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Report

Teaser, summary, work performed and final results

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)

Teaser

Summary

Despite the existence of an efficient vaccine, the hepatitis B virus (HBV) affects about 240 million people in the world who remain chronically infected. The HBV viral particle (virion) is composed of a nucleocapsid that is surrounded by lipids and envelope proteins. The capsid is ~30 nm in diameter and is made from 120 copies of the core protein (Cp) dimers. Cp contains an N-terminal domain (NTD) which is responsible for the capsid assembly, and a C-terminal domain (CTD), which interacts with the viral RNA called pregenomic RNA (pgRNA). The CTD undergoes dynamic phosphorylation and dephosphorylation events that regulate its function. Its conformation seems therefore to be central in several critical steps along the viral life cycle, including the specific encapsidation of pgRNA and the intracellular trafficking to the nucleus. However, it remains very challenging to observe the CTD conformation by X-ray diffraction, as it is highly flexible. Even high-resolution cryo-EM studies do not reveal sufficiently detailed information for the CTD. Instead, NMR is a powerful tool to study conformational and dynamical aspects of biomolecules, especially flexible domains like the CTD.
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.

Work performed

The first task of the project was the NMR characterization of HBV capsids from the CTD-truncated form (Cp149), which do not package any RNA. Cp149 was expressed in a bacterial expression system where it autoassembles into capsids. Capsids were then sedimented into an NMR rotor of 3.2 mm in diameter to be analyzed by solid-state NMR at a spinning frequency of 17.5 kHz magic-angle spinning (MAS). The sequential assignments of the capsid resonances were performed using 3D solid-state NMR carbon-detected experiments. We now have the full 13C and 15N chemical shift assignments which was the first step towards further investigations on the different conformations assessed by the capsid during the different steps of the viral life cycle. A comparison with the solution-NMR chemical shifts reported for the Cp149 dimer highlights conformational differences in three distinct regions of the protein which are structurally affected by the transition from the dimer state to the capsid state. These results were presented as oral presentation in national and international conferences, including the ANRS conference (national network of hepatitis) in Paris (France) in February 2017 and EUROMAR (international NMR conference) in Warsaw (Poland) in July 2017 and were published in early 2018 (Lecoq et al., Biomolecular NMR Assignments, 2018).

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.

Final results

We have introduced solid-state NMR as a complementary technique to study the HBV capsid, as it has a great potential to fill the missing pieces in the structure-function relationship of the virus. Indeed, solid-state NMR is highly complementary to electron microscopy in that it can trace conformational and dynamic changes of the protein, both identified as central in the viral life cycle, with highest sensitivity. With the here presented project, we aim to make an impact in structural virology by establishing approaches which allow to follow, on a residue specific level, the different functional conformations capsid protein domains can take along virus maturation.

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.