Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation

Abstract

Modulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures.

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

“…Typically, going from 800 MHz to 1.2 GHz increases the signal-to-noise ratio by nearly a factor of 2. But already today, the combination of 100 kHz MAS, high magnetic fields (>800 MHz) ( Figure 1 ) and improved sample preparation, e.g., by direct sedimentation [ 37 , 38 ], allows for high-resolution spectroscopy of large biomolecular assemblies like viral capsids or envelopes [ 35 , 39 , 40 , 41 ] if they show high local symmetry. If the protein-monomers forming the assemblies are structurally diverse, this results in the observation of multiple sets of NMR signals (see also below), and increases spectral crowding or line widths if the splittings are unresolved.…”

“…As demands in sample quantity are diminishing (see above), other production approaches have recently been developed, and a highly attractive compromise between expression in eukaryotic cells vs. E. coli is the use of eukaryotic cell-free systems, optimally one that combines high expression yields with an increased folding capacity and access to a range of post-translational modifications [ 58 ]. One such system is based on wheat germ cell-free protein synthesis (WG-CFPS) [ 40 , 41 , 59 , 60 ]. The high efficiency and selectivity of isotopic labeling constitutes a major asset of this system for NMR studies.…”

“…In this context, the use of cell-free synthesis systems for sample preparation is particularly interesting as assembly modulators can act directly on the capsid protein exiting from the ribosome, and hence in a preassembly state. The sub-milligram Cp183 capsids obtained through cell-free protein synthesis are fully sufficient for the smaller sample requirements of fast MAS NMR, and they can simply be sedimented into a 0.7 mm NMR rotor after purification on a sucrose gradient ( Figure 4 a) [ 40 ]. The capsids directly pack nucleic acids during cell-free synthesis, and only the full-length protein successfully assembles in the system.…”

“…The capsids directly pack nucleic acids during cell-free synthesis, and only the full-length protein successfully assembles in the system. In the presence of a heteroaryldihydropyrimidine (HAP) CAM, the cell-free Cp183 capsids opened up completely ( Figure 4 b) [ 40 ], indicating suitability of this system for solid-state NMR analysis. These data show how the combination of WG-CFPS and solid-state NMR can open the way for a better understanding of the action of CAMs under conditions that more closely mimic the situation in a cell.…”

Proton‐Detected Solid‐State NMR of the Cell‐Free Synthesized α‐Helical Transmembrane Protein NS4B from Hepatitis C Virus

“…WG‐CFPS thus presents an efficient alternative to cellular eukaryotic expression systems, and has the advantage that various NMR isotope labeling schemes, including amino‐acid selective labeling, can be easily implemented . Importantly, also deuteration in combination with complete amide protonation can be achieved directly during synthesis, avoiding a denaturation and refolding step, which can compromise the native fold of a membrane protein.…”