Department of Biology, Wake Forest University, Winston-Salem, North Carolina, USA.
Fralin Biomedical Research Institute, Roanoke, Virginia, USA.
J Virol. 2023 Feb 28;97(2):e0003923. doi: 10.1128/jvi.00039-23. Epub 2023 Feb 7.
Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility . To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2 versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2 formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2 factories had delayed fusion kinetics. Moreover, NSP2 and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2. Finally, NSP2 exhibited defects in its capacity to induce LLPS droplet formation when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.
许多病毒将其复制所需的材料隔离在离散的细胞内工厂中。对于轮状病毒（RV），这些工厂称为类病毒体，它们通过液-液相分离（LLPS）过程在宿主细胞质中形成。非结构蛋白 2（NSP2）及其结合伴侣非结构蛋白 5（NSP5）对于类病毒体的生物发生至关重要。然而，NSP2 和 NSP5 如何合作形成工厂还不完全清楚。NSP2 的 C 端结构域（CTR）（残基 291 至 317）具有柔韧性，使其能够参与结构域交换相互作用，促进八聚体相互作用，并可能促进类病毒体的形成。分子动力学模拟表明，位置 294 处的赖氨酸到谷氨酸的变化（K294E）降低了 NSP2 CTR 的柔韧性。为了测试感染过程中 NSP2 CTR 柔韧性降低的影响，我们设计了一种带有该变化的突变型 RV（rRV-NSP2）。单周期生长测定显示，rRV-NSP2 的终点滴度比野生型对照（rRV-WT）降低了>1.2 个对数。通过免疫荧光测定，我们发现 rRV-NSP2 形成的类病毒体比 rRV-WT 更小、更多。活细胞成像实验证实了这些结果，并表明 rRV-NSP2 工厂的融合动力学延迟。此外，与对照 NSP2 相比，NSP2 和其他几个 CTR 突变体在共表达 NSP5 的细胞中形成的类病毒体样结构更少。最后，当与 NSP5 一起孵育时，NSP2 诱导 LLPS 液滴形成的能力存在缺陷。这些结果强调了 NSP2 CTR 柔韧性在支持 RV 工厂生物发生中的重要性。
