Ruprecht-Karls-Universit├Ąt Heidelberg
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Reinstein0118 - Scientist (f/m) / PhD position
Project no:
Reinstein0118

Project leader:

Project supervisor:
Reinstein, Jochen
Application deadline:
30. Nov 2018
Start of PhD project:
1. Jan 2019

Project description:

Title:
Assembly, DNA loading and processing of virophages
Summary:
Virophages are small DNA viruses that co-infect unicellular organisms that contain giant viruses. They recruit the cytosolic reproduction and assembly machinery of the giant virus whose own propagation is then stalled. Up to now 3 different virophages, namely Sputnik, Mavirus and Zamilon were isolated (1-3). They contain about 20 coding regions in a DNA genome of approx. 20 kb. The most conserved components are the two capsid proteins, a protease that assists maturation and a FtsK-HerA type ATPase that supposedly is responsible for ATP dependent DNA transport into the assembled capsid.

The successful candidate will characterize and compare key features of Mavirus and Sputnik/Zamilon capsid assembly and processing (6) and active transport of DNA. Common themes as well as unique features correlated to adaption of the different virophages to their different hosts/environment should be elucidated. The assembly/disassembly pathways will be characterized with spectroscopic, kinetic and structural methods (5,6) with the aim to understand better the hierarchy and pathway of supramolecular organisation of capsid structure and what determines the exact size of the assembled particles in the absence of a molecular ruler. Key to address these questions is also development and application of mostly fluorescence based markers for FRET (4) and ideally also Fluorescence Microscopy based assays. Rationally modified and loaded Capsids should also be used for in vivo probing and as nano-containers.
References:
[1] Zhang X, Sun S, Xiang Y, Wong J, Klose T, Raoult D, et al. Structure of Sputnik, a virophage, at 3.5-A resolution. Proc Natl Acad Sci U S A. 2012;109:18431-6.

[2] Gaia M, Benamar S, Boughalmi M, Pagnier I, Croce O, Colson P, et al. Zamilon, a Novel Virophage with Mimiviridae Host Specificity. Plos One. 2014;9.

[3] Fischer MG, Suttle CA. A virophage at the origin of large DNA transposons. Science. 2011;332:231-4.

[4] Muller B, Anders M, Reinstein J. In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics. PLoS One. 2014;9:e99504.

[5] Zeymer C, Fischer S, Reinstein J. trans-Acting arginine residues in the AAA+ chaperone ClpB allosterically regulate the activity through inter- and intradomain communication. J Biol Chem. 2014;289:32965-76.

[6] Born D, Reuter L, Mersdorf U, Mueller M, Fischer MG, Meinhart A, et al. Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus. PNAS 2018. DOI 10.1073/pnas.1805376115
Methods that will be used:
Molecular Biology, Protein Biochemistry+Biophysics, Spectroscopy, transient kinetics, Electron Microscopy, Fluorescence microscopy
Cooperation partners:
Matthias Fischer, MPImR Heidelberg
Alexander Schug, Forschungszentrum J├╝lich
Personal qualifications:
A strong interest in quantitative and analytical techniques and molecular mechanisms, solid training in molecular biology and biochemistry, knowledge in biophysics and physical chemistry.
Keywords:
Capsid, folding & assembly, DNA transport ATPase, structure, fluorescence