Virus-Inspired Function in Engineered Protein Cages | Journal of the

Perspective Cite This: J. Am. Chem. Soc. 2019, 141, 9432−9443

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Virus-Inspired Function in Engineered Protein Cages Thomas G. W. Edwardson and Donald Hilvert*

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Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland that developing our own analogous nanomachines could solve practical challenges within all branches of science and technology. Due to the simple programmability of nucleic acid self-assembly, an abundance of artificial DNA and RNA structures and nanodevices has been created in the past few decades.1 Although it was believed that the folding complexity of polypeptides would hamper de novo design of protein nanotechnology,2 a combination of computational advances, protein engineering, and directed evolution has produced many novel and functional structures.3−10 In this Perspective, we focus on the engineering and evolution of self-assembling protein compartments.11−17 Specifically, the exploitation of nonviral protein cages for the capture, transport, and release of nucleic acids will be discussed, both in the context of viral evolution and as platforms for advanced applications in biotechnology and medicine.

ABSTRACT: The structural and functional diversity of proteins combined with their genetic programmability has made them indispensable modern materials. Well-defined, hollow protein capsules have proven to be particularly useful due to their ability to compartmentalize macromolecules and chemical processes. To this end, viral capsids are common scaffolds and have been successfully repurposed to produce a suite of practical protein-based nanotechnologies. Recently, the recapitulation of viromimetic function in protein cages of nonviral origin has emerged as a strategy to both complement physical studies of natural viruses and produce useful scaffolds for diverse applications. In this perspective, we review recent progress toward generation of virus-like behavior in nonviral protein cages through rational engineering and directed evolution. These artificial systems can aid our understanding of the emergence of viruses from existing cellular components, as well as provide alternative approaches to tackle current problems, and open up new opportunities, in medicine and biotechnology.

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VIRUSES Viruses are the most abundant biological entities on our planet, with an estimated population of >1031 particles.18,19 We are now beginning to understand the importance of the interactions between viruses and their hosts across all domains (archea, bacteria, eukarya) in the context of complex life and its evolution.20 For example, viral genes constitute much of the “non-coding” human genome, equating to more than five times the number of protein coding genes.21 Sequence and structural comparisons have also revealed that viruses have provided genes coding for important functional proteins in the course of natural evolution.22−24 Moreover, homologues of viral fusion proteins have been observed in eukaryotes, where they play an important role in cell−cell fusion.25 These findings in combination with the high percentage of the human genome that is derived from retroviral sequences identify viruses as potential drivers of divergence.26,27 Within the biosphere, viruses play an important evolutionary role by providing an ongoing source of selection pressure.28,29 The influence of viruses on human health cannot be overstated, with both historical pandemics and recent outbreaks of deadly strains highlighting our vulnerability to these pathogens.30−37 As such, bettering our understanding of viruses toward the prevention and treatment of infectious disease is a vital area of research.38−43 However, beyond this context, viruses also provide ideal systems to study the mechanistic principles of biomolecular self-assembly and molecular evolution,44−48 probe cell biology,49,50 and serve as platforms for the development of nanotechnology.51−57 Nevertheless, the origin of viruses remains a contentious issue, and there are various explanations for both their

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INTRODUCTION The ability of chemists to create synthetic molecules contributed greatly to the rapid technological development seen in the 20th century. Indeed, Earth’s human population now relies heavily on the fertilizers, plastics, and drugs made available through advances in chemical methodology. The second half of last century is notable for the structural elucidation of biomacromolecules, giving birth to the fields of biochemistry and molecular biology. Today, combining this knowledge with clever synthetic methods that produce sequence-defined biopolymers has enabled the current generation of chemists to tinker with the components of life itself. Molecular engineering of polynucleotides, polypeptides, and polysaccharides is an ever growing and important facet of the chemical sciences. Developing our capacity to engineer these information-rich and functional biopolymers is extremely powerful as it allows us to imitate, interface, interrogate, and, ultimately, influence biological processes. These capabilities have obvious implications for the diagnosis and treatment of disease, and much effort is focused in this direction. However, the myriad structural and functional roles served by biopolymers in nature hints that imagination and ambition are the only limiting factors on the broad impact of biomolecular technology. Proteins and RNA embody nature’s functional diversity and are constituents of many biological machines. Considering the variety of tasks natural proteins perform, it stands to reason © 2019 American Chemical Society

Received: April 6, 2019 Published: May 22, 2019 9432

DOI: 10.1021/jacs.9b03705 J. Am. Chem. Soc. 2019, 141, 9432−9443

Perspective

Journal of the American Chemical Society

Figure 1. (a) Backbone representations of AaLS-wt, AaLS-neg, and AaLS-13 monomers. Mutations introduced by engineering or evolution are shown as colored spheres. (b) Surface models of the three corresponding AaLS capsids show the very different sizes and geometries, which all assemble from pentameric capsomers. Red surfaces indicate the highly negatively charged lumen of the AaLS-neg and AaLS-13 capsids, which enables the efficient encapsulation of positively charged guest molecules. (c) Negative stain transmission electron micrographs of the three AaLS capsids. PDB IDs: 1HQK (AaLS-wt), 5MQ3 (AaLS-neg), and 5MQ7 (AaLS-13).

Viruses are arguably more diverse in their molecular biology than living systems, as they possess distinct RNA or DNA genomes and mechanisms of replication. However, all viruses share some features, such as lack of active metabolism and their own translational machinery. Another unifying characteristic is the use of a proteinaceous capsid to contain and protect the genome.80

emergence and importance in the context of evolutionary biology.58,59 There are currently three distinct hypotheses considered for the origin of viruses: (1) Viruses are examples of prebiotic selfreplicators.60−63 Considering the enzymatic activity of certain RNA sequences and their ability to duplicate,64 this model fits with the RNA world concept and is a viable explanation of how virus-like agents may have existed before the complexity of modern molecular biology.65,66 (2) Viruses represent genetic elements that “escaped” from their cognate organism and transformed into self-replicating entities by means of a transport vector.67 Satellite viruses with an ssRNA genome encoding only their capsid protein provide an example of how such simple virions may have looked.68 (3) Viruses are a regression from more complex living systems. In this case, it is likely that symbionts gradually evolved to become obligate parasites. Here, large nucleocytoplasmic DNA viruses are a convincing example, as their complexity blurs the lines between viruses and microorganisms.69,70 Of course, it is possible, and perhaps even likely, that a combination of these evolutionary routes resulted in the diversity of viruses that we see today.71,72 Due to implications for the origin of life as a whole,73 RNA sequences capable of self-replication have been extensively studied,74−77 supporting a virus-first scenario. However, until recently, key steps in the progressive and regressive hypotheses had not been tested in the laboratory.78,79 An experimental approach to test the progressive hypothesis is to use our knowledge of protein engineering to recapitulate certain aspects of the viral life cycle that may permit the emergence of self-replicating entities. Importantly, these mechanisms can then be understood and integrated in a controlled manner to be used in biomolecular technology.

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RNA PACKAGING IN VIVO Beyond viruses, proteinaceous compartments are found serving other purposes across the tree of life. These capsules are wellsuited to store and transport cargo, and they can be found harboring very different materials. For example, ferritins carry and control the release of metal ions,81 while gas vesicles are filled with air to control buoyancy in some bacteria.82 Another important class of protein containers are bacterial microcompartments, which are enzyme-containing protein-bounded organelles responsible for specific metabolic processes in prokaryotes.83,84 This natural diversity of function has inspired efforts to exploit protein compartments for our own purposes.85,86 To date, virus-like particles,87−91 ferritins,92−97 heat shock proteins,98−100 molecular chaperones,101−103 gas vesicles,104 vault ribonucleoproteins,105,106 and bacterial microcompartments107−110 have all been engineered and repurposed to create customized containers. More recently, wholly artificial protein cages, obtained through computational design, have also emerged as useful scaffolds.111−115 The cage-forming lumazine synthase from Aquifex aeolicus (AaLS) has proven to be a valuable starting point from which to engineer and evolve unique functional assemblies.17 Although the AaLS protein has a flavodoxin-like fold and neither sequence nor structure similarity to any known virus,116 it forms a virus-like icosahedral assembly, which is 9433


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These findings showcase how easily a protein capsule can be driven to exhibit basic virus-like behavior. A crucial property is the multimeric nature of the protein cage itself, which effectively amplifies any mutations gained by the individual protein. This is of important consequence to non-specific host−guest interactions, permitting rapid gain-of-function via minimal mutation and thus aiding evolution. Alongside accumulating point mutations, proteins can also evolve through fusion to entirely new domains at the genetic level. In many ssRNA viruses, capsid proteins bind their genome through terminal nucleic acid binding domains (NBDs).134−136 These lumenally presented arginine- and lysine-rich peptides play a crucial role in promoting the self-assembly of the complete virion, as the long RNA acts to nucleate relatively weakly interacting capsid proteins and stabilize the capsid.137−139 Inspired by this common motif, Azuma et al. developed a modular in vivo RNA packaging system using a circularly permuted variant of the AaLS protein.140 In wild-type AaLS capsids, both N- and C-termini are presented on the outer surface. This is beneficial for external modification141−144 but prohibits extensive changes to the internal cavity without affecting overall capsid structure. To overcome this limitation, circular permutation was employed to relocate the protein termini to the lumenal side, providing a variant, called cpAaLS, which can present genetically fused peptides to the interior cavity (Figure 3a).129 Exploiting the potential modularity of this design, five model RNA-binding peptides were trialed for their ability to recruit endogenous RNA upon production in E. coli (Figure 3b).140 Moreover, by means of a dual plasmid system using different inducers for the cpAaLS-NBD and wild-type AaLS proteins, patchwork capsids with defined ratios of each component could be produced (Figure 3c). Such control over the number and design of the NBDs presented enabled an investigation into the relationships between capsid charge, cavity volume, and the distribution of packaged RNA cargo. This study demonstrated that acquiring terminal peptides containing at least six arginines enabled efficient RNA packaging in vivo.140 Deep sequencing revealed that almost 70% of the encapsulated RNA originated from the plasmid-based expression system (unpublished data), consistent with the use of strong promotors for the expression of the capsid genes. Moreover, it was found that both overall lumenal charge and available cavity volume are critical factors that determine the maximal length of guest RNA encapsidated. The charge dependency is consistent with studies on ssRNA viruses,134,145−147 which show that genome length is linearly proportional to capsid charge. Unlike the RNA-length-dependent capsid morphologies observed in some viruses,146,148 AaLS:cpAaLS-NBD capsids have a strong preference for the T = 1 state. This imposes a strict limit on the amount of nucleic acid packaged due to available cavity volume. Indeed, deviation from optimal RNANBD charge compensation was observed upon the addition of uncharged linker peptides that fill up the lumen with steric bulk (Figure 3d).140 In many viruses, the relative free energies of capsomer− capsomer and capsomer−RNA interactions are crucial factors for concomitant genome packaging and virion assembly.138 By altering these relative affinities, both RNA packaging and capsid assembly efficiency are affected. Using two-component capsids, the relative capsomer−RNA affinity can be tuned by altering the ratio of AaLS to cpAaLS-NBD. Interestingly,

classified as T (triangulation number) = 1 by Caspar−Klug theory (Figure 1).117 In its natural role, the 60-mer cage encapsulates cognate riboflavin synthase via a short recognition peptide,118 forming an active complex that catalyzes the final two steps of riboflavin biosynthesis. Exploiting and improving upon this natural encapsulation capacity through a combination of protein engineering and directed evolution has afforded both efficient encapsulation systems and structural diversification.119−122 The resulting artificial compartments have been employed as proteasome-inspired nanoreactors,123 carboxysome mimics,124 templates for enzyme-mediated polymer synthesis,125 and model systems for investigations on the mechanics and self-assembly of porous protein capsules.126−128 The morphological plasticity of AaLS has been demonstrated through unprecedented structural changes observed in different variants of the protein,122,129 resulting in a family of unique protein cages based on similar pentameric capsomers (Figure 1). By providing ready access to new structure and function, such malleability may foster divergent evolution.130 For example, could such a protein compartment be directed to exhibit completely new, specifically viromimetic, function? All viruses rely on their host cell’s translational machinery for replication. However, the recognition, packaging, and protection of the viral genome are crucial steps in the life cycle of a virus that must be encoded within the virion components themselves. Modern viruses employ different strategies to selectively package the correct genome within their protein capsid.131 These range from the elaborate packaging motors of bacteriophages to sequence-specific and structure-dependent RNA-protein self-assembly.132 However, for the emergence of virus-like particles from naı̈ve starting scaffolds, it is likely that simpler intermediate steps must first be made. The AaLS capsids have proven to be useful scaffolds for investigating how simple nucleic encapsulation systems can be generated from nonviral proteins. For example, using wild-type AaLS as a starting point, Woycechowsky and co-workers showed that nucleic acid encapsulation could be achieved by introducing four mutations (T86R, D90N, T120R, and E122R) per monomer (Figure 2).133 These changes converted

Figure 2. (a) Four mutations were introduced to AaLS-wt to produce the variant AaLS-pos. (b) AaLS-pos co-assembles with RNA in vivo and protects its cargo from nuclease digestion. The capsid retains the same 16 nm diameter as its parent AaLS-wt and encapsulates RNAs of up to approximately 350 nt in length. PDB ID: 1HQK (AaLS-wt).

the overall negative charge (−60 e) of the capsid lumen to a highly positive charge (+240 e). Expression of the AaLS-pos mutant in Escherichia coli afforded capsids containing a mixture of RNAs with lengths up to 350 nucleotides. The resulting capsids retained their structural integrity and were able to protect their cargo from nuclease digestion, an important role for any viral capsid. 9434


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Figure 3. (a) Circular permutation of AaLS-wt and genetic fusion of nucleic acid binding domains (NBDs) to the new N-terminus afforded cpAaLS-NBD capsids. (b) Several different NBDs were tested to study the effect of charge and size on RNA encapsulation. (c) Co-expression of cpAaLS-NBD and AaLS-wt proteins provides patchwork capsids that encapsidate RNA upon assembly in vivo. (d) Patchwork capsids with 1:1 ratios of the two proteins have the same 16 nm external diameter. However, increasing the steric bulk of the NBDs decreases the capsid cavity volume and favors the packaging of shorter RNAs. PDB ID: 1HQK (AaLS-wt).

assemblies with higher cpAaLS-NBD content contained a population of larger capsids, although no apparent increase in maximal RNA length was observed. This effect may be due in part to strengthening the average capsomer−RNA interaction or to the interplay of charge compensation and available cavity volume, or may simply reflect the morphological preference of the cpAaLS protein, which is known to form both larger spheres and rods.129 Overall, this study shows how a conceptually simple model system can be used to probe complex relationships between carrier, cargo, and their coassembly.

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NUCLEOCAPSID EVOLUTION The immediate gain-of-function obtained by positive charging of nonviral protein cage lumen reveals a possible pathway for viruses to arise from suitable starting proteins. Moreover, the absolute size limitations imposed by the protein cage highlight an important criterion for the emergence of an infectious particle: its protein shell must have, or be capable of adopting, a large enough size to encapsulate its own coding genes. Due to structural plasticity, large protein cages can be obtained through rational engineering and directed evolution of smaller starting scaffolds.122,129 Alternatively, computational design provides a powerful means to generate artificial protein containers with large internal volumes.149,150 Recently, these approaches were exploited in two independent studies to afford nonviral protein compartments capable of encapsulating their own RNA genome in bacterial cells.78,79 Remarkably, these investigations demonstrate that virus-like behavior can emerge from innocuous starting scaffolds with relative ease. Butterfield et al. used artificial two-component icosahedra as a starting point to create synthetic nucleocapsids.78 These ca. 30 nm diameter “I53” capsids are comprised of a natural pentameric lumazine synthase from Mesorhizobium loti and a trimeric KDPG aldolase from Thermotoga maritima. However, the capsomer−capsomer interfaces were re-engineered to promote co-assembly into the artificial architecture (Figure 4a).149 Beyond the I53 capsids, this interface design approach has successfully produced other novel assemblies of varying size, geometry, and porosity.149−152

Figure 4. Two starting points for nucleocapsid evolution. (a) Design model for the I53-50 capsid showing pentamers (blue) and trimers (light blue). (b) Co-expressed pentamer- and trimer-forming proteins bearing positive charge co-assembled with capsid mRNA to produce nucleocapsids in vivo. (c) Circular permutation and N-terminal fusion of the λN+ peptide afforded λcpAaLS. (d) In vivo, λcpAaLS capsids assembled with short fragments of RNA, many of which contain BoxB tags. PDB IDs: 1HQK (AaLS-wt) and 1QFQ (λN peptide); I53-50 design model obtained from ref 149.

To drive RNA encapsidation in I53 cages, positively charged amino acids were introduced on the lumenal surface of both the trimer and pentamer capsomers.78 This immediately enabled the protein capsules to encapsulate their own encoding mRNA, albeit with low efficiency (Figure 4b). It is likely that strong overexpression of the requisite genes played an important role in driving packaging, providing many more copies of the capsid mRNA than other endogenous sequences. This is corroborated by analysis of RNA cargo in AaLS-based viral mimics.79,140 Together these findings suggest that transcriptional efficiency could significantly impact virion 9435


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increase in capsid volume or charge over the evolutionary trajectory, this increase in cargo length can be attributed to improved protection. Indeed, the best variants could protect their RNA cargo from digestion by RNase A, a small enzyme that very efficiently catalyzes the hydrolysis of 5′-3′ phosphodiester bonds in RNA. Upon production in E. coli, more than 10% of the most evolved capsids contained their own encoding full-length mRNA, which is comparable to some recombinantly produced natural viruses.162 Through similar rounds of mutagenesis and screening, the positively charged I53 variants also evolved into nucleocapsids, of which 1 out of every 11 packaged its own full-length RNA genome.78 Like λcpAaLS, little change in overall capsid size was observed over the course of evolution. However, based on the design model and deep mutational scanning data,163 the authors were able to show that residues lining the pores on the capsid surface were crucial for cargo protection (Figure 6a).

formation, in terms of both capsid protein production and cognate mRNA loading, without the need for sequence-specific protein−RNA interactions. Terasaka et al. employed a different strategy to generate artificial nucleocapsids capable of packaging their own coding mRNA.79 Exploiting a known protein−RNA interaction, a circularly permuted variant of AaLS was equipped with the λN+ peptide derived from bacteriophage lambda,215 giving the protein λcpAaLS (Figure 4c). The λN+ peptide recognizes a specific RNA hairpin, called BoxB, which allows selective packaging of BoxB-tagged mRNA within capsids presenting the λN+ peptide on their lumenal surface. Although the addition of the λN+ peptide caused protein cage expansion to diameters similar to I53 cages (27−29 nm) and the constituent mRNA displaying BoxB tags was highly overexpressed, only short fragments of RNA were encapsulated in this initial design (Figure 4d). Furthermore, λcpAaLS capsids present more positive charge and have shorter mRNA than their similarly sized I53 counterparts. Clearly other protein cage characteristics, such as stability, dynamics, and porosity, are also important to achieve efficient packaging and protection of mRNA. Compartmentalization enables Darwinian evolution by tethering genotype and phenotype. Ubiquitous in nature, this concept has been re-imagined and successfully applied in the laboratory for the generation of novel genetically encoded molecules, permitting the discovery of hard-to-predict mutations that give rise to useful properties.153−156 For this purpose, practical compartments range in scale and substance: from laboratory glassware or microfluidic droplets to singlecelled organisms or even individual virions.157−161 Thus, nonviral protein cages that encapsulate their own mRNA are a welcome complement to the current systems available for directed evolution. To investigate whether more virus-like characteristics could be generated from their artificial starting points, both I53 and λcpAaLS were subjected to laboratory evolution to improve packaging efficiency and cargo protection (Figure 5). In the case of λcpAaLS, error-prone PCR was used to introduce random mutations across the capsid mRNA.79 After only two rounds of mutagenesis combined with increasingly stringent in vivo selection and subsequent in vitro screening, the packaging capacity of λcpAaLS capsids was increased from ∼500 to ∼2000 nt per capsid. As there was no significant

Figure 6. (a) Model of the I53-50 capsid showing the surface electrostatics of the pore region before (top) and after (bottom) laboratory evolution, during which positively charged residues lining the pore were depleted. (b) Beneficial mutations introduced to the λcpAaLS protein over two rounds of evolution, many of which appear to disrupt the hydrophobic core. PDB IDs: 1HQK (AaLS-wt) and 1QFQ (λN peptide); the I53-50 design model and mutations were obtained from ref 149.

This finding is in contrast to λcpAaLS capsids, where beneficial mutations were distributed across the protein, even within the hydrophobic core (Figure 6b). This difference highlights how the choice of starting scaffold dictates possible evolutionary routes to successful structural adaptation. In this light, it is possible that proteins with high plasticity even favor divergent evolution. For example, viruses of the order Mononegavirales,164 which assume vastly different structures based on the same related protein, exemplify how structural diversification may proceed through a promiscuous intermediate.165 Our views of virion structure and assembly principles are historically protein-centric, mostly due to the lack of highresolution structural data for packaged genomes. However, recent studies have provided thought-provoking insights into the roles that viral genomes play in the assembly pathways of virions in host cells.136,139 Like most ssRNA viruses, the formation of artificial nucleocapsids requires co-assembly of RNA and protein components. As such, the effect of accrued mutations on mRNA secondary structure can affect both assembly efficiency and cargo loading selectivity. For instance, reversion of silent mutations accumulated during evolution on the λcpAaLS capsid mRNA completely abolished genome packaging without affecting capsid structure.79 Moreover, calculations suggested that these silent mutations improved RNA compactness,166 which is a known characteristic of ssRNA viral genomes.167 These findings demonstrate how the

Figure 5. General scheme for the laboratory evolution of nucleocapsids, which was successfully applied to generate I53 and λcpAaLS-based nucleocapsids that encapsulate their own RNA genomes. 9436


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Journal of the American Chemical Society co-evolution of RNA and protein within artificial systems sheds light on the interplay of these components and their effect on the structure and assembly of nucleocapsids.

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VIRUS-INSPIRED DELIVERY SYSTEMS Imitation of virion self-assembly and genome packaging in designer systems can provide new tools for in-cell RNA sampling, trapping of unstable intermediates, or novel RNA expression systems.140,168,169 However, these are not the only steps of the viral life cycle that can be exploited for the development of valuable technologies; molecular delivery is an area where viruses are especially instructive. Viral particles circulate in diverse biological environments, enter specific cell types, and release their payloads to specific subcellular compartments as a means to replication.80 Moreover, viruses achieve this through frugal design using minimal genetic information. Whether directly or indirectly inspired by these features,170−173 nanomedicine platforms aspire to such functional elegance.174−177 In fact, some viruses are used directly for therapeutic applications,178−180 and viral vectors are at the forefront of gene therapy.181 Virus-like particles have also been applied in proof-of-principle studies for the delivery of nucleic acids, such as antisense oligonucleotides,182 CRISPR guide RNA,183 and short interfering RNA (siRNA).168,184−186 An exciting prospect emerging from the area of artificial protein cages is the engineering of assemblies that recapitulate virus-like behavior but through alternative molecular mechanisms. These materials provide new opportunities and alternative solutions to important problems in biotechnology and medicine.174,187 We recently reported the first example of a nonviral protein cage capable of delivering oligonucleotides to mammalian cells to efficiently modulate gene expression.113 Starting from the O3-33 scaffold, which was computationally designed by Baker and co-workers,151 arginine mutations were introduced to the lumenal surface to afford a porous protein cage, called OP, with a highly positively charged interior cavity (Figure 7a,b). After isolation from E. coli, the OP protein is easily obtained as an empty cage, which encapsulates short oligonucleotides with high efficiency and affinity through its six surface pores. Similar to the rapid functionality gained by positive charging of the AaLS and I53 capsids, minimal mutation of a non-functional scaffold enabled significant biological activity. The OP cage is able to deliver siRNA cargo to mammalian cells and efficiently knock down a constitutively expressed protein through RNA interference (Figure 7c).113 This biological activity raises mechanistic questions, as the siRNA molecule must be delivered to the cytoplasm, requiring the protein cage to imitate a virion and overcome the many physical barriers in its path.188,189 Viruses employ myriad mechanisms for efficient cell entry190 and have co-evolved with their host’s physiology to take advantage of cellular properties and processes. Endocytosis is one such process that is exploited by many viruses to enter target cells.191 However, to successfully take advantage of this pathway, viral particles must also be able to escape from the endosomal compartment before eventual degradation. In the case of enveloped viruses, fusion of their encasing membrane with the endosomal membrane provides a means to release the viral capsid into the cytoplasm.192 For non-enveloped viruses there are two distinct mechanisms: formation of a pore through which the genome can be released into the cytosol, or wholesale disruption of the endosome to release the capsid.193

Figure 7. (a) Surface structure of the OP cage assembly, with blue highlighting the positively charged lumen. (b) OP monomer showing the six arginine mutations introduced with respect to the original O333 scaffold. (c) OP cages carrying GFP-targeting siRNA (OP:siRNA) knock down protein expression to the same degree as the commercial reagent lipofectamine (Lipo:siRNA). PBS buffer, naked siRNA, and OP carrying a scrambled sequence siRNA (OP:scramble) have no effect on GFP signal. OP cages lacking His6 tags (No His6) are less active. (d) While stable in the absence of other oligonucleotides, high concentrations of tRNA can displace the siRNA cargo from OP cages, freeing them to induce RNA interference. PDB ID: 6FDB (OP). Panel (c) adapted from ref 113.

The siRNA-delivering OP cage is not enveloped, nor is it known to have the capacity to form pores in the endosomal membrane. This suggests that a lysis-type endosomal escape mechanism is in operation. Although the exact mechanism has not yet been elucidated, the available data provide a plausible explanation based on the multiple histidine residues presented on the cage surface. The common appearance of histidine in the pH-dependent conformational switches of viral fusion machinery is a prime example of the co-evolution of host and parasite, as the sidechain pKa of histidine (6.0) is well matched to the acidification that occurs during endosomal maturation.194,195 This property also facilitates endosomal escape of histidine-rich peptides, due to both the proton sponge effect and the ability of protonated histidine to permeabilize endosomal membranes.196−200 The OP protein contains a C-terminal hexahistidine tag that is presented on the outer surface of the cage assembly. Like viruses, the polyvalent nature of the OP assembly amplifies the number of displayed histidines to a total of 144 residues on the exterior of each capsid. The importance of these histidine residues for effective gene knockdown was confirmed by the lower activity of an OP variant bearing no hexahistidine tags (Figure 7c).113 The importance of the histidine tags for other steps of siRNA delivery is still unknown, but this mechanism could provide a general approach to furnish protein cages with the ability to circumvent endosomal trapping. The final and crucial step to host cell infection is release of the viral genome from its protective capsid. Viruses have evolved to respond to a variety of specific cellular factors to induce uncoating,201,202 including conformational changes upon protein binding, enzymatic processing, chemical cues, and mechanical forces.203 Due to the porous nature of the artificial OP assembly, uncoating is not necessary, as the cargo 9437


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the added advantage of being able to steer the course toward solutions to our own scientific and technological problems.

can be released from cages with some degree of spatial control. Although the siRNA cargo is tightly bound within the OP lumen (Kd < 1 nM), it can be displaced in the presence of a threshold concentration of competitor nucleic acid (Figure 7d).113 The cytoplasm meets this criterion, as it contains high concentrations of tRNAs, which are small enough to enter the pores of the cage and displace the siRNA cargo. This strategy takes advantage of physicochemical properties unique to the cytosol, offering a simple solution to local release. Recreating biological activity in the image of simple viruses, such as Picornaviridae,204 sets a precedent for the construction of artificial nanodevices that can recapitulate the behaviors of more elaborate viral structures. For example, the introduction of membrane binding domains to designed protein cages enabled their escape from cells in a manner reminiscent of enveloped viruses.112 Eventually, integration of multiple virusinspired functions into such artificial systems should provide a means to optimize this unique class of materials for a broad range of applications.

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Thomas G. W. Edwardson: 0000-0001-8661-8036 Donald Hilvert: 0000-0002-3941-621X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the ETH Zurich and the European Research Council (Advanced ERC grant ERC-AdG2012-321295 to D.H.). T.G.W.E. is very grateful to the Human Frontier Science Program for a long-term fellowship.

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PERSPECTIVES With our ever growing ability to predictably engineer and deliberately evolve proteins, the most pertinent question may be: What do we want to create? Taking into account the diverse structural and functional roles played by natural proteins, it is evident that development of our own protein nanotechnology has unlimited potential.205 For example, the generation of designer enzymes is greatly improving and broadening the scope of biocatalysis,206 facilitating the production of existing and new chemicals in a sustainable manner.207 Proteins themselves are also important products, such as in the pharmaceutical industry, where “biologics” are a class of therapeutic agent that has steadily grown in significance.208 Due to their broad chemical functionality and sequence definition, natural and artificial polypeptides grant access to unique materials properties unattainable with traditional polymers.209 Perhaps most exciting is the so-called “dark matter” of the protein universe:210 unexplored structural space that can be accessed through fully de novo design techniques and provide unique materials that may find roles in technology not yet envisioned. However, as we continue to hone our abilities as architects of the molecular world, we still have much to learn from what nature has provided. Of the natural protein assemblies, viruses are particularly inspiring subjects for imitation, as their intertwining through all branches of life provides a unique lens through which to study biology. Importantly, as we gain insight into virus structure and function, we are taught lessons from self-sustaining nanotechnology that has lasted and improved over millennia. Discovery of the physical mechanisms underpinning virus function provides inspiration for our own biomolecular nanotechnology and enables the discovery of new targets for the development of suitable antiviral defenses.211 As such, knowledge of the origin and evolution of viruses at the molecular level is indispensable. Sequence information can be used to retrace phylogenetic paths, but the lack of truly ancient molecular fossils demands speculation.212−214 The recapitulation of, albeit artificial, evolutionary trajectories in the laboratory, combined with careful sequence and structural characterization, provides a complementary approach to investigate the molecular evolution of genetically encoded assemblies. As directors of such unnatural selection, we have

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