Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure

Ruszkowski, M.; Strugała, Aleksander; Indyka, Paulina; Tresset, Guillaume; Figlerowicz, Marek; Urbanowicz, Anna

doi:10.1039/d1nr05650f

Cited by 8 publications

(4 citation statements)

References 42 publications

(105 reference statements)

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“…To test this hypothesis, future experiments and analysis might focus on the growth process rather than nucleation. Recently, BMV and MS2 capsids of different sizes and symmetries have been observed ( 62 – 66 ). Experiments that determine how such malformed or overgrown capsids form would shed light on the interactions that control growth.…”

Section: Discussionmentioning

confidence: 99%

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Garmann

Goldfain

Tanimoto

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA–protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)—for which RNA–protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA–protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA–protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA–protein interactions, while the growth process is driven less by RNA–protein interactions and more by protein–protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

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Section: Discussionmentioning

confidence: 99%

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Garmann

Goldfain

Tanimoto

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…To test this hypothesis, future experiments and analysis might focus on the growth process rather than nucleation. Recently, BMV, MS2, and Qb capsids of different sizes and symmetries have been observed (40)(41)(42)(43)(44)(45). Experiments that determine how such malformed or overgrown capsids form would shed light on the interactions that control growth.…”

Section: Discussionmentioning

confidence: 99%

Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles

Garmann

Goldfain²,

Tanimoto³

et al. 2022

Preprint

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Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of over 500 individual particles of brome mosaic virus (BMV)—for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed until the assembly kinetics become indistinguishable from diffusion-limited adsorption. In contrast, the time to grow a capsid after nucleation varies weakly with both salt and protein concentration. These results show that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intra-protein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.RNA viruses first inspired the term “self-assembly.” Yet much is still not understood about how even the simplest such viruses assemble or if different viruses assemble in similar ways. Theoretical models suggest many possible assembly pathways, with many different roles for the RNA, but until recently measuring these pathways has not been possible. We use a sensitive microscopy technique to follow the assembly of individual particles of BMV, a plant virus. We find evidence of an RNA-mediated nucleation-and-growth pathway that is strikingly similar to that of MS2, a bacterial virus. The last common ancestor of BMV and MS2 existed only in ancient times, suggesting that their assembly pathway might be evolutionarily conserved and other viruses might follow a similar pathway.

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“…Structure determination of flexible viruses in general is a major challenge for both NMR and X-ray crystallography. The cryo-EM approach, which also allows to obtain high-resolution 3D structures, works better with icosahedral [ 22 , 23 , 24 ] or rigid rod-shaped helical plant viruses [ 25 ], while the virions of flexible viruses have mostly been studied by low-resolution structural methods, such as various optical methods, tritium planigraphy or negative staining TEM [ 9 , 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy

Shtykova,

Dubrovin,

Ksenofontov

et al. 2024

Viruses

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The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

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Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure

Abstract: Cryo-EM reconstructions of VLPs assembled from recombinant capsid protein of brome mosaic virus reveal a few classes of VLP with either icosahedral or non-icosahedral arrangements of capsomers.

Cited by 8 publications

References 42 publications

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy

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