Department of Molecular Biophysics, Humboldt University, 10115 Berlin, Germany.
Institute for Translational HIV Research, University Hospital Essen, 45147 Essen, Germany.
Viruses. 2022 Feb 23;14(3):457. doi: 10.3390/v14030457.
Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.
汉坦病毒是具有三片段、负义 RNA 基因组的包膜病毒。病毒的 S 片段编码多功能核衣壳蛋白（N），该蛋白参与基因组包装、细胞内蛋白运输、免疫调节以及汉坦病毒感染过程中的几个其他关键过程。在这项研究中，我们从普马拉病毒（PUUV）生成了荧光标记的 N 蛋白构建体，这是中欧、北欧和东欧的主要汉坦病毒。我们在啮齿动物细胞系 CHO-K1 中全面表征了该蛋白，监测 N 蛋白复合物形成的动力学，并研究了与宿主蛋白以及病毒糖蛋白 Gc 和 Gn 的共定位。我们观察到形成了大的、纤维状的 PUUV N 蛋白聚集体，从早期点状和刺状组装中迅速聚结。此外，我们发现 N 与波形蛋白、肌动蛋白和 P 体具有显著的空间相关性，但与微管没有相关性。N 构建体也与 Gn 和 Gc 共定位，尽管不如与彼此相关的糖蛋白强烈。最后，我们评估了 N 构建体的寡聚化，观察到有效的、浓度依赖性的多聚化，复合物包含超过 10 个单个蛋白。
