Molecular Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
J Virol. 2014 Jul;88(14):7998-8015. doi: 10.1128/JVI.00940-14. Epub 2014 May 7.
The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal α2 and α4 helices of each monomer. Pairing of α2-α2' creates a continuous hydrophobic surface, while the α4-α4' helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the α4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the α4-α4' helix pair conformation is critical in preserving the overall α-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the α4-α4' helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. Importance: Structure-based mutagenesis study of the dengue virus core protein (CP) reveals that the α4-α4' helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected. In vitro inefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation for in vivo virus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by α4-α4' helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the α4-α4' helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production.
登革病毒（DENV）的病毒体由包裹核衣壳的病毒包膜组成，核衣壳由病毒基因组 RNA 和核心蛋白（CP）组成的复合物构成。DENV CP 通过每个单体的内部α2 和α4 螺旋形成二聚体。α2-α2' 的配对形成连续的疏水面，而α4-α4' 螺旋对通过内侧残基的侧链相互作用将同源二聚体连接在一起。然而，DENV CP 二聚体构象及其与功能的关系的重要性仍知之甚少。由于突变导致 CP 与脂滴（LD）的结合丧失，表明 CP 疏水面未暴露，这为二聚体缺失提供了一种可能的解释。进一步的测定表明 CP 折叠与蛋白质稳定性之间存在联系。全长 RNA 衍生病毒产生的衰减与 CP 突变有关，因为在病毒翻译和复制中没有检测到明显缺陷。体外特征测定进一步强调了α4-α4' 螺旋对构象在维持 CP 整体α-螺旋含量、热稳定性和二聚体形成能力方面的关键作用，这些特征与核衣壳形成效率相关。添加 Tween 20 可改善体外核衣壳样颗粒的形成，表明 LD 在体内核衣壳形成中的作用。本研究首次直接将α4-α4' 螺旋对相互作用与 CP 二聚体构象联系起来，CP 二聚体构象是 CP 功能的基础，特别是在病毒体生产过程中的核衣壳形成。
登革热病毒核心蛋白（CP）的结构基突变研究表明，α4-α4' 螺旋对是维持其二聚体构象的关键，CP 功能的基础是在核衣壳形成和病毒产生中。全长 RNA 衍生病毒产生的衰减与 CP 突变有关，因为在病毒翻译和复制中没有检测到明显缺陷。体外核衣壳样颗粒（NLP）形成效率低且尺寸小，可能解释了 CP 突变时体内病毒产生效率低下的原因。此外，在非离子洗涤剂存在下，α4-α4' 螺旋对突变体显示出 NLP 形态从不完全状态向完整颗粒的转变，表明细胞内脂滴（LD）在 CP-LD 相互作用和促进核衣壳形成中的调节作用。本研究首次将α4-α4' 螺旋对相互作用与 CP 二聚体构象直接联系起来，CP 二聚体构象是 CP 功能的基本要求，特别是在病毒体生产过程中的核衣壳形成。
