核輸出訊息結合蛋白質NESI之肌動蛋白結合區之功能分析

Abstract

D型肝炎病毒(hepatitis delta virus; HDV)是個球狀的RNA病毒，其病毒顆粒的外套(envelope)由HBV的表面抗原與細胞膜組成，內部則為約1.7 kb單股負向的環狀RNA基因體及其轉譯產生的兩種delta抗原，分別為小型delta抗原(HDAg-S)，含195個胺基酸（約24kDa）；以及大型delta抗原(HDAg-L)，含214個胺基酸（約27 kDa）。在HDV的病毒生活史中，小型delta抗原為病毒複製所必須，而大型delta抗原可與HBV的表面抗原結合，對病毒顆粒的包裹是重要的。 本實驗室先前的研究發現，大型delta抗原C端第198至210個胺基酸為proline-rich的核輸出訊號(nuclear export signal; NES)，命名為NES(HDAg-L)，此NES藉由CRM1-independent pathway進行大型delta抗原的細胞核輸出，且宿主蛋白質NESI 【NES(HDAg-L)-interacting protein】，可調控病毒基因體-抗原複合體的核輸出及病毒顆粒的包裹。 NESI蛋白質具有467個胺基酸（約53kDa），經由序列分析，其第4至13個胺基酸可能會與肌動蛋白（actin）結合，而第193至209個胺基酸可能為核位訊號（nuclear localization signal; NLS）。先前的研究證實NESI胺基酸1-185片段可與肌動蛋白結合，且knock down NESI的表現可觀察到細胞的移動能力增加，但其分子機制未明。本論文利用免疫沈澱法再次證明NESI與肌動蛋白間的專一性結合，並以GST pull-down assay發現NESI N端的30個胺基酸為兩者的交互作用所必須。而帶有肌動蛋白結合區突變的NESI蛋白質則喪失與肌動蛋白的能力，顯示其actinin-type actin binding domain 之consensus sequence對NESI-actin複合體形成的重要性。以免疫螢光染色觀察到NESI分布在細胞核中及細胞質裡靠近核的位置，且在細胞質的NESI與F-actin有共位現象(co-localization)，但是在活體外（in vitro）結合實驗中，並沒有觀察到NESI(1-30)胜肽與純化的F-actin共同沈降，對溶液中G-actin與F-actin的動態平衡也沒有明顯影響。

Hepatitis delta virus (HDV) is a spherical RNA virus, which is enveloped by the surface antigens of the hepatitis B virus. Inside the viral particles, HDV contains a single-stranded, negative sense, circular RNA genome of 1.7 kb and its only known products, small delta antigen (HDAg-S) and large delta antigen (HDAg-L). Previous studies from our laboratory identified a novel proline-rich nuclear export signal at the C-terminus of HDAg-L, designated NES(HDAg-L). In addition, host protein NESI which specifically interacts with NES(HDAg-L) participated in nuclear export of HDV RNP complex and virus assembly. NESI consists of 467 amino acid residues. It bears a putative actin binding site and a bipartite nuclear localization signal. Association of NESI(1-185) and actin was demonstrated in our laboratory. In addition, In NESI-knockdown cells, the cell mobility was increased in the scratch assay. However, regulations of the putative actin-binding domain from amino acid residues 4 to 13 in the cell mobility are unclear. In this study, the specific interaction between NESI and actin was further confirmed by co-immuoprecipitation analysis. GST pull-down assay indicates that the N-terminal 30 amino acid residues are required for NESI to interact with actin. The actin-binding ability of NESI protein was lost when mutations were introduced into the actin binding domain. These results indicate that the consensus sequence of the actinin-type actin binding domain is important for NESI-actin complex formation. Immunofluorescence staining demonstrated that NESI localized in the nucleus and the perinuclear region. The cytoplasmic NESI co-localized with F-actin. However, no co-sedimentation of NESI(1-30) peptide with purified F-actin was detected, and no significant changes in the equilibrium between G-actin and F-actin was observed in a in vitro binding assay. These results suggest that, unlike alpha-actinin, NESI may not function as F-actin bundling.