Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA
Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA
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DOI：
10.1038/s41467-020-18447-z
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发表时间：
2020-09-17
影响因子：
16.6
通讯作者：
Kielian, Margaret
中科院分区：
文献类型：
--
作者：
Brown, Rebecca S.;Anastasakis, Dimitrios G.;Kielian, Margaret
甲病毒衣壳蛋白（Cp）选择性地将基因组RNA（gRNA）包装到病毒核衣壳中以产生感染性病毒。使用可光活化的核糖核苷交联和创新的生物素化Cp修复方法，在这里，我们全面定义了gRNA上Semliki森林病毒（SFV）Cp的结合位点。虽然感染细胞中的数据证明Cp与所提出的基因组包装信号（PS）结合，但诱变实验表明PS不是产生感染性SFV或基孔肯雅病毒所必需的。相反，我们确定了多个Cp结合位点，这些位点富集在gRNA特异性区域上，并促进感染性SFV产生和gRNA包装。细胞质与病毒核衣壳中结合位点的比较表明，出芽引起Cp-gRNA相互作用的离散变化。值得注意的是，Cp的顶部结合位点在整个病毒组装过程中保持不变，并且在体外与Cp特异性结合并组装成核心样颗粒。我们的数据一起提出了一个选择性甲病毒基因组识别和组装的模型。
The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.