Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.
Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.
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DOI：
10.1128/jvi.02024-21
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发表时间：
2022-03-23
影响因子：
5.4
通讯作者：
Broadbent AJ
中科院分区：
文献类型：
--
作者：
Reddy VRAP;Campbell EA;Wells J;Simpson J;Nazki S;Hawes PC;Broadbent AJ
为了获得更多关于鸟导航病毒科病毒工厂(VFS)性质的信息，我们使用了一种表达Split-GFP 11的重组传染性法氏囊病病毒(IBDV)，该病毒标记在先前我们已经证明是VFS的感染细胞中表达GFP 1-10的标记。我们发现，在放线菌素存在的情况下，VFS与5-乙炔基尿苷共定位，表明它们含有新合成的病毒RNA；在洋地黄素固定和渗透的感染细胞中可见VFS，表明它们不是膜结合的。光漂白后的荧光恢复(FRAP)VFS内的一个感兴趣的区域发生了迅速的恢复，从大约25%恢复到原始强度的87%，超过146 S，VFS被1，6-己二醇处理溶解，表明它们显示出与液-液相分离一致的性质。VF GFP信号与衣壳蛋白VP2的共定位(Manders系数[MC]0.6)低于VP3(MC，0.9)，这促使我们用透射电子显微镜(TEM)研究VF的超微结构。在被感染的细胞中，在细胞质中观察到病毒粒子的晶状排列(PA)，以及离散的电子致密区。利用相关光学和电子显微镜(CLEM)，我们观察到电子致密区与VFS的GFP信号相关，这与PAS不同。综上所述，鸟导航病毒科VFS含有新合成的病毒RNA，不受膜结合，表现出与液-液相分离一致的性质，与透射电子显微镜观察到的PAS不同。禽科的重要成员感染鸟类、鱼类和昆虫，并对家禽业和水产养殖业具有重大经济意义的疾病负责。尽管它们很重要，但它们如何在细胞中复制仍然知之甚少。在这里，我们展示了BirNaviridae病毒工厂不受膜限制，表现出与液-液相分离一致的特性，并且不同于通过透射电子显微镜观察到的病毒粒子的准晶阵列，从而增强了我们关于病毒复制的基础知识，这些病毒复制可以用于制定控制疾病的策略，或优化其治疗应用。
To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders’ coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.
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影响因子：
5.4
作者：
Jobe F;Simpson J;Hawes P;Guzman E;Bailey D
通讯作者：
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影响因子：
3.8
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