Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
J Virol. 2019 Dec 12;94(1). doi: 10.1128/JVI.01341-19.
Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as "swollen" endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of "budding" occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication. In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes.
许多在细胞质中复制的病毒通过尚未完全了解的机制显著重塑和刺激宿主细胞膜的积累，以实现有效的复制。对于轮状病毒来说，病毒粒子组装的一个关键步骤需要在称为 viroplasm 的病毒复制中心附近积累膜。早期的电子显微镜研究将 viroplasm 相关膜描述为“肿胀”的内质网 (ER)。我们之前证明轮状病毒感染会引发细胞自噬，并且含有自噬标记蛋白 LC3 和轮状病毒 ER 合成的跨膜糖蛋白 NSP4 的膜会运输到 viroplasms，表明 NSP4 必须离开 ER。这项研究旨在解决 NSP4 从 ER 中逸出的机制，并确定与 viroplasms 相关的膜是否来自 ER。我们报告说：(i) NSP4 通过 COPII 囊泡从 ER 中逸出，导致 COPII 囊泡运输和 ER 出口位点受损；(ii) COPII 囊泡被 LC3 II 劫持，LC3 II 与 NSP4 相互作用；(iii) 与 NSP4/LC3 II 相关的膜在 viroplasms 附近积累。此外，在与 viroplasms 相关的膜中未检测到内质网跨膜蛋白 SERCA 和钙网蛋白，这提供了证据表明，轮状病毒成熟过程中的“出芽”是通过自噬劫持的 COPII 囊泡膜发生的。这些发现揭示了一种新的轮状病毒成熟机制，该机制依赖于细胞内宿主蛋白运输和自噬来积累病毒复制所需的膜。在一种对于非包膜病毒来说极其罕见的形态发生步骤中，不成熟的轮状病毒颗粒在称为 viroplasms 的复制中心组装，并通过细胞质细胞膜出芽，以获得用于感染性颗粒组装的外壳蛋白。从历史上看，用于颗粒出芽的细胞内膜被认为是内质网 (ER)，因为与不成熟颗粒相互作用以触发出芽的轮状病毒非结构蛋白 NSP4 作为 ER 跨膜蛋白合成。本研究表明，NSP4 通过 COPII 囊泡从 ER 中逸出，并且含有 NSP4 的 COPII 囊泡被细胞自噬机制劫持，该机制介导 NSP4 向 viroplasms 的运输。改变轮状病毒成熟的范例，我们提出不成熟轮状病毒颗粒出芽所需的细胞膜不是 ER 的延伸，而是 COPII 衍生的自噬隔离膜。
