Design and self-assembly of simple coat proteins for artificial viruses

Hernández-García, Armando; Kraft, Daniela J.; Janssen, Anne; Bomans, Paul H. H.; Sommerdijk, Nico A. J. M.; Thies‐Weesie, Dominique M. E.; Favretto, Marco E.; Brock, Roland; Wolf, Frits A. de; Werten, Marc W. T.; Schoot, Paul van der; Stuart, Martien Cohen; Vries, Renko de

doi:10.1038/nnano.2014.169

Cited by 153 publications

(261 citation statements)

References 30 publications

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“…We study the formation of capsids from a recombinant coat protein inspired by the structure of the tobacco mosaic virus (TMV), whose design and production is described in detail elsewhere 7 and in the Supporting Information. Coat proteins of the TMV feature three distinct functionalities: 8 (I) a hydrophilic domain that protects the capsid and its cargo against aggregation, misfolding, and enzymatic attack, (II) a binding domain with high affinity for the nucleic acids, and (III) a domain that, when folded, provides a specific attraction between neighboring capsid proteins.…”

Section: Resultsmentioning

confidence: 99%

Illuminating the Reaction Pathways of Viromimetic Assembly

et al. 2017

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The coassembly of well-defined biological nanostructures relies on a delicate balance between attractive and repulsive interactions between biomolecular building blocks. Viral capsids are a prototypical example, where coat proteins exhibit not only self-interactions but also interact with the cargo they encapsulate. In nature, the balance between antagonistic and synergistic interactions has evolved to avoid kinetic trapping and polymorphism. To date, it has remained a major challenge to experimentally disentangle the complex kinetic reaction pathways that underlie successful coassembly of biomolecular building blocks in a noninvasive approach with high temporal resolution. Here we show how macromolecular force sensors, acting as a genome proxy, allow us to probe the pathways through which a viromimetic protein forms capsids. We uncover the complex multistage process of capsid assembly, which involves recruitment and complexation, followed by allosteric growth of the proteinaceous coat. Under certain conditions, the single-genome particles condense into capsids containing multiple copies of the template. Finally, we derive a theoretical model that quantitatively describes the kinetics of recruitment and growth. These results shed new light on the origins of the pathway complexity in biomolecular coassembly.

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Section: Resultsmentioning

confidence: 99%

Illuminating the Reaction Pathways of Viromimetic Assembly

et al. 2017

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“…The thickness and width of the core, as obtained from SAXS data, would seem compatible with some sort of solenoid configuration. We also studied an uncharged variant with X = Q, which also showed a strong self-assembling tendency; when conjugated with a cationic binding block and a random coil, it assembled with DNA in a manner reminiscent of rod-like viruses 13 .…”

Section: Introductionmentioning

confidence: 99%

Dock ‘n roll: folding of a silk-inspired polypeptide into an amyloid-like beta solenoid

2016

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Polypeptides containing the motif ((GA)mGX)n occur in silk (we refer to them as ‘silk-like’) and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II’ β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for molecules to reach the folded state.

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“…virus-like particle | engineered nanoparticles | disulfide stabilization | hepatitis core protein | cell-free protein synthesis V irus-like particles (VLPs) are probably the most precisely defined and, therefore, potentially the most useful complex nanometer-scale scaffolds (1). VLPs mimic the capsid structure of real viruses, but lack infectious genetic material.…”

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Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery

Chan

et al. 2015

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

119

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Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.virus-like particle | engineered nanoparticles | disulfide stabilization | hepatitis core protein | cell-free protein synthesis V irus-like particles (VLPs) are probably the most precisely defined and, therefore, potentially the most useful complex nanometer-scale scaffolds (1). VLPs mimic the capsid structure of real viruses, but lack infectious genetic material. Selected VLPs derived from pathogens have already provided major advances in the development of vaccines that have known and relatively homogeneous structures as well as enhanced immunogenicity (2). Such nanoparticles provide comparable cellular uptake and intracellular trafficking compared with natural viruses (3), and also have repetitive surfaces for the high-density display of vaccine antigens (4). In addition, VLPs offer favorable trafficking from the injection site to lymph nodes (5). Since the first reported use of a hepatitis B core protein (HBc) VLP as an antigen carrier in 1987 (6), at least 110 VLP vaccine candidates have been constructed by using capsid proteins from 35 different viral families (7).Among different types of VLPs, the HBc VLP is the most flexible and promising model for fundamental and applied immunological studies (8). One advantage of the HBc VLP platform is that the capsids can be produ...

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Design and self-assembly of simple coat proteins for artificial viruses

Cited by 153 publications

References 30 publications

Illuminating the Reaction Pathways of Viromimetic Assembly

Illuminating the Reaction Pathways of Viromimetic Assembly

Dock ‘n roll: folding of a silk-inspired polypeptide into an amyloid-like beta solenoid

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery

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