Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA, USA.
Translational Biology, Medicine, and Health Graduate Program, Roanoke, VA, USA.
Sci Rep. 2017 Aug 24;7(1):9301. doi: 10.1038/s41598-017-08068-w.
Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.
轮状病毒（RV）可以通过重组过程进化，在共感染过程中，11 个双链 RNA 基因组片段在菌株之间交换。然而，当共感染株的基因或编码蛋白在功能上不相容时，重组受到限制。在这项研究中，我们采用了一种基于辅助病毒的反向遗传学系统，以鉴定与限制进入猿猴 RV 株 SA11 的重组相关的 NSP2 基因区域。我们表明，具有来自人类 RV 株 Wa 或 DS-1 的 NSP2 基因的 SA11 重组体可以有效地被拯救，并且没有检测到明显的复制缺陷。然而，我们无法拯救具有人类 RV 株 AU-1 NSP2 基因的 SA11 重组体，该基因与 SA11 的差异为 186 个核苷酸（36 个氨基酸）。为了绘制限制决定因素图谱，我们设计了包含嵌合 NSP2 基因的病毒，其中 AU-1 序列的特定区域被 SA11 序列取代。我们表明，跨越 AU-1 NSP2 基因核苷酸 784-820 的区域对于观察到的限制至关重要；然而，其他决定因素存在于其他基因区域。我们使用计算机模拟和体外分析来预测 784-820 区域如何影响 NSP2 基因/蛋白功能，从而为理解重组限制机制提供信息。
