Scaffold Properties Are a Key Determinant of the Size and Shape of Self-Assembled Virus-Derived Particles

Abstract

Controlling the geometry of self-assembly will enable a greater diversity of nanoparticles than now available. Viral capsid proteins, one starting point for investigating self-assembly, have evolved to form regular particles. The polyomavirus SV40 assembles from pentameric subunits and can encapsidate anionic cargos. On short ssRNA (≤814 nt), SV40 pentamers form 22-nmdiameter capsids. On RNA too long to fit a T=1 particle, pentamers forms strings of 22-nm particles and heterogeneous particles of 29 to 40 nm diameter. However, on dsDNA SV40 forms 50 nm particles composed of 72 pentamers. A 7.2-Å resolution cryo-EM image reconstruction of 22-nm particles shows that they are built of twelve pentamers arranged with T=1 icosahedral symmetry. At threefold vertices, pentamers each contribute to a three-helix triangle. This geometry of interaction is not seen in crystal structures of T=7 viruses and provides a structural basis for the smaller capsids. We propose that the heterogeneous particles are actually mosaics formed by combining different geometries of interaction from T=1 capsids and virions. Assembly can be trapped in novel conformations because SV40 interpentamer contacts are relatively strong. The implication is that by virtue of their large catalog of interactions, SV40 pentamers have the ability to self-assemble on and conform to a broad range of shapes. KeywordsSV40; self-assembly; capsid; scaffold; capsid assembly; virus assembly Interest in the in vitro assembled SV40 particle stems from its use as a model for selfassembly,(1, 2) for production of protein-coated nanoparticles,(3, 4) and its potential as a vector for gene and drug delivery.(5) SV40 has human tropism, transduces many cell types and organs, and infects non-dividing cells. Furthermore, SV40 is non-immunogenic(6, 7) and non-pathogenic for humans.(8-10) Recombinant SV40 capsid proteins spontaneously assemble into virus-like particles (VLPs) that package SV40 or plasmid DNA. (5,(11)(12)(13) Corresponding author: Adam Zlotnick (azlotnic@indiana.edu). § These authors contributed equally to this work Supporting InformationExperimental method details and supplementary figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Accession codesThe cryo-EM density map has been deposited to EMDataBank.org and the accession number is EMD-2375. (20,23) Under milder conditions chaperones were required for assembly of 50 nm particles,(24) which we suggest indicates a kinetic rather than thermodynamic barrier to assembly under more physiological ionic conditions. NIH Public AccessThe mechanism of in vitro assembly of nucleic acid-filled VLPs is the subject of continued investigation.(25) With SV40, short dsDNA of 600 bp yields virus-like ~50 nm particles even in the absence of chaperones.(26) Short RNA molecules, up to 814 nt long, led exclusively to assembly of 22 nm particles composed of twelve pentamers.(27) These data suggest that nucleic acid serves as a scaffold and also a nucleating fac...

Mechanisms of Virus Assembly

“…To understand the effect of perturbing parameters from these optimal values, in vitro assembly products were characterized by electron microscopy (e.g. (76, 78, 98, 108, 109)) or SAXS (16, 17) as functions of subunit and RNA concentrations, subunit-subunit interactions (controlled by pH, ionic strength, and protein sequence), and subunit-polymer interactions (controlled by ionic strength and protein-RNA binding domains). In addition, several groups have performed Brownian dynamics simulations in which coarse-grained triangular or pentameric subunits assemble around flexible polyelectrolytes (49, 50, 99, 110–112), semi-flexible polyelectrolytes (112), or model NAs (99).…”

“…5D), triplets, and quadruplets. An early study likely also observed doublets, although the structures could not be confirmed (72) and recently doublet dodedecadron capsids were observed for assembly of SV40 capsid proteins around certain lengths of RNA (17). Simulations independently predicted the formation of doublets around polyelectrolytes with about twice the optimal length (49, 99, 114) (Fig.…”

Cryo‐electron microscopy and cryo‐electron tomography of nanoparticles

“…Virus‐based nanoparticles and virus‐like particles (VLPs) that lack the viral genetic material required for replication are potential platforms for vaccine design . Simian virus 40 (SV40), which is a polyomavirus found in both monkeys and humans, can be induced to form differently sized icosahedral particles depending on the size and composition of the nucleic acid component (DNA or RNA) . Assembly assays with the SV40 VP1 protein and short (1.9 knt) ssRNA molecules produce 22 nm diameter VLPs that are smaller than mature virions.…”

“…(a) Protein‐RNA nanoparticles (T = 1 polyomavirus SV40 VP1 virus‐like particles) 22 nm in diameters (arrows) and larger assemblies, scale bar, 50 nm. (Reprinted with permission from Ref . Copyright 2013 American Chemical Society) (b) Lipid‐based hexosome nanoparticles, scale bar, 50 nm.…”

Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry

“…In vitro, SV40, and other polyomaviruses, VP1 pentamers can assemble into a broad array of structures from 20 nm T=1 capsids to 40 nm T=7 capsids to 40 nm diameter tubes 19, 20 . Different nucleic acid substrates yield a similar range of structures with relatively stiff dsDNA favoring T=7 particles 21, 22 . On ssRNA substrates, VP1 forms uniform 22 nm diameter T=1 virus-like particles (VLPs) under mild solution conditions (50 mM MOPS, pH 7.2, 125 mM NaCl) in the absence of cellular or external factors 23 .…”