Ruprecht-Karls-Universität Heidelberg
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Chlanda0117 - Scientist (f/m) / PhD position
Project no:
Chlanda0117

Project leader:

Project supervisor:
Chlanda, Petr
Application deadline:
25. Jan 2018
Start of PhD project:
1. Apr 2018

Project description:

Title:
Structural analysis of Ebola virus entry intermediates by cryo-electron tomography
Summary:
The main objective of this project is to structurally characterize Ebola virus (EBOV) entry route in cells focusing on virus disassembly and membrane fusion. EBOV outbreak in 2013-2016, which caused more than 11,000 deaths, triggered an effort to develop a vaccine and to better understand EBOV virus replication cycle. EBOV assembles into long filamentous virions (1-15 μm) at the plasma membrane. The negative single stranded RNA genome is coiled across a length of ~0.9 μm and protected by the nucleocapsid composed of the nucleoprotein (NP), VP35, VP40, and VP24 proteins. Released EBOV virions enter epithelial cells by macropinocytosis. The Ebola fusion glycoprotein (GP) is proteolytically processed in the late endosome by low-pH sensitive cathepsin proteases to a 19 kDa fragment, which binds to niemann-pick-C1 receptor (NPC1). The 19 kDa fragment bound to NPC1 together with yet unknown factor(s) is able to induce membrane fusion allowing release of the genome [1]. Both the disassembly of the filamentous virus and the mechanism underlying GP mediated membrane fusion in the endosomes are poorly understood processes and have not been structurally characterized yet. PhD student will establish production of non-infectious EBOV virus-like particles (VLP), which are composed of EBOV major structural proteins and are structurally similar to EBOV [2]. The EBOV VLP will be subjected to different chemical conditions mimicking the environment of the endosomes to determine critical factors necessary for membrane binding, fusion and virus disassembly. In addition, EBOV VLP internalization and disassembly will be studied in cells to structurally characterize entry intermediates by cryo-electron tomography and correlative-light and electron microscopy [3]. The study will be validated by comparing the VLP results to the infectious virus, which will be performed under BSL3/4 conditions by our collaborator.
References:
1 Lee, J.E. and Saphire, E.O. (2009). Ebolavirus glycoprotein structure and mechanism of entry. Future Virol 4, 621-635.

2 Bharat, T.A., Noda, T., Riches, J.D., Kraehling, V., Kolesnikova, L., Becker, S., Kawaoka, Y. and Briggs, J.A. (2012). Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci U S A 109, 4275-80.

3 Chlanda, P. et al. (2016). The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes. Nat Microbiol 1, 16050.
Methods that will be used:
PhD student will mainly learn and use confocal microscopy and variety of electron microscopy techniques including the state-of-the-art cryo-electron tomography, image processing and correlative light and electron microscopy techniques. In addition, she/he will be performing cell culture, molecular biology and basic biochemistry techniques.
Cooperation partners:
Dr. Katrik Chandran (Albert Einstein College of Medicine, NY, USA)
Personal qualifications:
We are looking for a motivated and inquisitive PhD student who is interested in virology, microscopy techniques and image processing.
Previous experience in microscopy techniques is desirable but not required.
Keywords:
Ebola virus, cryo-electron tomography