virus-based organelles - enzyme and dna-based virus nanostructures and their cellular interactions
Mark de Ruiter is a PhD student in the research group Biomolecular Nanotechnology. His supervisor is prof.dr. J.J.L.M. Cornelissen from the Faculty of Science and Technology.
Various protein-based organelles exist in nature that are involved in a wide variety of different metabolic pathways. However, the exact benefit and function of protein-based organelles is still not completely understood. In the first part of this thesis, we create virus-based nanoreactors to mimic these organelles, to gain more understanding about the benefits of performing reactions inside a protein capsid. Furthermore, we investigated their potential use for medical applications. To improve on the current design, it is important to understand both the assembly of the virus(-like) particles and their cellular interaction. Therefore, in the second part of this thesis we aim to understand the assembly processes of a virus protein capsid and to find the optimal shape for virus-based nanostructures for cellular uptake.
This thesis starts with a literature review on nanoreactors, evaluating some natural protein-based organelles and the strategies to encapsulate non-native enzymes in protein cages. Here, interesting (assembly) properties of the cowpea chlorotic mottle virus (CCMV) are described. These properties indicated that it is a good candidate for the fabrication of nanoreactors and nanostructures. We therefore used the capsid proteins (CPs) of CCMV for the fabrication of our nanoreactors. This was done by encapsulating various enzymes with different sizes using a charge-based encapsulation approach. In this approach the used enzymes: thrombin, luciferase, horseradish peroxidase, glucose oxidase, L-asparaginase and β-galactosidase, were modified with either polystyrene sulfonate or single stranded DNA (ssDNA). When these are mixed with free CPs at neutral pH and physiological ionic strength this results in a cargo directed encapsulation, where approximately one enzyme is encapsulated per capsid. The formed particles were monodisperse with average diameters between 16 and 24 nm, depending on the size of the encapsulated enzyme. Further analysis with Cryo-EM 3D reconstruction revealed that the particles were well-defined T = 1 or pseudo T = 2 particles with icosahedral symmetry. These icosahedral structures have large and dynamic pores that allow reactants to diffuse into and out of the protein capsid, which was utilized during catalytic evaluation. Most, but not all, of the enzymes retain their activity and we show that the capsid around the enzymes can change the catalytic behaviour. The degree to which the activity is affected depends on the used substrate.
To explore their medical applications, the fabricated virus-based nanoreactors were evaluated with cancer cells to show their intracellular and extracellular functioning. The intracellular function was evaluated using the β-galactosidase based nanoreactor, where the uptake, activity and stability of these particles was monitored in cancer cells. The nanoreactors show an increased intracellular enzymatic activity compared to non-encapsulated enzymes. This beneficial effect of the capsid was confirmed in a test with a protease substrate, which showed that the virus shell protects the enzymatic cargo when entering the cell.
For the extracellular function, the L-asparaginase based nanoreactor was used. This nanoreactor outperformed the free enzyme in killing acute lymphoblastic leukemia cells and can therefore be a potential new treatment for this type of cancer. The results from these two cases show that CCMV-based nanoreactors are useful for the delivery and protection of enzymes, with clear potential for medical use.
To understand the assembly of virus protein capsids, we investigated how the capsid proteins (CP) of CCMV assemble around DNA into well-defined structures at neutral pH. Therefore, different types of DNA with varying lengths were used during the assembly with CP. This study includes ssDNA to form spherical structures and double stranded DNA (dsDNA) to form rods. We found that increasing the lengths of the used DNA resulted in faster assembly and in the formation of larger spheres or longer rod-like structures. We also found that for ssDNA a length of 14 nucleotides is the minimum length required to induce the assembly of full virus-like particles under the used conditions. The spherical structures were determined to be a mixture of icosahedral structures. They also contain slightly elongated non-icosahedral structures, which have not been observed before for CCMV or other viruses.
To further study the assembly of viruses, we developed a new FRET-based probing strategy. Three different fluorescent ATTO dyes were linked to three different ssDNA oligomers, which were hybridized to a longer ssDNA strand. Upon assembly of the CPs this resulted in a 16-fold increase in FRET efficiency. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This makes it an effective probe for assembly, which potentially can be designed with the natural nucleic acid cargo of viruses.
The cellular uptake route of the CCMV virus and shape dependency of viruses during their uptake are still not completely understood. Therefore, we investigated the cellar uptake route and intracellular positioning of spherical and rod-like CCMV-based nanostructures into multiple cell lines. The results show that multiple routes are possible for CCMV-based nanostructures and the pathways for uptake depend only slightly on the shape of the viral particle and cell type. The uptake mechanism in Hela cells was investigated in more detail and we discovered that almost all the nanostructures show clathrin-mediated endocytosis as the main uptake route. With confocal analysis, we confirmed that all the nanostructures are indeed endocytosed by cells because the structures colocalize with the endosomes after 4 hours. Further in vivo evaluation in zebra fish embryos revealed that most of the native CCMV is taken up by neutrophils. This triggered us to perform an initial study to use CCMV-based nanostructures for gene delivery to macrophages, with potential use in vaccine development.
Overall, we showed the potential of using the CPs of CCMV in medical applications. The used nanoreactors show a clear benefit over non-encapsulated enzymes during the cell studies. This opens up the possibility of using them for the treatment of cancer, various enzyme deficiency diseases and their use as ‘on site’ drug producers, either using pro-drugs or natural metabolites as substrates. Additionally, the results from the DNA-based assemblies give insight in the encapsulation and the cell uptake process, which can be used to encapsulate various nucleic acid materials including mRNA, siRNA and plasmids. This can be used in vaccination, gene regulation and gene therapy. The results on the assembly and uptake of CCMV into cells are also relevant to discover trends in other viruses. These discoveries can help in the development of anti-viral drugs. However, not all applications of a virus are in the medical field. The virus-based nanoreactors and can, for example, also be used as industrial catalysts, in sensors and in other functional nanomaterials. The nanostructures can be used to create nanowires, to stabilize nanoparticles and to fabricate highly ordered assemblies. To realize this potential, more research needs to be done before CCMV and its different nano-constructs can find its way into the various applications. For example, the purity needs to be improved and the production needs to be upscaled. Also, more in vitro and in vivo tests are required to show the medical effects of these structures, because op their potential immunogenicity. The reaction of the immune system on the plant virus should be studied.
We can conclude that the results presented in this thesis form a solid basis for further research in the use of CCMV for medical and other applications. The benefits of the virus-based nanoreactors and nanostructures will hopefully lead to the development of new drug formulations. Thus viruses will not only make us ill, but eventually make us better instead.
