Institute of Biotechnology and Research Programme in Molecular and Integrative Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, 00790, Helsinki, Finland.
MRC Laboratory of Molecular Biology, Francis Crick Ave, Cambridge, CB2 OQH, UK.
Sci Rep. 2018 Apr 11;8(1):5820. doi: 10.1038/s41598-018-23552-7.
Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.
人类肠道孤儿病毒（HPeV）是小 RNA 病毒科肠道病毒属的成员，其基因组 RNA 为正链单股，衣壳呈二十面体对称。最近的研究发现，有序的 RNA 结构可与衣壳蛋白 VP1 和 VP3 相互作用，并促进 HPeV1 的病毒装配。本研究采用一种结合了可逆交联、RNA 亲和纯化和肽质量指纹图谱（RCAP）的方法，在溶液中对完整 HPeV1 病毒粒子的衣壳蛋白 RNA 相互作用区域进行了作图。衣壳蛋白 VP1 和 VP3 的无规则 N 端，以及出人意料的 VP0，被鉴定为与 RNA 相互作用。将这些结果与使用重组表达的 VP0 和 VP1 获得的结果进行比较，证实了病毒粒子结合区域，并揭示了在 HPeV1 的晶体结构中未观察到的 VP0 的分离 RNA 结合区域。我们使用 RNA 荧光各向异性来确认每个衣壳蛋白 N 端的 RNA 结合能力。这些发现表明，病毒 RNA 与衣壳蛋白之间的动态相互作用调节病毒装配，并提示 VP0 具有新的作用。
