Periodic Reporting for period 1 - HBVssNMR (Investigating the structural role of the Hepatitis B virus core proteinC-terminal domain in assembly and maturation using solid-stateNMR)
Okres sprawozdawczy: 2017-06-01 do 2019-05-31
The global aim of the project was to use solid-state NMR to study the HBV capsids, in particular to reveal structural and dynamic features of its C-terminal domain.
We also looked at Cp149 capsids using the newly fast-MAS approach which enables to spin at 100 kHz MAS frequency and record proton-detection experiments on small amounts of sample. We have shown that these data are complementary to the previous carbon-detected experiments and that subtle conformational details can be probed by NMR (manuscript accepted for publication). For example, the capsid geometry is detected in the form of four distinct NMR signals corresponding to the four asymmetric subunits in the capsid. We compared the advantages of both NMR approaches to study HBV capsids and showed the impact of protein deuteration at different spinning frequencies. The success of amide-proton assignments shall open the way for the analysis of the HBV capsid in the presence of partner molecules, notably those only available in small quantities.
To understand the impact of RNA-content and phosphorylation state of the core protein, various capsid samples were prepared and analyzed by a combination of NMR methods, including proton, carbon and phosphorus-detected solid-state NMR experiments, as well as solution-state NMR on the core protein dimer and capsid. These data will be used to gain deeper understanding on the capsid-RNA interactions and the role of phosphorylation in the HBV life cycle.
Overall we have shown with this project that the combination of NMR methods is a powerful tool to shed light on the conformational changes occurring in the HBV core protein and it will pave the way for further functional studies on the HBV capsid.
We have demonstrated that the different types of HBV capsids can be studied by solid-state NMR at the molecular level. The various conformational states of the HBV capsids were simulated by in vitro preparations involving different nucleic acids partners and different phosphorylation states. The site-selective information from NMR is of particular importance to further analyze in detail the conformations HBV capsid is thought to access during particle maturation. In addition, capsid assembly as well as the dynamical behavior of the core protein C-terminal domain are key functions which are targeted for modulation or inhibition. NMR is a highly powerful method to study interactions, our data will therefore be used to probe the impact of antivirals on the capsids. In particular, the effect of capsid assembly modulators which are currently in development by pharmaceutical industries will be assessed using solid-state NMR.
The knowledge provided by NMR shall provide important basis to the ongoing effort of the community in drug development by expanding the present molecular description to include the role of the core protein CTD in virus maturation. It will add rationale to the design of molecules which inhibit or modulate essential functions the core protein has in virus proliferation. The answers we seek shall represent a considerable step towards hepatitis B virus molecular understanding, and will bring significant contribution in the broader research for virus elimination and cure, that will have an impact on the 240 million people chronically infected.