Visualization of Single Molecules Building a Viral Capsid Protein Lattice through Stochastic Pathways

Abstract

Direct visualization of pathways followed by single molecules while they spontaneously self-assemble into supramolecular biological machines may provide fundamental knowledge to guide molecular therapeutics and the bottom-up design of nanomaterials and nanodevices. Here, high-speed atomic force microscopy is used to visualize self-assembly of the bidimensional lattice of protein molecules that constitutes the framework of the mature human immunodeficiency virus capsid. By real-time imaging of the assembly reaction, individual transient intermediates and reaction pathways followed by single molecules could be revealed. As when assembling a jigsaw puzzle, the capsid protein lattice is randomly built. Lattice patches grow independently from separate nucleation events whereby individual molecules follow different paths. Protein subunits can be added individually, while others form oligomers before joining a lattice or are occasionally removed from the latter. Direct real-time imaging of supramolecular self-assembly has revealed a complex, chaotic process involving multiple routes followed by individual molecules that are inaccessible to bulk (averaging) techniques.

Physics of viral dynamics

“…With further refinement of the existing techniques, as well as the development of new techniques, in the coming years we expect large steps in terms of understanding the dynamical properties of viruses. Examples of potential new physical virology approaches in the experimental fields include developments in optical microscopy that allow for nanometre resolution imaging 158 , or in AFM, allowing the acquisition of millisecond to microsecond height spectroscopy data 159 for real-time assembly studies 43 , 160 . Beyond using such techniques for an improved understanding of assembly and steady-state dynamics, it also would be interesting to perform assembly kinetics experiments in crowded environments, to mimic the actual situation in cells and to reveal whether depletion attraction and other crowding effects alter the results.…”

“…Assembly starts at specific sites on the surface, followed by the growth of the lattice and fusion of the different patches, eventually producing full coverage of the surface. By increasing both the spatial and temporal resolution, it is possible to identify the nature of the nucleation complex and to follow assembly in real time 43 . The necessary increase in resolution has been achieved by using high-speed AFM (HS-AFM) [44][45][46] .…”

“… In atomic force microscopy (AFM), a sharp tip scans the substrate yielding a sub-nanometre-scale topographic profile of the surface-adhered particles 182 , 183 . Recent technical advances have increased the scanning velocity by several orders of magnitude, and sub-second frame rates are now possible with high-speed AFM 43 – 46 . Optical tweezers capture dielectric, micrometre-sized beads in a focused laser beam.…”

In Vitro Assembly of Virus-Like Particles and Their Applications

“…VLP morphology and possible intermediate aggregates formed during an in-vitro assembly reaction can be visualized using transmission electron microscopy (TEM) or atomic force microscopy (AFM). TEM can also be used to distinguish between empty and encapsulated VLPs [52,59] and AFM can assess the VLP height profile [19,80,81]. A VLP high-resolution structure can be determined using Cryo-EM [52] or crystallography [82].…”

“…Different methods have been used to characterize intermediate assemblies and the assembly pathways of virus capsids, such as electron microscopy [83][84][85][86], X-ray crystallography [57], atomic force microscopy [57,58,85], small-angle X-ray scattering [87][88][89][90][91][92], mass spectrometry [93][94][95], size-exclusion chromatography [84], resistive-pulse sensing [84,96], interferometric scattering microscopy [97], single-molecule fluorescence correlation spectroscopy [98], optical tweezers in combination with confocal fluorescence microscopy and acoustic force spectroscopy [58,99]. Recently, high-speed atomic force microscopy (HS-AFM), a powerful single-molecule technique for real-time visualization of biomolecules in dynamic action [100], has been used to visualize self-assembly of HIV capsid protein lattice [81]. This physical virology technique will enable real-time capsid assembly studies of other viruses in the future.…”