Imaging and manipulation of single viruses by atomic force microscopy

Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a λ-like phage of which the maturationinduced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: (i) an increase of the Young's modulus, (ii) a strong rise of the capsid's ultimate strength, and (iii) a growth of the resistance against material fatigue. The Young's modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young's modulus isn't dependent on the crosslinking between capsomers. In contrast, the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage λ, the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage λ. These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers.AFM | elastic network model | nanoindentation | normal mode analysis | virus structural mechanics H K97 is a double stranded DNA bacteriophage infecting Escherichia coli. The capsid protein of HK97 has a subunit fold that is shared by phages λ, P22, T4, and phi29, among others, as well as the eukaryotic virus, Herpes (1). In addition, the maturation pathways of these viruses show similar morphological changes (2). HK97 provides a unique model system to study capsid maturation, as large quantities of isometric particles can be produced in an E. coli expression system. These particles are expressed without the portal and terminase proteins used for genome packaging, but can be isolated as procapsids and matured in vitro using chemical agents instead of DNA, and maturation can be trapped in various expansion states (3, 4). In vitro expansion can be effectively induced with a variety of chemical agents, including lowering the pH below 4. The particles expand from the Prohead II (P-II) form, which differs from Prohead I (P-I) by the absence of the 104AA N-terminal polypeptide (termed the Δ-domain) from each subunit (gp5). The Δ-domain is presumed to function in scaffolding capsid assembly (5, 6). The Δ-domain is cleaved by an internally packaged protease (gp4) upon assembly, allowing the particle transition to the ...

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“…Before starting experiments we image the viral particles by AFM (23,24). The morphologies of the particles in these images, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Mechanics of bacteriophage maturation

Roos¹,

Gertsman

May

et al. 2012

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

107

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“…The Young moduli for a number of DNA phages (l, f29, etc.) were measured by a groundbreaking AFM nano-indentation technique, 441 with elastic constants of up to B1.8 GPa detected 442 close to that of hard plastics. A bimodal distribution of the elastic constants identified for phi29 phage has been attributed to weaker protein-protein contacts in the equatorial regions of the shell.…”

Section: B Capsid Self-assembly and Shell Elasticitymentioning

confidence: 99%

Electrostatic interactions in biological DNA-related systems

Cherstvy

2011

Phys. Chem. Chem. Phys.

164

148

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In this perspective article, we focus on recent developments in the theory of charge effects in biological DNA-related systems. The electrostatic effects on different levels of DNA organization are considered, including the DNA-DNA interactions, DNA complexation with cationic lipid membranes, DNA condensates and DNA-dense cholesteric phases, protein-DNA recognition, DNA wrapping in nucleosomes, and inter-nucleosomal interactions. For these systems, we develop a theoretical framework to describe the physical-chemical mechanisms of structure formation and anticipate some biological consequences. General biophysical principles of DNA compaction in chromatin fibers and DNA spooling inside viral capsids are discussed in the end, with emphasis on electrostatic aspects.

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“…New techniques, reagents, and methods that will further enhance the value of AFM to structural biologists are currently under development (3). Finally, the resolution of the technology and the unique insights into virus structure that it yields will be highly influenced by advances in tip acuity and improvements in instrument design.…”

Section: What Does Afm Offer the Structural Biologist?mentioning

confidence: 99%

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Kuznetsov

McPherson

2011

Microbiol Mol Biol Rev

126

101

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SUMMARY Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number ( T ). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.

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Imaging and manipulation of single viruses by atomic force microscopy

Cited by 70 publications

References 148 publications

Mechanics of bacteriophage maturation

Mechanics of bacteriophage maturation

Electrostatic interactions in biological DNA-related systems

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

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