School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan.
Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan.
Viruses. 2022 Apr 18;14(4):839. doi: 10.3390/v14040839.
Positive-stranded RNA viruses modify host organelles to form replication organelles (ROs) for their own replication. The enteroviral 3A protein has been demonstrated to be highly associated with the COPI pathway, in which factors operate on the ER-to-Golgi intermediate and the Golgi. However, Sar1, a COPII factor exerting coordinated action at endoplasmic reticulum (ER) exit sites rather than COPI factors, is required for the replication of foot-and-mouth disease virus (FMDV). Therefore, further understanding regarding FMDV 3A could be key to explaining the differences and to understanding FMDV's RO formation. In this study, FMDV 3A was confirmed as a peripheral membrane protein capable of modifying the ER into vesicle-like structures, which were neither COPII vesicles nor autophagosomes. When the C-terminus of 3A was truncated, it was located at the ER without vesicular modification. This change was revealed using mGFP and APEX2 fusion constructs, and observed by fluorescence microscopy and electron tomography, respectively. For the other 3A truncation, the minimal region for modification was aa 42-92. Furthermore, we found that the remodeling was related to two COPII factors, Sar1 and Sec12; both interacted with 3A, but their binding domains on 3A were different. Finally, we hypothesized that the N-terminus of 3A would interact with Sar1, as its C-terminus simultaneously interacted with Sec12, which could possibly enhance Sar1 activation. On the ER membrane, active Sar1 interacted with regions of aa 42-59 and aa 76-92 from 3A for vesicle formation. This mechanism was distinct from the traditional COPII pathway and could be critical for FMDV RO formation.
正链 RNA 病毒修饰宿主细胞器，形成复制细胞器 (RO) 以进行自身复制。肠病毒 3A 蛋白已被证明与 COPI 途径高度相关，该途径中的因子作用于内质网 (ER) 到高尔基体中间和高尔基体。然而，在 FMDV 的复制中，Sar1（一种在 ER 出口位点发挥协调作用的 COPII 因子，而不是 COPI 因子）是必需的。因此，进一步了解 FMDV 3A 可能是解释差异和理解 FMDV RO 形成的关键。在这项研究中，FMDV 3A 被确认为一种能够将 ER 修饰成囊泡样结构的外周膜蛋白，这些囊泡既不是 COPII 囊泡也不是自噬体。当 3A 的 C 端被截断时，它位于 ER 中而没有囊泡修饰。这种变化通过 mGFP 和 APEX2 融合构建体来揭示，并分别通过荧光显微镜和电子断层扫描来观察。对于另一个 3A 截断，修饰的最小区域为 aa 42-92。此外，我们发现重塑与两种 COPII 因子 Sar1 和 Sec12 有关；两者都与 3A 相互作用，但它们在 3A 上的结合域不同。最后，我们假设 3A 的 N 端将与 Sar1 相互作用，因为其 C 端同时与 Sec12 相互作用，这可能增强 Sar1 的激活。在 ER 膜上，活性 Sar1 与 3A 的 aa 42-59 和 aa 76-92 区域相互作用形成囊泡。这种机制不同于传统的 COPII 途径，可能对 FMDV RO 形成至关重要。
