Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology

Abstract

Within biology, molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nanosystems that exist at the interface of living organisms and nonliving biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. Composed of self-assembling protein subunits, VLPs provide both a model for studying materials' assembly at the nanoscale and useful building blocks for materials design. The robustness and degree of understanding of many VLP structures allow for the ready use of these systems as versatile nanoparticle platforms for the conjugation of active molecules or as scaffolds for the structural organization of chemical processes. Lastly the prevalence of viruses in all domains of life has led to unique activities of VLPs in biological systems most notably the immune system. Here we discuss recent efforts to apply VLPs in a wide variety of applications with the aim of highlighting how the common structural elements of VLPs have led to their emergence as paradigms for the understanding and design of biological nanomaterials.

Keywords: Bioconjugation; Biomaterials; Biomimicry; Confined catalysis; Confined polymerization; Nanomaterials; Vaccine; Virus-like particle.

Fig. 1

Self-assembly of a virus-like particle (VLP). The final structure of a VLP is programed into the genetic sequence of a single or a few structural proteins that self-assemble to form a cage structure. Illustration was generated from PDB: 2XYY (P22 procapsid) but does not authentically reflect the assembly process of P22.

Fig. 2

VLP structures cover a range of sizes and morphologies providing a library of geometric tools for materials applications. Displayed are some of the VLPs discussed in this review with relative size scale approximately preserved except for TMV, which is shown at half scale compared to the rest of the VLPs. PDB: 2XYY, 2B2G, 1QBE, 5A33, 1ZA7, 3J6R, 1SHS, 2IY4, 3AJO, and 4UDV.

Fig. 3

In silico design can produce novel VLP structures by engineering novel subunit interfaces. Three example crystal structures of two component VLPs designed by Baker and coworkers. Cages are designated as (A) T33-15 (PDB: 4NWO), (B) T32-28 (PDB: 4NWN), and (C) T33-21 (PDB: 4NWP). In each cage, subunits are colored by subunit type (King et al., 2014).

Fig. 4

Hierarchical assembly of VLPs can be facilitated through direct VLP contacts or molecular mediators in the form of dendrimers, inorganic nanoparticles, or other VLPS. VLPs, which are themselves an assembly of subunit monomers, can be made to assemble into extended structures and under certain conditions the structures can be ordered. The use of molecular mediators is a common route toward assembly as it allows for the agglomeration of identical particles through properties such as charge.

Fig. 5

The interior space of the P22 VLP can be utilized by introducing a scaffold via atom-transfer radical polymerization. The radical initiator 2-bromoisobutyryl aminoethyl maleimide was coupled to an internal cysteine of the P22 coat protein. Polymerization of the capsid interior with 2-aminoethyl methacrylate (AEMA) introduced as many as 9000 amine sites within the intracapsid space. These sites could then be functionalized with Gd-DPTA-NCS resulting in high particle loading (Lucon et al., 2012). Figure used with permission from Lucon, J., et al., 2012. Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading. Nat. Chem. 4, 781–788.

Fig. 6

The symmetry of VLPs reflects a single change over the entire particle. (A) Nonspecific labeling of lysines in the MS2 capsid has the potential to label six sites (shown as black spheres) per subunit (left), which are reflected as 1080 total sites on both the interior and exterior of the capsid the assembled capsid (right) (Anderson et al., 2006). (B) Greater specificity can be introduced through site-directed mutagenesis such as the cysteine-containing loops inserted by Finn and coworkers into the both the large and small subunit of CPMV (asymmetric unit at left). These mutations are translated about the assembled capsid resulting in spatially precise labeling of the capsid (right) (Wang et al., 2002a). PDB: 2B2G and 5FMO.

Fig. 7

The structural similarity of BMCs and VLPs. (A) A model of the carboxysome based on subunit crystal structures and EM analysis of global structure shows a T = 75 icosahedron (740 hexamers, 12 pentamers) approximately 115 nm in diameter. (B) A cryo-EM reconstruction of the P22 VLP in the expanded form (PDB: 2XYZ) shows a T = 7 icosahedron (60 hexamers, 12 pentamers) approximately 60 nm in diameter. Images are approximately to scale. (C and D) Transmission electron micrographs showing the carboxysome and the expanded P22 VLP, respectively. Scale bars are 50 nm. Figure adapted from Tanaka, S., et al., 2008. Atomic-level models of the bacterial carboxysome shell. Science 319, 1083–1086.

Fig. 8

Statistical vs directed encapsulation. In a statistical encapsulation process (A), the capsid assembly is triggered in the presence of a cargo and some of the cargo ends up in the capsid as a function of the cargo and coat protein concentrations. In a directed encapsulation process (B), the cargo contains a specific tag or directly fused to the coat protein. The cargo associates with the coat protein prior to assembly and, ideally, triggers or directs the encapsulation process resulting in a more controlled particle formation and higher density packing.

Fig. 9

Encapsulation of genetic cargo within a VLP can be pursued using synthetic or genetic means. Direct genetic fusion of a cargo to the primary coat protein of the VLP can lead to successful encapsulation but has been shown to interrupt the assembly of the VLP. Bioconjugate approaches utilizing small-molecule cross-linkers as a means of attachment (a CLICK linker is shown) can allow for encapsulation either before or after VLP assembly but require either a reversible assembly process or a cargo that is small enough to enter the assembled VLP. Genetic or synthetic fusion of a cargo to a specific encapsulation signal such as a secondary structural protein or a VLP-specific nucleic acid tag allows for recruitment of cargo during VLP assembly. This approach is limited by the availability of such a tag and the ability to incorporate the tagged cargo into the assembly process.

Fig. 10

Encapsulation of the heterodimeric hydrogenase within the P22 VLP leads to a more than 100× increase in the turnover of the enzyme. At left a schematic of gene production in this system showing production of the two-hydrogenase subunits (red and green) as fusions to the P22 scaffold (SP yellow) and subsequent expression of the P22 coat (CP blue) allowing for folding and maturation of the cargo before encapsulation. This strategy protects and enhances the cargo as evidenced by the increase in hydrogen production shown in the reaction plot at right where the optimized encapsulated sample (red) drastically out performs the free enzyme (green) and an unoptimized encapsulated construct (blue). Figure adapted from Jordan, P.C., et al., 2016. Self-assembling biomolecular catalysts for hydrogen production. Nat. Chem. 8, 179–185.

Fig. 11

Multienzyme encapsulation in a single VLP using the P22 system. (A) An artistic representation of the CelB-GLUK-SP fusion protein encapsulated in P22VLP. P22-CP, CelB, GLUK, linker regions (PDB: 2GP8, 1UA4, 3APG, and 2XYY). (B) A negatively stained transmission electron micrograph showing P22 VLPs with the multienzyme fusion protein encapsulated. Scale bar 100 nm. (C) Maximum initial turnover of the coencapsulated enzyme construct (CelB-GLUK-P22) compared to a 1:1 stoichiometric mixture of the individually encapsulated enzymes (GLUK-P22:CelB-P22) under substrate saturating conditions for enzyme 1 (CelB). No pathway advantage is observed under normal conditions but an advantage can be induced by changing the kinetic balance. In this case the balance is altered by selectively inhibiting CelB to the same degree in coencapsulated construct and the control. Figure adapted from Patterson, D.P., Schwarz, B., Waters, R.S., Gedeon, T., Douglas, T., 2013. Encapsulation of an enzyme cascade within the bacteriophage P22 virus-like particle. ACS Chem. Biol. 9, 359–365.

Fig. 12

iBALT readily forms in the murine lung after i.n. administration of sHSP. (A) iBALT structures, indicated by arrows, emerge adjacent to airways and blood vessels after five administrations of sHSP compared to a PBS control (B). Stained fluorescence microscopy reveals that, compared to a control (C), iBALT structures contain (D) CD4+ T cells, B220+ B cells, and (E) CD21+ follicular DC. Adapted from Wiley, J.A., et al., 2009. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 4, e7142.

Fig. 13

Biomimetic encapsulation of influenza nucleoprotein can elicit CD8+-mediated immunity. (A) Nucleoprotein (NP) was encapsulated in the P22 VLP (left) via genetic fusion to the scaffolding protein (SP) mimicking the natural position of the NP in Influenza an artist's rendering of which is shown (right). (B) Mice vaccinated with NP163-P22 (the first third of the NP gene encapsulated in P22) showed improved survival after subsequent challenge with PR8 and X31 compared to the empty P22 or a PBS control. Protection could be negated by the addition of the CD8+ T cell-depleting IgG (TIB210). (C) The initial weight loss of all the immunized mice suggests that even the NP163-P22 mice initially are infected and that the mechanism of protection is not humoral. Figure adapted from Patterson, D.P., Rynda-Apple, A., Harmsen, A.L., Harmsen, A.G., Douglas, T., 2013. Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza. ACS Nano 7, 3036–3044.

Fig. 14

Polyvalent presentation of whole proteins can be pursued using a variety of strategies. Direct genetic fusion of a cargo to the primary coat protein of the VLP has been extensively pursued with small peptides but can become problematic for larger proteins with nontrivial quaternary structure. Bioconjugate approaches utilize small-molecule cross-linkers as a means of attachment (a CLICK linker is shown) allowing for attachment after the VLP has assembled but requiring processing of the VLP. Genetic fusion of a cargo to a secondary structural protein such as a decoration protein retains the appeal of genetic system while avoiding direct fusion to the primary coat protein; however, this strategy requires an available decoration protein with a stable binding interaction.

Fig. 15

Biomimetic display of influenza hemagglutinin (HA) on HpFn leads to improved protection compared to a traditional trivalent vaccine (TIV). A transmission electron micrograph showing 1999 (NC) HA genetically fused to HpFn and displayed as the native trimer at the threefold axis of the VLP. At right, a structural model is arranged to match the orientation of representative particles from the EM. Adapted from Kanekiyo, M., et al., 2013. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499, 102–106.