Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
PLoS One. 2022 Mar 1;17(3):e0264643. doi: 10.1371/journal.pone.0264643. eCollection 2022.
Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.
登革热病毒（DENV）引起一种主要的虫媒病毒病，有 25 亿人生活在有风险的地区。DENV 由 50nm 直径的包膜颗粒组成，表面蛋白呈二十面体对称排列，而核衣壳（NC）结构组织的信息则缺乏。DENV NC 由病毒基因组，即一条正链单链 RNA，由衣壳（C）蛋白包装组成。在这里，我们使用重组 DENVC 建立了可重复的 DENV 核衣壳样颗粒（NCLP）体外组装的条件。我们使用小角度 X 射线散射、瑞利光散射以及荧光各向异性分析了在溶液中有无寡核苷酸存在时 NCLP 的形成情况，并用原子力和透射电子显微镜成像技术对颗粒结构特性进行了表征。在溶液中进行的 2-、5-和 25- 核苷酸的实验确定，2-核苷酸太小，而 5-核苷酸足以形成 NCLP。组装过程依赖于浓度，表现出饱和曲线，DENVC 与寡核苷酸的摩尔比为 1:1，表明存在涉及 DENVC 二聚体和与 NCLP 兼容的有序结构的平衡。成像方法证明，在 DENVC 浓度降至亚纳摩尔浓度时，在云母或碳表面上沉积蛋白质后，即使在没有寡核苷酸的情况下，也能形成规则的球形 NCLP，在后者情况下，这是表面驱动的。总之，结果表明，DENV NCLP 的体外组装依赖于 DENVC 电荷中和，这必须是一个非常协调的过程，以避免非特异性聚集。我们的假设是，DENV α4-α4'中的一个特定的高正点是 DENVC-RNA 结合的主要位点，该位点首先需要中和，以允许 NC 形成。
