PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

 

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

Department: Virology

Title of the PhD project: : Structure-function studies on yellow fever virus

Name of the lab: Virologie Structurale

Head of the lab: Felix Rey

PhD advisor: Giovanna Barba Spaeth

Email address: gbarba@pasteur.fr

Web site address of the lab: http://www.pasteur.fr/ip/easysite/pasteur/en/research/scientific-departm...

Doctoral school affiliation and University: iViv (interdisciplinaire pour le Vivant), Université Pierre et Marie Curie - Paris 6 (UPMC), Université Paris Diderot, Paris 7

 

 

 

Presentation of the laboratory and its research topics:

 

Our laboratory, the « Unite de Virologie Structurale », is interested in comparative structural studies in order to understand molecular mechanisms involved in the various steps of the virus cycle. The scientific goals of our research are:

1- to provide a structural basis for understanding the molecular mechanisms of membrane fusion used by enveloped viruses to enter a target cell;
2- to understand the assembly of viral capsids and nucleocapsids;
3- to understand the molecular mechanism used by RNA viruses to replicate its genome by both structural and functional studies of RNA-dependent RNA polymerases and associated replication enzymes;
4- to combine the crystallographic data on isolated proteins with the structure of entire viruses obtained by cryomicroscopy in order to acquire insight about their function.

We study mostly viruses of global public health and/or of veterinary concern. The knowledge gained can be used for translational structure-based design of preventive or curative antiviral agents. Furthermore, these structural studies often provide crucial information about evolutionary relations between apparently unrelated viruses.

 

 

 

Description of the project:

(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))

 

Membrane fusion is a crucial biological process. It is essential for communication between cells, tissue differentiation, intracellular organelle trafficking, and pathogen infection. Functional and structural studies have highlighted the role of fusion proteins in the underlying mechanisms of membrane fusion, but the picture is far to be complete. 

Our laboratory, the Unité de virologie structurale, discovered and participates to the unravelling of the mechanism of action of class II viral fusion proteins. These viral proteins drive fusion by changing their conformation from a metastable dimeric form on the surface of the infectious virus to an irreversible trimeric form inserted into the endosome membrane to allow delivering of the viral genome in the cell. The progressive acidification of the endosomal compartment during endocytosis is the trigger of the dimer-to-trimer conformational change. Although the structure of the pre and post-fusion forms of this class of protein has been solved, the dynamic of the process is largely unknown. Understanding the steps of virus fusion will allow the identification of important targets to block the first steps of infection as well as shed light on a significant biological event. 

 

The objective of the PhD project is the analysis of the fusion properties of yellow fever virus envelope protein. Yellow fever virus is a highly pathogenic human virus but it is also the only Flavivirus for which a highly efficient vaccine exists. Twelve of the 31 amino acids that are mutated in the attenuated strain cluster in the envelope (E) protein. We have solved the structure of wild-type and vaccine E proteins in the pre-fusion form, showing that most of the mutations are localized in regions that play a crucial role during the pH-induced conformational changes involved in viral fusion.

Preliminary results suggest that the triggering of the fusion for the two strains could occurs at different pH, within different cellular compartments, which could induce different responses of the cells to the infections.  

During the PhD of the candidate, we will analyze the effect of the mutations observed between the two forms on the pH dependence of the fusion. For this we will combine functional and structural approaches. From the functional point of view, we will generate different forms of the virus harboring different sets of mutations and analyze their ability to fuse with liposomes in controlled pH conditions. Through this, we should be able to identify the amino acids that are responsible for the variation in the virus sensitivity to the pH and to analyze the interplay between them. From the structural point of view, we will approach the system by different ways and favor the most promising in view of the preliminary results. The laboratory currently develops a technique enabling the enrichment in stable isotopes (15N, 13C, 2H) of fusion proteins produced in insect cells. This will allow us to study the transition between the pre- and post- fusion states of different mutants dynamically, by nuclear magnetic resonance, using in particular ultrafast recording technics. NMR is particularly adapted to look at the stability and flexibility of proteins, accordingly it could help to select the mutants best suitable for crystallization of the protein in the post-fusion state. Finally, we also dispose of the competence and equipment to look at the wild-type and mutant viruses by electron-cryo-microscopy.

 

The Unité de virologie structurale is a leading laboratory in its field and the candidate will have access to all required equipment, including a P3 laboratory. The NMR part, leaded by dr. Francois Bontems, will be performed in collaboration with the Laboratoire de chimie et biologie structurales at the CNRS center of Gif-sur-Yvette.

 

 

References:

 

The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. Nature. 375:291-298. (1995)

 

Class II enveloped viruses. Vaney MC, Rey FA. Cell Microbiol. 2011 Oct;13(10):1451-1459. Review.

 

Keywords:

 

Yellow fever virus, membrane fusion, crystal structure, electroncryomicroscopy, NMR

 

 

Expected profile of the candidate (optional):

 

 

 

Contact: Giovanna Barba Spaeth, gbarba@pasteur.fr; Felix Rey, rey@pasteur.fr

 

Mis à jour le 16/09/2013