Oropouche virus glycoprotein topology and cellular requirements for virus assembly
Oropouche virus glycoprotein topology and cellular requirements for virus assembly
复制标题
奥罗普切病毒糖蛋白拓扑结构和病毒组装的细胞要求
DOI：
10.1101/2020.05.28.122689
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Barbosa N
中科院分区：
文献类型：
--
作者：
Barbosa N
奥罗普什病毒（Oropouche virus，Orobunyavirus）是奥罗普什热（Oropouche fever）的病原体，奥罗普什热是南美洲常见的一种使人衰弱的发热性疾病。我们使用的重组表达的OSTOM多蛋白，编码的表面糖蛋白Gn和Gc加上非结构蛋白NSm，探测OSTOM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OST-Gn具有两个预测的跨膜结构域，这两个跨膜结构域对于糖蛋白转运到高尔基复合体和糖蛋白分泌至关重要，并且与相关的正布尼亚病毒不同，这两个跨膜结构域在Gn成熟期间被保留。使用布雷菲德菌素A和莫能菌素抑制糖蛋白分泌的药物破坏高尔基体功能。感染研究先前已经表明，细胞内体分选复合物所需的运输（ESCRT）的机器被招募到高尔基体膜在Oscillus组装和ESCRT活性是所需的病毒分泌。ESCRT相关的ATP酶VPS 4的显性负性形式显著降低重组OX 3糖蛋白分泌并阻断病毒从感染细胞释放，并且VPS 4与OX 3糖蛋白和与高尔基体标记共染色的膜部分共定位。此外，免疫沉淀和荧光显微镜实验表明，Ocephalus糖蛋白与ESCRT-III组分CHMP 6相互作用，CHMP 6的显性负性形式的过表达显著降低Ocephalus糖蛋白分泌。综上所述，我们的数据突出了M多聚蛋白加工在正布尼亚病毒之间的差异，高尔基体和ESCRT功能是糖蛋白分泌所必需的，并确定CHMP 6作为ESCRT-III组分，与Oupuche糖蛋白相互作用。ImportanceOropouche病毒引起Oropouche热，一种在南美洲常见的衰弱性疾病，其特征是高烧，头痛，肌痛和呕吐。这种人畜共患病毒的三重基因组能够重配，在过去50年中，南美发生了多次奥罗波什热流行，使奥罗波什病毒感染成为对公共卫生的重大威胁。然而，这种虫媒病毒的分子特征知之甚少。我们开发了一个重组蛋白表达系统，以研究卵巢糖蛋白成熟和分泌的细胞决定因素。我们发现，蛋白水解处理的M多肽，它编码的表面糖蛋白（Gn和Gc）加上非结构蛋白（NSm），不同的Ospirum和它的近亲布尼亚姆韦拉病毒。此外，我们表明，OSPUM糖蛋白分泌需要细胞ESCRT膜重塑机制，并确定OSPUM糖蛋白与ESCRT蛋白CHMP 6相互作用。
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.ImportanceOropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterised by high fever, headache, myalgia and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a non-structural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular ESCRT membrane-remodelling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.