Rotavirus (RV) replication occurs in cytoplasmic compartments, known as viroplasms, that are composed of viral and cellular proteins. Viroplasm formation requires RV nonstructural proteins NSP2 and NSP5 and cellular lipid droplets (LDs); however, the mechanisms required for viroplasm assembly remain largely unknown. We previously identified two conformationally-distinct forms of NSP2 (dNSP2, vNSP2) found in RV-infected cells that interact differentially with hypo- and hyperphosphorylated NSP5, respectively, and indicate a coordinated phosphorylation-dependent mechanism regulating viroplasm assembly. We also reported that phosphorylation of dNSP2 on serine 313 by the cellular kinase CK1α triggers the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. To directly evaluate the role of CK1α-mediated NSP2 phosphorylation on viroplasm formation, we used a recently published plasmid-based reverse genetics method to generate a recombinant rotavirus (rRV) with a phosphomimetic NSP2 mutation (rRV NSP2 S313D). The rRV NSP2 S313D virus is significantly delayed in viroplasm formation, virus replication, and interferes with wild type RV replication during co-infection. The rRV NSP2 S313A virus was not rescued. Taking advantage of the delay in viroplasm formation, the NSP2 S313D phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (1) viroplasm assembly correlates with NSP5 hyperphosphorylation, and (2) that vNSP2 S313D co-localizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly.

轮状病毒 (RV) 复制发生在细胞质区室中，称为病毒质，由病毒和细胞蛋白组成。病毒质的形成需要 RV 非结构蛋白 NSP2 和 NSP5 以及细胞脂滴 (LDs)；然而，病毒质组装所需的机制仍然很大程度上未知。我们之前在 RV 感染的细胞中发现了两种构象不同的 NSP2（dNSP2、vNSP2），它们分别与低磷酸化和高磷酸化的 NSP5 相互作用，并表明调节病毒质组装的协调的磷酸化依赖性机制。我们还报道了细胞激酶 CK1α 对丝氨酸 313 上的 dNSP2 的磷酸化触发了 vNSP2 定位到病毒质组装位点及其与过度磷酸化的 NSP5 的关联。为了直接评估 CK1α 介导的 NSP2 磷酸化对病毒质形成的作用，我们使用最近发表的基于质粒的反向遗传学方法来生成具有拟磷 NSP2 突变 (rRV NSP2 S313D) 的重组轮状病毒 (rRV)。rRV NSP2 S313D 病毒在病毒质形成、病毒复制方面显着延迟，并在共感染期间干扰野生型 RV 复制。rRV NSP2 S313A 病毒未获救。利用病毒质形成的延迟，NSP2 S313D 拟磷突变体被用作观察病毒质组装中非常早期事件的工具。我们表明（1）病毒质组装与 NSP5 过度磷酸化相关，（2）vNSP2 S313D 与 RV 诱导的无 NSP5 的 LD 共定位，表明 vNSP2 磷酸化 S313 足以与 LD 相互作用，并且可能是 RV 诱导的 LD 形成所需的病毒因子。对 rRV NSP2 S313D 病毒的进一步研究有望揭示病毒质和 LD 起始和组装的新方面。