Oropouche virus (OROV; Genus: Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, that encodes the surface glycoproteins Gn and Gc plus the non-structural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion and, unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibit glycoprotein secretion. Infection studies have previously shown that the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly co-localizes with OROV glycoproteins and membranes co-stained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlights differences in M polyprotein processing across orthobunyaviruses, that Golgi and ESCRT function are required for glycoprotein secretion, and identifies CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins.

中文翻译：

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Oropouche 病毒糖蛋白拓扑结构和病毒组装的细胞要求

奥罗普切病毒（OROV；属：Orthobunyavirus) 是 Oropouche 热的病原体，这是一种在南美洲常见的使人衰弱的发热性疾病。我们使用编码表面糖蛋白 Gn 和 Gc 以及非结构蛋白 NSm 的 OROV M 多蛋白的重组表达来探测 OROV 组装和出芽的细胞决定因素。Gn 和 Gc 自组装并独立于 NSm 分泌。成熟的 OROV Gn 具有两个预测的跨膜结构域，这对于糖蛋白易位到高尔基复合体和糖蛋白分泌至关重要，并且与相关的原布尼亚病毒不同，这两个跨膜结构域在 Gn 成熟期间都保留。使用布雷菲德菌素 A 和莫能菌素破坏高尔基体功能抑制糖蛋白分泌。感染研究先前已经表明，运输所需的细胞内体分选复合物 (ESCRT) 机制在 OROV 组装过程中被募集到高尔基体膜上，并且 ESCRT 活性是病毒分泌所必需的。ESCRT 相关 ATP 酶 VPS4 的显性阴性形式显着降低重组 OROV 糖蛋白分泌并阻止病毒从感染细胞中释放，并且 VPS4 部分与 OROV 糖蛋白共定位，膜与高尔基标记共染色。此外，免疫沉淀和荧光显微镜实验表明，OROV 糖蛋白与 ESCRT-III 组分 CHMP6 相互作用，显性阴性形式的 CHMP6 过表达显着降低了 OROV 糖蛋白的分泌。综合起来，