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

Abstract

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.

Amino Acid Side Chains Buried along Intersubunit Interfaces in a Viral Capsid Preserve Low Mechanical Stiffness Associated with Virus Infectivity

“…This result is consistent with the established fact that a conformational rearrangement of the trimeric building blocks is required for MVM capsid assembly. 54,67 Stiffening the trimers and the capsid being assembled from them, 69 as reflected in stiffening the S3 region at the center of each trimer and S2 regions at their borders, may likely impair such conformational rearrangement.…”

Structural determinants of mechanical resistance against breakage of a virus-based protein nanoparticle at a resolution of single amino acids

“…These versions differ in the length of their structurally disordered N-terminal segments (Nt), which do not contribute to capsid assembly or architecture. VP2-only MVM capsids, structurally indistinguishable from natural MVM capsids, can be assembled both ex vivo 59 and in vitro, 60 and have been used in this study.…”

“…Trimers of protein monomers constitute stable capsid building blocks (CBBs) from which the MVM capsid is assembled [60][61][62][63] (Fig. 1).…”

“…51,52 The MVM capsid ( Fig. 1A and B), one of the smallest (25 nm in diameter) and structurally simplest viral NPs known, is made of a minimum number (60) of structurally equivalent protein monomers that have the same sequence and fold. There are no conformational switches related to quasi-equivalence that occur in icosahedral capsids made of more than 60 subunits.…”

Real-time assembly of an artificial virus elucidated at the single-particle level

“…Single molecule techniques such as electron microscopy and atomic force microscopy (AFM) provide for high resolution images of viruses, but typically yield images with only limited information on viral dynamics (16,17). High resolution AFM imaging and electron microscopy have recently shown transient capsid intermediates, from which assembly kinetic parameters can be estimated (18). New approaches such as resistive-pulse sensing ,optical tweezers and acoustic force spectroscopy have been reported to study the assembly of the icosahedral viruses Hepatitis B Virus and SV40 virus (19,20).…”