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

• Applicant: Dr. Imke Steffen
• Subject Area: Virology
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 232325249

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.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Dr. Graham Simmons