘基必需同時存在，才能使S-HDAg位於細胞核仁。在本論文研究中我們利用免疫螢光染色觀察五個賴胺酸(lysine)皆突變成精胺酸 (arginine)的S-HDAg，也就是在lysine-38,39,40,42,43突變成arginine-38,39,40,42,43的S-HDAg蛋白質(S-HDAg-K38-43R)與在lysine-38,39,40,42,43,72突變成arginine-38,39,40,42,43,72的S-HDAg蛋白質(S-HDAg-K38-43R/K72R) 在細胞內的分佈形態。結果顯示，不論在有HDV 基因體RNA或是反基因體RNA的存在下，突變型S-HDAg-K38-43R主要分佈在細胞核質，而S-HDAg-K38-43R/K72R則主要分佈在細胞質，且突變型S-HDAg-K38-43R與S-HDAg-K38-43R/K72R 皆無法協助HDV RNA 的複製。有趣的是，突變型S-HDAg-K72R在有HDV RNA存在下主要分佈在細胞質，且可以幫助HDV RNA的複製。為了進一步研究HDAg 蛋白N 端第35-65、第35-50及第51-65胺基酸殘基是否對於其細分佈於核仁是必需的，我們將HDAg 蛋白這三段胺基酸殘基融合於DsRed-hnRNPC1蛋白，分別命名為HDAg(35-65)-DsRed-hnRNPC1、HDAg(35-50)-DsRed-hnRNPC1及HDAg(51-65)-DsRed-hnRNPC1。結果發現， HDAg 蛋白N 端第35-65、第35-50或第51-65胺基酸殘基無法使原本位於細胞核質內的hnRNPC1變成位於核仁內。最後，在本研究中我們建立了一套細胞核仁內純化方式，利用分離細胞不同的部份，包括細胞質、細胞核質以及細胞核仁，結合陽離子交換樹酯管柱成功地純化了突變型S-HDAg，期望在未來的研究中能夠藉由質譜儀分析鑑定S-HDAg的乙醯化胺基酸的位置，以了解S-HDAg是否藉由乙醯化這種轉譯後修飾作用調控其在細胞內的分佈位置。

關鍵字

核仁位置訊息區域； D 型肝炎病毒抗原；乙醯化

並列摘要

Abstract Hepatitis delta virus (HDV) is a single-stranded RNA virus that contains a 1.7-kb circular genome. The single open reading frame of HDV encodes two viral nucleocapsid proteins named the 24-kDa small delta antigen (S-HDAg) and the 27-kDa large delta antigen (L-HDAg). The S-HDAg is essential for viral RNA replication, whereas L-HDAg is necessary for virion assembly. Previous studies have demonstrated that the HDAg is localized predominantly in the nucleoli of cultured cells transfected with a plasmid encoding the HDAg, however, the functional importance of such a phenomenon is not clear. Previous studies in our lab have identified Lys-72 as an acetylation site of S-HDAg. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. In addition, we found that one of the type III HDACs enzyme, SIRT, and the substitution of other lysine residues to arginine at the N-terminus of S-HDAg influence the subcellular localization of S-HDAg. Based on these findings, we proposed that the acetylation of HDAg may modulate its subcellular localization. It has been shown that the nucleolin binding site at amino acid residues 51–65 contributes to nucleolar localization of HDAg. By looking for sequence similarities between the NH2-terminal domain of S-HDAg and other published sequences mediating nucleolar localization of different cellular and viral proteins, we aligned those sequences with the NH2-terminal domain of S-HDAg. The alignment revealed a (R/K) (R/K)X(R/K) motif appearing as three copies in a region enriched with basic amino acids. Moreover, amino acid sequence analysis of the NH2-terminal domain of S-HDAg revealed the presence of two copies of candidate acetylation (K/R)XKK motif appearing in this region. Together, these findings imply that the NH2-terminal domain of S-HDAg may play a role in nucleolar retention of S-HDAg. Therefore, we proposed that while the amino acid residues 51–65 may act as nucleolar translocation sequence of S-HDAg, the amino acid residues 35–50 may act as nucleolar retention sequence and the retention signal may be regulated by acetylation. In this study, we found that two small delta antigen mutant, S-HDAg-K38-43R and S-HDAg-K38-43R/K72R translocate into the nucleoplasm and cytoplasm, respectively, and decrease viral replication. Interestingly, in contrast to our previous finding that the S-HDAg-K72R mutant was detected not only in the nucleolus but also in the cytoplasm in the absence of genome replication, this mutant was detected predominantly in the cytoplasm in the presence of genome replication. Moreover, in order to elucidate whether the the amino acid residue 35-65 、35- 50 and 51- 65 of S-HDAg was necessary for retaining S-HDAg in the nucleolus, we construct DsRed-hnRNPC1 fusion protein, referred to HDAg(35-65)-DsRed-hnRNPC1、HDAg(35-50)-DsRed-hnRNPC1 and HDAg(51-65)-DsRed-hnRNPC1. The results have shown that none of three fusion proteins can be detected in the nucleolus. Finally, we have set up a nucleoli isolation method and can therefore isolate S-HDAg from the nucleolar, nucleoplasmic, and cytoplasmic fraction of HeLa S3 containing mutants S-HDAg cell lines. The S-HDAg from each of these fractions can then be furthered purified by cation exchange column. In the future, the post-translational modifications, especially acetylation, may then be characterized by LC/MS/MS from these purified cellular S-HDAg.

並列關鍵字

Nucleolus Localization Sequence ； Hepatitis Delta Antigen ； Acetylation

參考文獻

Hsieh, S. Y., Chao, M., Coates, L., and Taylor, J. (1990). Hepatitis delta virus genome

Lin, J. H., Chang, M. F., Baker, S. C., Govindarajan, S. and Lai, M. M. (1990). Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA. J Virol 64, 4051-4058.

Chang, M. F., Chang, S. C., Chang, C.I., Wu, K. and Kang, H.Y. (1992). Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen. J Virol 66, 6019-6027.

Chang, F. L., Chen, P. J., Tu, S. J., Wang, C. J., and Chen, D. S. (1991). The large

Mu, J. J., Chen, D. S., and Chen, P. J. (2001). The conserved serine 177 in the delta

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D 型肝炎病毒抗原的核仁位置訊息區域：可能乙醯化的影響

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