Lee C H, Chang S C, Chen C J, Chang M F
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Biol Chem. 1998 Mar 27;273(13):7650-6. doi: 10.1074/jbc.273.13.7650.
Hepatitis delta antigens (HDAgs) are important for the replication and assembly of hepatitis delta virus (HDV). To understand the association between HDAgs and cellular proteins and the mechanism of viral multiplication, we have studied the interaction between HDAgs and nucleolin, a major nucleolar phosphoprotein. The interaction between HDAgs and nucleolin was first demonstrated by immunofluorescence staining studies. HDAgs and endogenous nucleolin were colocalized in the nucleoli of cultured cells transfected with plasmids encoding the small and large HDAg. Coimmunoprecipitation results indicated that the NH2-terminal domain of HDAg was essential for its binding to nucleolin. In vitro ligand binding assays revealed two nucleolin binding sites, NBS1 and NBS2. Each spanned amino acid residues 35-50 and 51-65, respectively, with a conserved core sequence K(K/R)XK. HDV replication was modulated by exogenous human nucleolin. In addition, a small HDAg mutant S-d65/75, which possesses both NBS1 and NBS2, was capable of transactivating HDV replication, whereas the small HDAg mutant S-d50/75, which retained NBS1 but not NBS2, was unable to support the replication of HDV. Thus, the nucleolin binding activity of HDAg is critical for its nucleolar targeting and is involved in the modulation of HDV replication.
丁型肝炎抗原（HDAgs）对于丁型肝炎病毒（HDV）的复制和组装至关重要。为了了解HDAgs与细胞蛋白之间的关联以及病毒增殖的机制，我们研究了HDAgs与核仁素（一种主要的核仁磷蛋白）之间的相互作用。HDAgs与核仁素之间的相互作用首先通过免疫荧光染色研究得以证实。在转染了编码小HDAg和大HDAg质粒的培养细胞的核仁中，HDAgs与内源性核仁素共定位。免疫共沉淀结果表明，HDAg的NH2末端结构域对于其与核仁素的结合至关重要。体外配体结合试验揭示了两个核仁素结合位点，即NBS1和NBS2。每个位点分别跨越氨基酸残基35 - 50和51 - 65，具有保守的核心序列K(K/R)XK。外源性人核仁素可调节HDV复制。此外，同时拥有NBS1和NBS2的小HDAg突变体S - d65/75能够反式激活HDV复制，而仅保留NBS1但不具有NBS2的小HDAg突变体S - d50/75则无法支持HDV的复制。因此，HDAg的核仁素结合活性对于其核仁靶向至关重要，并参与HDV复制的调节。
