Hepatitis Delta Antigen Requires a Minimum Length of the Hepatitis Delta Virus Unbranched Rod RNA Structure for Binding
Hepatitis Delta Antigen Requires a Minimum Length of the Hepatitis Delta Virus Unbranched Rod RNA Structure for Binding
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DOI：
10.1128/jvi.02467-08
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发表时间：
2009-05-01
影响因子：
5.4
通讯作者：
Casey, John L.
中科院分区：
文献类型：
--
作者：
Defenbaugh, Dawn A.;Johnson, Matthew;Casey, John L.
丁型肝炎病毒 (HDV) 是一种亚病毒病原体，可增加乙型肝炎病毒引起的肝脏疾病的严重程度。小环状 RNA 基因组及其互补体（反基因组）形成特有的无分支杆状结构，其中大约 70% 的核苷酸是碱基配对的。这些 RNA 与受感染细胞中唯一的病毒编码蛋白——丁型肝炎抗原 (HDAg) 相关。然而，核糖核蛋白复合物（RNP）的性质尚不清楚。先前使用天然细菌表达的 HDAg 进行的体外结合分析因缺乏 HDV RNA 特异性而受到阻碍。在这里，我们证明去除 HDAg C 端 35 个氨基酸会产生天然的细菌表达蛋白 HDAg-160，它以高亲和力特异性结合 HDV 无分支杆状 RNA。在电泳迁移率变动测定中，该蛋白质产生了一种离散的、微球菌核酸酶抗性复合物，其具有类似于 HDV 无支链杆状 RNA 的 400 个核苷酸 (nt) 片段。与 HDV RNA 的多个片段发生结合，但亲和力和效率各不相同。对删除无分支杆片段的影响的分析表明，结合不需要这些 RNA 片段内的一两个特定结合位点。相反，最小长度约 311 nt 的 HDV RNA 无分支杆对于 RNP 的形成至关重要。结果与 HDAg 以固定大小的多聚体而不是单个亚基结合 HDV 无分支杆状 RNA 的模型一致。
Hepatitis delta virus (HDV) is a subviral pathogen that increases the severity of liver disease caused by hepatitis B virus. Both the small circular RNA genome and its complement, the antigenome, form a characteristic unbranched rod structure in which approximately 70% of the nucleotides are base paired. These RNAs are associated with the sole virally encoded protein, hepatitis delta antigen ( HDAg), in infected cells; however, the nature of the ribonucleoprotein complexes (RNPs) is not well understood. Previous analyses of binding in vitro using native, bacterially expressed HDAg have been hampered by a lack of specificity for HDV RNA. Here, we show that removal of the C-terminal 35 amino acids of HDAg yields a native, bacterially expressed protein, HDAg-160, that specifically binds HDV unbranched rod RNA with high affinity. In an electrophoretic mobility shift assay, this protein produced a discrete, micrococcal nuclease-resistant complex with an similar to 400-nucleotide (nt) segment of HDV unbranched rod RNA. Binding occurred with several segments of HDV RNA, although with various affinities and efficiencies. Analysis of the effects of deleting segments of the unbranched rod indicated that binding did not require one or two specific binding sites within these RNA segments. Rather, a minimum-length HDV RNA unbranched rod approximately 311 nt was essential for RNP formation. The results are consistent with a model in which HDAg binds HDV unbranched rod RNA as multimers of fixed size rather than as individual subunits.