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

2009年，在刚果民主共和国偏远村庄曼加拉的一名出血热患者中首次发现新型横纹病毒曼加拉病毒(MANV)。它还被怀疑在上述患者患病之前造成该村两名青少年的出血性疾病和死亡。弹状病毒是一大类病毒，可感染植物、脊椎动物和无脊椎动物以及人类。然而，像狂犬病病毒这样的弹状病毒很少被发现对人类有致病性，唯一已知的导致出血性疾病的弹状病毒会感染鱼类。因此，MANV的发现不仅定义了一组新的弹状病毒，而且还在导致人类出血热的病原体名单上增加了一个新成员。病毒糖蛋白通过与细胞受体结合建立与宿主细胞的第一次接触，其功能对于生产性感染是必不可少的。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中对膜融合和生产性感染至关重要的单一氨基酸残基将通过定点突变进行鉴定。这包括预测的N-糖基化位点和内部融合环中的芳香族氨基酸，这是横纹病毒糖蛋白的共同特征。此外，将产生截断变体，与敏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。基于水泡性口炎病毒糖蛋白融合前后构象的已知晶体结构的分子模拟将有助于确定MANV糖蛋白序列中的关键残基和结构域。巨核细胞系Meg-01将成为鉴定细胞MANV受体的有用工具。MEG-01细胞对MANV-G感染不耐药，但在佛波醇12-肉豆蔻酸酯13-醋酸酯诱导分化后变得敏感。因此，分化和未分化的Meg-01细胞的mRNA池将通过深度测序和/或消减杂交进行比较，以确定差异表达的蛋白质。或者，通过慢病毒载体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。基于初步数据揭示了MANV-G广泛的物种和组织嗜性，候选分子将因已知的性质而被缩小范围，如质膜定位、高度的物种间保守性和在各种组织中的表达。这项研究将大大增加我们对新发现的横纹肌肉病毒及其细胞进入机制的了解，并为MANV感染的潜在治疗干预提供一个起点。

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.