Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. Here we show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T = 4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation.

Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis†B Virus Capsid C-Terminal Domain.

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

A pocket-factor-triggered conformational switch in the hepatitis B virus capsid.

Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid-envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the "synergistic double interaction" hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR.

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T 1 and T 1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds.

Nonsymmetrical Dynamics of the HBV Capsid Assembly and Disassembly Evidenced by Their Transient Species.

As with many protein multimers studied in biophysics, the assembly and disassembly dynamical pathways of hepatitis B virus (HBV) capsid proteins are not symmetrical. Using time-resolved small-angle X-ray scattering and singular value decomposition analysis, we have investigated these processes in vitro by a rapid change of salinity or chaotropicity. Along the assembly pathway, the classical nucleation-growth mechanism is followed by a slow relaxation phase during which capsid-like transient species self-organize in accordance with the theoretical prediction that the capture of the few last subunits is slow. By contrast, the disassembly proceeds through unexpected, fractal-branched clusters of subunits that eventually vanish over a much longer time scale. On the one hand, our findings confirm and extend previous views as to the hysteresis phenomena observed and theorized in capsid formation and dissociation. On the other hand, they uncover specifics that may directly relate to the functions of HBV subunits in the viral cycle.

Protein NMR Spectroscopy at 150†kHz Magic-Angle Spinning Continues To Improve Resolution and Mass Sensitivity.

Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic-angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.

100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids.

We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ~50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and HN back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170-110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several HN protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.

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

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.

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.

Solid-state [13C-15N] NMR resonance assignment of hepatitis B virus core protein.

Each year, nearly 900,000 deaths are due to serious liver diseases caused by chronic hepatitis B virus infection. The viral particle is composed of an outer envelope and an inner icosahedral nucleocapsid formed by multiple dimers of a ~ 20 kDa self-assembling core protein (Cp). Here we report the solid-state 13C and 15N resonance assignments of the assembly domain, Cp149, of the core protein in its capsid form. A secondary chemical shift analysis of the 140 visible residues suggests an overall alpha-helical three-dimensional fold matching that derived for Cp149 from the X-ray crystallography of the capsid, and from solution-state NMR of the Cp149 dimer. Interestingly, however, at three distinct regions the chemical shifts in solution differ significantly between core proteins in the capsid state versus in the dimer state, strongly suggesting the respective residues to be involved in capsid assembly.

Thermal factors influencing detection of Vibrio vulnificus using real-time PCR.

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.

Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR.

We used a rapid DNA extraction and purification method to obtain the DNA from Vibrio vulnificus seeded into clam tissue homogenates for real-time PCR quantification of the organism. Without enrichment, the limit of detection was 1 x 10(2) cfu/g of tissue with a linear detection range of 1 x 10(2) to 1 x 10(8) cfu/g. With a 5 h non-selective enrichment, the limit of detection was 1 cfu/g of tissue with a linear detection range of 1 to 1 x 10(6) cfu/g of tissue. We found a 10-fold higher detection limit with seeded clam tissue homogenates compared to pure culture in TSB(+). The detection limits with pure broth culture and seeded tissue homogenates were identical, 1 cfu/ml and 1 cfu/ml, respectively, following 5 h non-selective enrichment. However, the Ct value with tissue homogenates was about 3 threshold cycles higher than with pure culture.

Haemolytic-uraemic syndrome caused by a non-O157 : H7 Escherichia coli strain in experimentally inoculated dogs.

Both O157 : H7 and non-O157 : H7 Escherichia coli strains are reported to cause haemolytic-uraemic syndrome (HUS). This study was carried out to explore the pathogenicity of O157 : H7 and non-O157 : H7 E. coli strains in experimentally inoculated dogs. Twenty 40-day-old dogs were randomly divided into four groups, and the groups (n=5) were administrated orally with E. coli O157 : H7 strains HJ2001-1 (from a patient with serious haemorrhagic diarrhoea) and HZ2001-4 (from a domestic sheep kept in the house of a patient who died from diarrhoea and subsequent acute renal failure), HZ2001-9 (a non-O157 : H7 strain, from a 6-month-old child who died from diarrhoea and subsequent acute renal failure) or a control strain, EC8099. HJ2001-1 and HZ2001-4 caused slight diarrhoea, and the dogs recovered without any complications. However, HZ2001-9 resulted in watery diarrhoea accompanied with slightly bloody stools, followed by death on the fifth or sixth day. In the fatally infected experimental animals, necrotic lesions in the liver and bacterial embolism in the kidney were observed. The primary cause of death was microvascular thrombosis caused by the bacteria, leading to renal and multiple organ failure. Therefore, the non-O157 : H7 E. coli strain HZ2001-9 causes clinical signs and pathological lesions in dogs that are consistent with those in acute renal failure or HUS in humans.

Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR.

Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.