Characterization of the structure and function of the glycoprotein of the novel rhabdovirus Mangala virus and identification of its cellular receptor

新型弹状病毒曼加拉病毒糖蛋白的结构和功能表征及其细胞受体的鉴定

• 批准号: 232325249
• 负责人: Dr. Imke Steffen
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232325249?language=en
• 关键词: Characterization; structure; function; glycoprotein; novel

The novel rhabdovirus Mangala virus (MANV) was first identified in 2009 in a patient suffering from hemorrhagic fever in the remote village of Mangala in the Democratic Republic of Congo. It is further suspected to have caused the hemorrhagic disease and death of two teenagers in the village prior to the illness of the mentioned patient. Rhabdoviruses comprise a large family of viruses infecting plants, vertebrate and invertebrate animals and humans. However, few rhabdoviruses like rabies virus have been found to be pathogenic in humans and the only rhabdoviruses known to cause hemorrhagic disease infect fish. The discovery of MANV therefore not only defines a novel group of rhabdoviruses, but also adds a new member to the list of agents causing hemorrhagic fever in humans. The viral glycoprotein establishes the first contact with the host cell by binding to a cellular receptor and its function is essential for productive infection. It is therefore an important target for therapeutic intervention with viral infection. In the present proposal I suggest a detailed analysis of the MANV glycoprotein (MANV-G) structure and function. Single amino acid residues within MANV-G that are essential for membrane fusion and productive infection will be identified by site-directed mutagenesis. This includes predicted N-glycosylation sites and aromatic amino acids in the internal fusion loops, a common feature of rhabdovirus glycoproteins. Furthermore, truncation variants will be generated and binding studies with susceptible cell lines will reveal the minimal receptor-binding domain required for cellular attachment. Molecular modeling based on the known crystal structures of the pre- and post-fusion conformations of the vesicular stomatitis virus glycoprotein will aid to determine critical residues and domains within the MANV glycoprotein sequence. The megakaryocytic cell line MEG-01 will be a helpful tool for the identification of the cellular MANV receptor. MEG-01 cells are refractory to MANV-G driven infection, but become susceptible upon differentiation with phorbol 12-myristate 13-acetate. Consequently, mRNA pools from differentiated and undifferentiated MEG-01 cells will be compared by deep sequencing and/or subtractive hybridization to identify differentially expressed proteins. Alternatively, a cDNA library from a permissive cell line will be transduced into undifferentiated MEG-01 cells with the help of lentiviral vectors to select for potential receptors by genetic complementation. Based on preliminary data revealing a broad species and tissue tropism of MANV-G, candidate molecules will be narrowed down for known properties like plasma membrane localization, high inter-species conservation and expression in a variety of tissues. This study will substantially increase our understanding of the newly identified rhabdovirus MANV and its mechanism of cell entry and presents a starting point for potential therapeutic intervention in MANV infection.

新型横纹肌病毒曼加拉病毒（Mangala virus， MANV）于2009年在刚果民主共和国偏远的曼加拉村的一名出血热患者身上首次发现。在上述病人患病之前，它还被怀疑引起了该村两名青少年的出血性疾病和死亡。横纹肌病毒是一大类感染植物、脊椎动物和无脊椎动物以及人类的病毒。然而，很少有像狂犬病毒这样的横纹肌病毒被发现能对人类致病，而唯一已知能引起出血性疾病的横纹肌病毒会感染鱼类。因此，MANV的发现不仅定义了一种新的横纹肌病毒，而且在引起人类出血热的病原体名单上增加了一个新成员。病毒糖蛋白通过结合细胞受体与宿主细胞建立第一次接触，其功能对生产性感染至关重要。因此，它是病毒感染治疗干预的重要靶点。在本提案中，我建议详细分析MANV糖蛋白（MANV- g）的结构和功能。MANV-G中对膜融合和生产感染至关重要的单氨基酸残基将通过定点诱变确定。这包括预测的n -糖基化位点和内部融合环中的芳香氨基酸，这是横纹肌病毒糖蛋白的共同特征。此外，截断变异体将产生，与易感细胞系的结合研究将揭示细胞附着所需的最小受体结合域。基于水疱性口炎病毒糖蛋白融合前后构象的已知晶体结构的分子建模将有助于确定MANV糖蛋白序列中的关键残基和结构域。巨核细胞系MEG-01将为细胞MANV受体的鉴定提供一个有用的工具。MEG-01细胞对MANV-G驱动的感染不敏感，但在与phorbol 12-肉豆蔻酸13-乙酸分化后变得敏感。因此，分化和未分化的MEG-01细胞的mRNA池将通过深度测序和/或减法杂交进行比较，以鉴定差异表达的蛋白。或者，在慢病毒载体的帮助下，将来自允许细胞系的cDNA文库转导到未分化的MEG-01细胞中，通过遗传互补选择潜在的受体。基于揭示MANV-G广泛的物种和组织趋向性的初步数据，候选分子将根据质膜定位、高物种间保守性和多种组织表达等已知特性缩小范围。这项研究将大大增加我们对新发现的横纹肌病毒MANV及其进入细胞机制的理解，并为MANV感染的潜在治疗干预提供了一个起点。