The genescorresponding either to the full-length core protein (Cp183) or to its truncated form Cp149 were cloned into the pEU-E01-MCS vector (CellFree Sciences, Japan) for WGE-CF expression. The plasmids were amplified in DH5α bacteria, and purified using a NucleoBond Xtra Maxi kit (Macherey-Nagel, France). An additional purification step was performed with a phenol/chloroform extraction to ensure the purity of the plasmid according to the recommendations of CellFree Sciences (Yokohama, Japan).

Transcription was performed according to Takai et al. (2010) in 1.5 mL Eppendorf tubes using 100 μg/mL plasmid, 2.5 mM NTP mix (Promega), 1 U/μL SP6 RNA Polymerase (CellFree Sciences), and 1U/μL RNase inhibitor (CellFree Sciences) in transcription buffer (CellFree Sciences) containing 80 mM Hepes-KOH pH 7.6, 16 mM magnesium acetate, 10 mM DTT and 2 mM spermidine. After incubation for 6 h at 37°C, mRNA was used directly for translation.

Non-treated durum wheat seeds (Sud Céréales, France) were used to prepare home-made WGE as described in Fogeron et al. (2017), according to the protocol of Takai et al. (2010) with minor modifications. Translation was performed using the bilayer method as described in Takai et al. (2010), Fogeron et al. (2017) for small scale expression tests in the presence of compounds, or using the dialysis mode as described in David et al. (2018) for larger scale production followed by isolation on a sucrose density gradient. For the bilayer method, the bottom layer (20 μL) corresponding to the translation mixture contains per well 10 μL of mRNA, 10 μL of WGE, 40 ng/μL of creatine kinase and 6 mM of amino-acid mix (0.3 mM per amino acid, average concentration). The upper layer (200 μL) corresponding to the feeding buffer contains SUB-AMIX NA (CellFree Sciences; 30 mM Hepes-KOH pH 7.6, 100 mM potassium acetate, 2.7 mM magnesium acetate, 16 mM creatine phosphate, 0.4 mM spermidine, 1.2 mM ATP, 0.25 mM GTP, and 4 mM DTT), and 6 mM of amino acid mix (0.3 mM per amino acid, average concentration). For Cp183 expression in the presence of different compounds, 10 nmol of antiviral (dissolved in DMSO at a concentration of 10 mM) was added into 200 μL feeding buffer and translation was performed at 22°C for 16 h.

For large-scale production, dialysis cassettes with a volume of either 500 μL or 3 mL, depending on the production scale, and a MWCO of 10 kDa were used. The translation mixture contained ½ by volume of feeding buffer, 1/3 of mRNA, 1/6 of WGE, 40 ng/μL of creatine kinase, 0.3 mM of amino-acid mix. The feeding buffer (either 20 mL or 124 mL for a 500-μL or a 3-mL dialysis cassette, respectively) contains SUB-AMIX NA (CellFree Sciences) as described above, supplemented with 0.3 mM of amino-acid mix. The dialysis cassette containing the translation mix was soaked in the feeding buffer, and incubated for 16 h under shaking at 60 rpm, 22°C. A mix containing all twenty isotopically labeled amino acids (Cambridge Isotope Laboratory) was used for the production of ¹³C-¹⁵N-Cp183 for NMR studies in a 3 mL-translation reaction experiment.

The total cell-free reaction mixture (CFS) was treated with 25,000 units/mL of benzonase for 30 min at room temperature before centrifugation at 20,000 g, 4°C for 30 min. The supernatant (SN) was loaded onto a discontinuous sucrose gradient with layers of 10, 20, 30, 40, 50, and 60% sucrose (w/v), each with a volume of 350 μL for a production in a 500-μL cassette. For the production of a ¹³C-¹⁵N-Cp183 sample in a 3-mL dialysis cassette, the supernatant (SN) was split into two fractions and loaded onto two sucrose gradients with layers of 10, 20, 30, 40, 50, and 60% sucrose (w/v), each with a volume of 1.5 mL. The gradients were centrifuged at 200,000 g, 4°C for 12 h. After centrifugation, the different sucrose fractions were harvested and analyzed by SDS-PAGE and Western blotting, as well as by electron microscopy after negative staining as described below.

Cp183 capsids used as reference for negative stain EM with CAMs were obtained from BL21^*-CodonPlus (DE3) cells using plasmid pRSF-T7-HBc183opt. Expression and purification were done as previously reported (Heger-Stevic et al., 2018a; Lecoq et al., 2018b). In brief, protein was expressed overnight after induction with 1 mM IPTG at 20°C, and cell lysate was separated with 10–60% sucrose gradient. Cp183 capsids were precipitated after the sucrose gradient by 40% saturation ammonium sulfate, and resuspended in final buffer (50 mM Tris pH 7.5, 5 mM DTT, 1 mM EDTA, 5% sucrose). The interaction between preformed capsids and compounds was performed with a molar ratio of Cp183 monomer: compound of 1:4, at 37°C for 2 h.

Four different Cp183 NMR samples were prepared: two from cell-free protein synthesis, one synthesized using ¹³C/¹⁵N, and the other one ²H/¹³C/¹⁵N amino acids, resulting in a protonated sample, and a deuterated, but 100% protonated on exchanging protons, as synthesis is carried out in H₂O; and for reference two samples from E. coli expression, one deuterated and back exchanged on exchangeable sites, and one protonated (Heger-Stevic et al., 2018a; Lecoq et al., 2018b). NMR samples were filled into 0.7 mm rotors as sediment obtained by ultracentrifugation directly into the rotor (Böckmann et al., 2009) at 200,000 g for approximately 16 h at 4°C, yielding approximately 0.5 mg of sediment. As an internal chemical-shift reference, about 30 μL of saturated (0.3 M) 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added to the protein solution before sedimentation.

On each of the samples a two-dimensional (2D) fingerprint hNH spectrum was recorded. On the protonated, uniformly ¹³C-¹⁵N labeled cell-free produced sample, an hCANH 3D (Penzel et al., 2015) was recorded in addition. All spectra were acquired on a wide-bore 850 MHz Bruker Avance III spectrometer with a 0.7 mm triple-resonance MAS probe (Bruker Biospin) operated at 100 kHz MAS. Magic angle and shim for this probe were set using a 0.7 mm rotor with glycine ethylester by optimizing the intensity and J-coupling based splitting of the CO resonance (Penzel et al., 2018). The sample was cooled with a BCU (Bruker Cooling Unit) gas flow of 400 l/h with a VT (Variable Temperature) set to 272 K, corresponding to a sample temperature of approximately 22°C, extrapolated from the water chemical shift in a ¹H 1D (Gottlieb et al., 1997; Böckmann et al., 2009). Detailed acquisition parameters can be found in Supplementary Table 1.

TopSpin 4.0.3 (Bruker Biospin) was used for the data acquisition and processing. 2D hNH spectra were processed with 1,024 points in ¹H dimension (corresponding to 12.9 ms of acquisition time) and zero filling was applied to, respectively, 4,096 points in ¹H and 1,024 points in ¹⁵N dimension. The 3D hCANH was processed with zero filling to, respectively 2m048 points in ¹H, 128 points in ¹⁵N, and 256 points in ¹³C dimensions. All spectra were apodized with a shifted sine-bell window function using SSB = 3.5 in TopSpin. Linear prediction to twice the recorded number of points was applied in the ¹⁵N dimension for 2D hNH spectra of the protonated capsids produced by CFPS, and the deuterated E. coli capsids, in order to reach a similar number of points as acquired for the other samples. Spectral analyses were performed using the CcpNmr Analysis package 2.4.2 (Stevens et al., 2011). The proton linewidths were obtained using the parabolic fit function integrated on CcpNmr on six isolated peaks in the hNH spectra. The errors given represent the standard deviations between the six values. Signal-to-noise ratio were calculated on the bulk signals from 1D hNH spectra recorded and processed with similar parameters and divided by the square root of the number of scans.

The expression of Cp183 was assessed by 15% Coomassie blue stained SDS–PAGE and Western blotting as described in Fogeron et al. (2015). A polyclonal rabbit antiserum against the N-terminal domain of the HBV core protein (a-c149) was used to detect both Cp149 and Cp183 on blots.

Samples for electron microscopy were negatively stained as described in Lecoq et al. (2018b). Briefly, 5 μL of each fraction were loaded on a carbon-coated grid (EMS Microscopy) and incubated for 2 min at room temperature. Remaining liquid was drained using Whatman paper. Grids were negatively stained on a 50-μL drop of 2% phosphotungstic acid (pH = 7) for 2 min at room temperature and observed with a JEM-1,400 transmission electron microscope operating at 100 kV.

CFPS of the core protein was performed for both Cp149 and Cp183. The protein was found mainly in the soluble fraction after centrifugation, as indicated in Western blots in Figures 1A,B. The protein band is partly visible in the total CFS fraction of the Coomassie blue gel. Enrichment via a sucrose gradient reveals that Cp149 stays mainly in the load and in the 10% sucrose fraction, indicating the protein remained in an unassembled, probably dimeric state. Accordingly, the electron micrograph of the 10% fraction (Figure 1A, blue asterisk), showed only very few capsids. In contrast, Cp183 sedimented largely into the 50 and 60% sucrose fractions (Figure 1B, red asterisk), as expected when capsids have been formed. EM inspection revealed numerous auto-assembled Cp183 capsids with a diameter of about 30 nm, as also observed for capsids assembled in E. coli (Gallina et al., 1989; Lecoq et al., 2018b).

Upon expression in E. coli, both Cp183 and the CTD-less Cp149 variant auto-assemble into capsids. Only full-length protein packages RNA, while Cp149 capsids remain empty (Birnbaum and Nassal, 1990). Both types of capsids can be isolated from bacteria by a set of purification steps (Heger-Stevic et al., 2018b; Lecoq et al., 2018b), with Cp149 giving particularly high yields (100 mg per liter of culture, compared to 20 mg/L for Cp183). The capsids can be disassembled using either urea (Cp149) or guanidinium chloride (Cp183) (Zlotnick et al., 1997; Porterfield et al., 2010). Reassembly is concentration dependent, and in vitro assembly of the full-length protein needs addition of nucleic acids which are non-sequence specifically packaged (Porterfield et al., 2010). Failure of Cp149 to assemble upon WGE-CF synthesis is likely due to the higher concentrations this protein needs for assembly, while the interaction between the positively-charged Cp183 CTD with the negatively-charged nucleic acids enables Cp183 assembly at concentrations as low as 5 nM (Klein et al., 2004). Failure of Cp149 to assemble has also been observed in rabbit reticulocyte extract (Ludgate et al., 2016).

For large-scale production (~1 milligram) needed for NMR sample preparation, CFPS was carried out in dialysis reactions, as described for the duck HBV envelope subviral particle synthesis (David et al., 2018). Either protonated or deuterated, HN protonated Cp183 was prepared, with the latter referred to in the following as dCp183. In the large-scale synthesis, more protein was found in the pellet compared to the small-scale synthesis, likely due to higher concentrations. On sucrose gradient isolation, migrated to the 60 % fraction (Figures 2A,B). The preparation using the deuterated amino acids shows higher purity, which might be due to a slightly different migration behavior of the deuterated protein in the sucrose gradient. EM inspection revealed abundant capsids in both preparations (Figures 2C,D).

Conformational details can be revealed by NMR in so-called fingerprint spectra, which show either in two (2D) or three dimensions (3D) the typical signature of the protein preparation. Structural variations can be sensitively identified by comparing spectra recorded under different conditions, and analyzing the differences in the observed chemical shifts, i.e., the NMR frequencies (Williamson, 2013). An opportunity of the combination of CFPS and NMR is the fact that only the synthesized protein, which is the sole isotopically labeled protein, will be observed in the spectra. The use of a simple sucrose gradient concentration step thus might not produce perfectly pure protein; still, only the protein of interest will produce signal in the spectra. A possible drawback might lie in a loss of signal-to-noise ratio (SNR) in the spectra, since the NMR sample container (rotor) also might contain residual contaminating proteins (Figure 2A). It is thus important to establish whether protein samples prepared by CFPS are indeed compatible with the recording of 2D and in particular 3D spectra in a reasonable amount of time.

The hNH 2D correlation spectrum recorded in 16 h on the protonated cell-free Cp183 displays a highly similar spectrum to the one recorded on the capsids purified from E. coli (Figure 3A and Figure S1) in 10 h. The NMR signal amplitude of the sample from CFPS is about 35% of the spectra obtained on the preparation from purified E. coli protein recorded under the same experimental conditions. As both rotors were full with protein sediment, this means that the contaminating unlabeled proteins from the WGE fill almost 2/3 of the rotor. A 3D hCANH spectrum was recorded on the sample in 4 days and 15 h, and an overlay of all 3D NH planes onto the 2D NH plane shows that most signals in the 2D hNH spectrum are also observed in the 3D (Figure 3B).

The 2D spectrum recorded on the deuterated sample is shown in Figures 3C,D. SNR is very favorable in this sample, since the deuterated protein surprisingly showed better purity (Figure 2B). The spectrum reveals narrower lines than the spectrum from the protonated sample, as also observed in model systems (Penzel et al., 2019) and, in particular, also in capsid preparations purified from E. coli (Lecoq et al., 2019): 140 Hz on average for the protonated vs. 100 Hz for the deuterated sample, as measured on six isolated resonances. The SNR and proton linewidths for the four samples are summarized in Figures 3E,F, respectively. It reveals that CFPS samples show a greater variability in sample amounts than the well-established E. coli samples; further experience is needed to evaluate parameters allowing reproducible sample preparation using CFPS. The proton linewidths are virtually similar between the two protonated and two deuterated samples, indicating that production by CFPS or E. coli expression does not make a difference with respect to linewidth and therefore conformational homogeneity.

Importantly, several peaks are present in the cell-free synthesized dCp183 which could not be observed in the deuterated sample purified from E. coli, as emphasized in Figure 3D. The origin of this observation lies in the incomplete back-exchange in E. coli produced samples. Indeed, when deuterated protein is expressed in E. coli, synthesis takes place in D₂O, and exchange of deuterons to protons is achieved during the subsequent purification steps, carried out in H₂O. Still, solvent-inaccessible deuterons can remain in the protein over long periods of time, and often denaturation/renaturation of the protein is applied to complete proton exchange important for NMR observation. However, this step can be very difficult for more complex proteins, and the present experiment highlights this interesting feature of CFPS, where the protein is synthesized from the beginning in H₂O, and deuteration is achieved not via metabolism, but by addition of deuterated amino acids to the cell-free reaction. This results in fully protonated amide (and exchangeable sidechain) protons in the synthesized protein, which is essential for the recording of NMR spectra showing resonances for all amino acids.

CFPS proceeds in an open system, and a variety of substances can be added to the reaction mixture. We added different capsid assembly modulators to the reaction, in order to analyze whether this produces comparable phenotypes to those observed on capsids purified from E. coli. Figure 4A shows the Coomassie blue stained gels of the cell-free solutions without compounds, in the presence of DMSO used for solubilization of the antiviral, and in presence of AT-130, JNJ-623 (CAM-N), and JNJ-890 (CAM-A). The corresponding Western blots are shown in Figure 4B. None of the compounds inhibited protein synthesis. We analyzed the total cell-free solutions, without any concentration or purification, under the electron microscope, and compared the observed capsids as shown in Figures 4C–G with the ones obtained from addition of compounds to capsids purified from E. coli, shown in Figures 4H–J. One can see in the micrographs that the resulting objects closely resemble those obtained by addition to preformed capsids: DMSO vehicle and CAM-Ns produced no visible effect, whereas CAM-As showed the typical disruption of capsids also reported in the literature (Berke et al., 2017; Lahlali et al., 2018). Notably, the presence of AT-130 lead to poorer contrast in the EM micrographs of both preparations.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.