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Hepatitis B virus (HBV) is one of the most serious human pathogens: >250 million people suffer from chronic HBV infection and hundreds of thousands die from it annually. HBV is an enveloped virus with an icosahedral nucleocapsid packaging a partially double-stranded DNA (RC-DNA). The basic building

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General information

Reference : UMR9198-STEBRE-002
Workplace : SACLAY
Date of publication : Tuesday, June 30, 2020
Scientific Responsible name : Stéphane Bressanelli & Guillaume Tresset
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 October 2020
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly

Description of the thesis topic

Hepatitis B virus (HBV) is one of the most serious human pathogens: >250 million people suffer from chronic HBV infection and hundreds of thousands die from it annually. HBV is an enveloped virus with an icosahedral nucleocapsid packaging a partially double-stranded DNA (RC-DNA). The basic building block of the capsid is a dimer of a 183- (or 185- depending on the genotype) residue protein (Core): the N-terminal 149 residues (Cp149) comprise the assembly domain that self-assembles in vivo and in vitro into icosahedral capsids with mainly T = 4 (120 dimers) icosahedral symmetry. The 34 (or 36) remaining arginine-rich amino acids form the C-terminal domain (CTD) which is essential for the packaging of pregenomic RNA (pgRNA) by Core.
In this project, the graduate student will apply state of the art structural methods, particularly time-resolved small-angle X-ray scattering (TR-SAXS), small-angle neutron scattering (SANS) and cryo-electron microscopy (cryo-EM), to clarify the kinetics of assembly of the HBV capsid and how capsid assembly modulators (CpAM) interfere with these processes. Using recombinant proteins, in vitro-transcribed RNA and commercially available CpAM, the grad student will
1. Identify the pathways and intermediates in the assembly of the HBV capsid protein (Core) with or without pre-genomic RNA;
2. Produce the first direct structural data on the ways these out of equilibrium processes are derailed by CpAM.

Work Context

The PhD student will be co-supervised at Paris-Saclay University, in two collaborating research groups, IMAPP at Institute for Integrative Biology of the Cell (I2BC, Gif-sur-Yvette) https://www.i2bc.paris-saclay.fr/spip.php?article180&lang=en and SOBIO at the Laboratory for solid-state Physics (LPS, Orsay). https://www.equipes.lps.u-psud.fr/sobio/spip.php?article5 . I2BC is a large institute, ca 700 people in 5 departments for 67 research groups with 14 core facilities (including a cryo-EM facility) and 11 support services (including an IT and scientific computing service). LPS comprises ca 230 people conducting top level research in soft and condensed matter physics. Both institutes are located near Paris, France.

Constraints and risks

We are looking for a graduate student with a background in structural biology, biochemistry or biophysics, with experience in nucleoprotein complex purification and/or handling and with abilities in complex data processing. The successful candidate will
(i) produce and purify recombinant capsid proteins and RNA (I2BC)
(ii) disassemble recombinant capsids to purify the dissociated dimers
(iii) prepare the rapid mixing experiments to find best conditions to trigger capsid assembly with and without CpAM and/or RNA (LPS, I2BC)
(iv) take part in the TR-SAXS experiments to acquire time-resolved high-resolution data at synchrotrons (SOLEIL (Saint-Aubin, France), ESRF (Grenoble, France)), in the SANS experiment at neutron sources (ILL (Grenoble)) and in the cryo-EM experiments (I2BC, LPS)
(v) take part in the generation of physical and structural models from the data to characterize the kinetic pathways of capsid assembly and disassembly and the effects of CpAM.

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