Department of Microbiology and Immunology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20007, USA.
J Virol. 2010 Feb;84(3):1406-13. doi: 10.1128/JVI.01723-09. Epub 2009 Nov 18.
Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.
乙型肝炎 delta 病毒（HDV）RNA 形成无分支杆状结构，与细胞内复制 HDV 的乙型肝炎 delta 抗原（HDAg）相关。先前使用细菌表达的 HDAg 进行的体外结合实验表明，最小核糖核蛋白复合物的形成至少需要大约 300 个核苷酸（nt）的 HDV 无分支杆状 RNA，并表明 HDAg 作为固定大小的多聚体结合 RNA。本研究专门研究了 HDAg 多聚化在 HDV 核糖核蛋白复合物（RNP）形成中的作用。通过定点突变破坏 HDAg 多聚化，发现 RNP 形成的性质发生了深刻变化。多聚化缺陷的突变 HDAg 蛋白既不表现出结合所需的 300nt RNA 大小要求，也不表现出对无分支杆状结构的特异性。结果明确表明，HDAg 作为多聚体结合 HDV RNA，并且 HDAg 多聚体在结合 RNA 之前形成。发现 RNP 形成是温度依赖的，这与结合时发生的构象变化一致。最后，用长度依次超过最小长度的无分支杆状 RNA 构建的 RNP 分析表明，多聚体结合不仅限于第一个结合的 HDAg，并且需要 604 至 714 个核苷酸之间的最小 RNA 长度来结合第二个多聚体。结果证实了先前关于 HDAg 作为大多聚体结合的提议，并表明多聚体是 HDV RNP 结构的关键决定因素。
