Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

Mechanical disassembly of single virus particles reveals kinetic intermediates predicted by theory.

New experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T=1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly.

Systematic analysis of biological roles of charged amino acid residues located throughout the structured inner wall of a virus capsid.

Structure-based mutational analysis of viruses is providing many insights into the relationship between structure and biological function of macromolecular complexes. We have systematically investigated the individual biological roles of charged residues located throughout the structured capsid inner wall (outside disordered peptide segments) of a model spherical virus, the minute virus of mice (MVM). The functional effects of point mutations that altered the electrical charge at 16 different positions at the capsid inner wall were analyzed. The results revealed that MVM capsid self-assembly is rather tolerant to point mutations that alter the number and distribution of charged residues at the capsid inner wall. However, mutations that either increased or decreased the number of positive charges around capsid-bound DNA segments reduced the thermal resistance of the virion. Moreover, mutations that either removed or changed the positions of negatively charged carboxylates in rings of acidic residues around capsid pores were deleterious by precluding a capsid conformational transition associated to through-pore translocation events. The results suggest that number, distribution and specific position of electrically charged residues across the inner wall of a spherical virus may have been selected through evolution as a compromise between several different biological requirements.

Quantitatively probing propensity for structural transitions in engineered virus nanoparticles by single-molecule mechanical analysis.

Viruses are increasingly being studied from the perspective of fundamental physics at the nanoscale as biologically evolved nanodevices with many technological applications. In viral particles of the minute virus of mice (MVM), folded segments of the single-stranded DNA genome are bound to the capsid inner wall and act as molecular buttresses that increase locally the mechanical stiffness of the particle. We have explored whether a quantitative linkage exists in MVM particles between their DNA-mediated stiffening and impairment of a heat-induced, virus-inactivating structural change. A series of structurally modified virus particles with disrupted capsid-DNA interactions and/or distorted capsid cavities close to the DNA-binding sites were engineered and characterized, both in classic kinetics assays and by single-molecule mechanical analysis using atomic force microscopy. The rate constant of the virus inactivation reaction was found to decrease exponentially with the increase in elastic constant (stiffness) of the regions closer to DNA-binding sites. The application of transition state theory suggests that the height of the free energy barrier of the virus-inactivating structural transition increases linearly with local mechanical stiffness. From a virological perspective, the results indicate that infectious MVM particles may have acquired the biological advantage of increased survival under thermal stress by evolving architectural elements that rigidify the particle and impair non-productive structural changes. From a nanotechnological perspective, this study provides proof of principle that determination of mechanical stiffness and its manipulation by protein engineering may be applied for quantitatively probing and tuning the conformational dynamics of virus-based and other protein-based nanoassemblies.

Noribogaine (12-hydroxyibogamine): a biologically active metabolite of the antiaddictive drug ibogaine.

Ibogaine (IBO) is a plant-derived alkaloid that is being evaluated as a possible medication for substance use disorders. When administered peripherally to monkeys and humans, IBO is rapidly converted to an o-demethylated metabolite, 12-hydroxyibogamine (NORIBO). We have found in rats that peak blood levels of NORIBO can exceed those of the parent compound, and NORIBO persists in the bloodstream for at least 24 h. Surprisingly few studies have examined the in vivo biological activity of NORIBO. In the present series of experiments, we compared the effects of intravenous (i.v.) administration of IBO and NORIBO (1 and 10 mg/kg) on unconditioned behaviors, circulating stress hormones, and extracellular levels of dopamine (DA) and serotonin (5-HT) in the nucleus accumbens of male rats. IBO caused dose-related increases in tremors and forepaw treading, whereas NORIBO did not. Both IBO and NORIBO produced significant elevations in plasma corticosterone and prolactin, but IBO was more potent as a stimulator of corticosterone secretion. Neither drug affected extracellular DA levels in the nucleus accumbens. However, both IBO and NORIBO increased extracellular 5-HT levels, and NORIBO was more potent in this regard. The present data demonstrate that NORIBO is biologically active and undoubtedly contributes to the in vivo pharmacological profile of IBO in rats. Most importantly, NORIBO appears less likely to produce the adverse effects associated with IBO (i.e., tremors and stress-axis activation), suggesting that the metabolite may be a safer alternative for medication development.

Noribogaine stimulates naloxone-sensitive [35S]GTPgammaS binding.

Noribogaine is formed in vivo by the O-demethylation of the indole alkaloid ibogaine. We report here that noribogaine acts as a full agonist at the mu-opioid receptor. Noribogaine-stimulated guanylyl 5'gamma-[35S]thio]triphosphate ([35S]GTPgammaS) was studied in rat thalamic membranes to measure activation of guanine nucleotide binding proteins (G-proteins) in the presence of excess GDP. Noribogaine caused a 170% increase above basal [35S]GTPgammaS binding at sub-micromolar effective concentrations (EC50) in a naloxone-sensitive manner, confirming that this effect was an opioid receptor-mediated process. The level of intrinsic activity for noribogaine in these assays was comparable to the full agonists DAMGO and morphine. In contrast, ibogaine had no significant effect on [35S]GTPgammaS binding over a similar concentration range. The efficacy of noribogaine as a full mu-opioid agonist may explain ibogaine's ability to block the acute signs of opiate withdrawal and its suppressive effects on morphine self-administration.

Properties of ibogaine and its principal metabolite (12-hydroxyibogamine) at the MK-801 binding site of the NMDA receptor complex.

The putative anti-addiction alkaloid ibogaine and its principal metabolite 12-hydroxyibogamine appear to act at the (+)-5 methyl-10,11,dihydro-5H- dibenzo[a,d]cycloheten-5-10-imine maleate (MK-801) binding site in the N-methyl-D-aspartate (NMDA)-receptor cation channel. This conclusion is based on findings that both compounds competitively displaced specific [3H]MK-801 binding to membranes from postmortem human caudate and cerebellum and from frog spinal cord. Ibogaine was 4-6-fold more potent than its metabolite and both compounds were less potent (50-1000-fold) than MK-801 binding to the NMDA receptor. In addition, ibogaine (100 microM) and 12-hydroxyibogamine (1 mM) blocked (85-90% of control) the ability of NMDA (100 microM, 5 s) to depolarize frog motoneurons in the isolated frog spinal cord. The prevention of NMDA-depolarizations in frog motoneurons showed use-dependency and was very similar to the block produced by MK-801. In view of the abilities of MK-801 to affect the responses to addictive substances in pre-clinical investigations, our results are compatible with the idea that the ability of ibogaine and 12-hydroxyibogamine to interrupt drug-seeking behavior may, in part, result from their actions at the MK-801 binding site.

In vivo neurobiological effects of ibogaine and its O-desmethyl metabolite, 12-hydroxyibogamine (noribogaine), in rats.

Ibogaine is a naturally occurring compound with purported antiaddictive properties. When administered to primates, ibogaine is rapidly o-demethylated to form the metabolite 12-hydroxyibogamine (noribogaine). Peak blood levels of noribogaine exceed those of ibogaine, and noribogaine persists in the bloodstream for at least 1 day. Very few studies have systematically evaluated the neurobiological effects of noribogaine in vivo. In the present series of experiments, we compared the effects of i.v. administration of ibogaine and noribogaine (1 and 10 mg/kg) on motor behaviors, stress hormones, and extracellular levels of dopamine (DA) and serotonin (5-HT) in the nucleus accumbens of male rats. Ibogaine caused dose-related increases in tremors, whereas noribogaine did not. Both ibogaine and noribogaine produced significant elevations in plasma corticosterone and prolactin, but ibogaine was a more potent stimulator of corticosterone secretion. Neither drug altered extracellular DA levels in the nucleus accumbens. However, both drugs increased extracellular 5-HT levels, and noribogaine was more potent in this respect. Results from in vitro experiments indicated that ibogaine and noribogaine interact with 5-HT transporters to inhibit 5-HT uptake. The present findings demonstrate that noribogaine is biologically active and undoubtedly contributes to the in vivo pharmacological profile of ibogaine in rats. Noribogaine is approximately 10 times more potent than ibogaine as an indirect 5-HT agonist. More importantly, noribogaine appears less apt to produce the adverse effects associated with ibogaine, indicating the metabolite may be a safer alternative for medication development.