Conserved structures and dynamics in 5'-proximal regions of Betacoronavirus RNA genomes.

Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.

An artificial protein cage made from a 12-membered ring.

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.

Shape-Morphing of an Artificial Protein Cage with Unusual Geometry Induced by a Single Amino Acid Change.

Artificial protein cages are constructed from multiple protein subunits. The interaction between the subunits, notably the angle formed between them, controls the geometry of the resulting cage. Here, using the artificial protein cage, "TRAP-cage", we show that a simple alteration in the position of a single amino acid responsible for Au(I)-mediated subunit-subunit interactions in the constituent ring-shaped building blocks results in a more acute dihedral angle between them. In turn, this causes a dramatic shift in the structure from a 24-ring cage with an octahedral symmetry to a 20-ring cage with a C2 symmetry. This symmetry change is accompanied by a decrease in the number of Au(I)-mediated bonds between cysteines and a concomitant change in biophysical properties of the cage.

Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles.

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.

Programmable polymorphism of a virus-like particle.

Virus-like particles (VLPs) have significant potential as artificial vaccines and drug delivery systems. The ability to control their size has wide ranging utility but achieving such controlled polymorphism using a single protein subunit is challenging as it requires altering VLP geometry. Here we achieve size control of MS2 bacteriophage VLPs via insertion of amino acid sequences in an external loop to shift morphology to significantly larger forms. The resulting VLP size and geometry is controlled by altering the length and type of the insert. Cryo electron microscopy structures of the new VLPs, in combination with a kinetic model of their assembly, show that the abundance of wild type (T = 3), T = 4, D3 and D5 symmetrical VLPs can be biased in this way. We propose a mechanism whereby the insert leads to a change in the dynamic behavior of the capsid protein dimer, affecting the interconversion between the symmetric and asymmetric conformers and thus determining VLP size and morphology.

Chemically induced protein cage assembly with programmable opening and cargo release.

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo­electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.

A single residue can modulate nanocage assembly in salt dependent ferritin.

Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.