The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.
The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.
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布尼亚病毒目（Bunyavirales）是最大的一组负义RNA病毒，含有许多致命的人类病原体，这些病原体尚未获得批准的抗感染措施。布尼亚病毒基因组由多个负义RNA片段组成，其被病毒编码的核衣壳蛋白（NP）包围，核衣壳蛋白与病毒聚合酶一起形成核糖核蛋白（RNP）。RNP代表RNA合成和病毒体组装的底物，其需要固有的柔性，与从病毒体溢出的RNP的外观一致。这些观察结果导致了描述整个RNP架构的相互冲突的模型。在这里，我们从Bunyamwera病毒（BUNV）（原型正布尼亚病毒）中纯化RNP。通过负染色成像的纯化的RNP的长度产生3个RNP群体，表明RNP具有一致的缩合方法。采用显微镜的方法，我们最终表明，NP部分的BUNV RNP是螺旋形的。此外，我们提出了一个伪原子模型，这部分的基础上，在13 μ m分辨率的冷冻电子显微镜平均，这使我们能够适应BUNV NP晶体结构的分子动力学。通过使用微型基因组系统的NP诱变证实了该模型。该模型表明，相邻的NP单体在RNP链的相互作用，通过灵活的N-和C-末端臂横向只有，没有纵向螺旋稳定的相互作用，从而提供了一个潜在的模型的RNP灵活性的分子基础。过量的RNA酶处理破坏了天然RNP，表明RNA是维持RNP结构的关键。总的来说，这项工作将为布尼亚病毒RNP组装，包装和RNA复制的研究提供信息，并有助于未来的抗病毒策略。
The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies.
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影响因子：
14.9
作者：
Ariza A;Tanner SJ;Walter CT;Dent KC;Shepherd DA;Wu W;Matthews SV;Hiscox JA;Green TJ;Luo M;Elliott RM;Fooks AR;Ashcroft AE;Stonehouse NJ;Ranson NA;Barr JN;Edwards TA
通讯作者：
Edwards TA
影响因子：
3
作者：
Briegel, Ariane;Pilhofer, Martin;Mastronarde, David N.;Jensen, Grant J.
通讯作者：
Jensen, Grant J.
影响因子：
28.3
作者：
Coloma, Rocio;Arranz, Rocio;Martin-Benito, Jaime
通讯作者：
Martin-Benito, Jaime
影响因子：
3
作者：
Kremer, JR;Mastronarde, DN;McIntosh, JR
通讯作者：
McIntosh, JR
影响因子：
4.5
作者：
Mohl, Bjorn-Patrick;Barr, John N.
通讯作者：
Barr, John N.