Griffin Brittany L, Chasovskikh Sergey, Dritschilo Anatoly, Casey John L
Global Infectious Disease Program, Georgetown University Medical Center, Washington, DC, USA Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA.
Radiation Medicine, Georgetown University Medical Center, Washington, DC, USA.
J Virol. 2014 Jul;88(13):7402-11. doi: 10.1128/JVI.00443-14. Epub 2014 Apr 16.
The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg.
RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.
丁型肝炎病毒（HDV）的环状基因组RNA和反基因组RNA形成特征性的无分支、准双链RNA二级结构，其中短的双链螺旋片段穿插着内环和凸起。这些RNA与病毒编码的蛋白质丁型肝炎抗原（HDAg）形成的核糖核蛋白复合体（RNP）在病毒生命周期中发挥着重要作用，包括病毒复制和病毒粒子形成。对于这些复合体的形成、结构以及它们在这些关键过程中的功能，我们了解得还很少。在此，我们研究了HDAg结合所需的特定RNA特征以及所形成复合体的拓扑结构。应用于游离和HDAg结合的HDV RNA的引物延伸选择性2'-羟基酰化分析（SHAPE）表明，RNA与HDAg结合时其特征性二级结构得以保留。值得注意的是，分析表明RNA中预测的未配对位置在RNP中仍保持动态。对内部环和凸起发生突变的RNA以及合成设计的RNA的体外结合活性分析表明，独特的二级结构而非RNA一级序列是HDAg RNA结合特异性的主要决定因素。对体外形成的RNP的原子力显微镜分析揭示了HDV RNA通过弯曲或缠绕而显著压缩的复合体。我们的结果支持这样一种模型，即HDV RNA中的内环和凸起为准双链结构提供了灵活性，使得RNA能够被HDAg弯曲和压缩。
由丁型肝炎病毒RNA和蛋白质HDAg形成的核糖核蛋白复合体（RNP）在病毒复制中发挥关键作用。这些RNP的结构以及形成它们所需的RNA特征均未得到表征。HDV RNA不同寻常之处在于它形成一种无分支的准双链结构，其中短的碱基配对片段穿插着内环和凸起。我们分析了HDV RNA序列和二级结构在最小RNP形成中的作用，并使用原子力显微镜观察了该RNP的结构。我们结果表明，HDAg不识别RNA的一级序列；相反，HDV RNA中未配对碱基对HDAg结合的主要贡献在于允许无分支准双链RNA结构具有灵活性。通过原子力显微镜对RNP的观察表明，RNA在复合体中显著弯曲或压缩。
