Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories
Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories
复制标题
通过反向遗传学拯救的重组轮状病毒揭示了 NSP5 过度磷酸化在病毒工厂组装中的作用
作者：
G. Papa;Luca Venditti;F. Arnoldi;E. Schraner;Christiaan A. Potgieter;A. Borodavka;C. Eichwald;O. Burrone
轮状病毒（RV）双链RNA基因组复制并包装成细胞质结构中的病毒子代，称为病毒囊。非结构蛋白NSP 5在RV感染期间经历复杂的过度磷酸化过程，是形成这些病毒诱导的细胞器所必需的。然而，它在病毒质形成和RV复制中的作用从未被直接评估，因为缺乏一个完全易处理的轮状病毒反向遗传学（RG）系统。在这里，我们展示了一个新的应用程序，最近开发的RG系统，通过建立一个稳定的反式互补NSP 5生产细胞系所需的救援轮状病毒与突变的NSP 5。这种方法使我们能够提供该蛋白在RV复制过程中的关键作用的第一个直接证据。此外，使用重组RV突变体，我们揭示了感染过程中NSP 5过度磷酸化的分子机制及其参与复制型病毒的组装和成熟。轮状病毒（RV）在圆形的细胞质病毒工厂中复制，尽管它们如何组装仍然未知。在RV感染期间，NSP 5经历过度磷酸化，其由单个丝氨酸残基的磷酸化引发。这种翻译后修饰在病毒质形成中的作用及其对病毒复制的影响仍然不清楚。在这里，我们研究了NSP 5在RV感染过程中的作用，利用一个修改后的完全易处理的反向遗传学系统。使用稳定产生NSP 5的反式互补细胞系来产生和表征在NSP 5中具有突变的几种重组轮状病毒（rRV）。我们证明了缺乏NSP 5的rRV完全不能组装病毒颗粒和复制，证实了其在轮状病毒复制中的关键作用。产生了许多NSP 5磷酸化受损的突变体，以进一步询问这种翻译后修饰在复制型病毒载体组装中的功能。我们发现，rRV突变株表现出受损的病毒复制和MA 104细胞中组装圆形病毒颗粒的能力。此外，我们使用NSP 5磷酸化阴性的rRV菌株以及表达野生型NSP 5或选定的NSP 5缺失突变体的MA 104衍生的稳定转染细胞系研究了RV感染期间NSP 5过度磷酸化的机制。我们的研究结果表明，NSP 5过度磷酸化是圆形病毒颗粒组装的关键步骤，突出了NSP 5的C-末端尾部在形成复制能力的病毒工厂中的关键作用。这种复杂的NSP 5磷酸化级联反应可以作为在其他RNA病毒中组装功能性病毒工厂的范例。重要性轮状病毒（RV）双链RNA基因组复制并包装成病毒子代的细胞质结构，称为病毒囊。非结构蛋白NSP 5在RV感染期间经历复杂的过度磷酸化过程，是形成这些病毒诱导的细胞器所必需的。然而，它在病毒质形成和RV复制中的作用从未被直接评估，因为缺乏一个完全易处理的轮状病毒反向遗传学（RG）系统。在这里，我们展示了一个新的应用程序，最近开发的RG系统，通过建立一个稳定的反式互补NSP 5生产细胞系所需的救援轮状病毒与突变的NSP 5。这种方法使我们能够提供该蛋白在RV复制过程中的关键作用的第一个直接证据。此外，使用重组RV突变体，我们揭示了感染过程中NSP 5过度磷酸化的分子机制及其参与复制型病毒的组装和成熟。
The rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse-genetics (RG) system for rotaviruses. Here, we show a novel application of a recently developed RG system by establishing a stable trans-complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants, we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms. ABSTRACT Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, although how they assemble remains unknown. During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this posttranslational modification in the formation of viroplasms and its impact on virus replication remain obscure. Here, we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse-genetics system. A trans-complementing cell line stably producing NSP5 was used to generate and characterize several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that an rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. A number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this posttranslational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibited impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wild-type NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyperphosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replication-competent viral factories. Such a complex NSP5 phosphorylation cascade may serve as a paradigm for the assembly of functional viral factories in other RNA viruses. IMPORTANCE The rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse-genetics (RG) system for rotaviruses. Here, we show a novel application of a recently developed RG system by establishing a stable trans-complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants, we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms.
影响因子：
--
作者：
John T. Patton;Lynn S. Silvestri;M. Tortorici;R. V. Carpio;Z. Taraporewala
通讯作者：
John T. Patton;Lynn S. Silvestri;M. Tortorici;R. V. Carpio;Z. Taraporewala