The Assembly and Confinement Properties of the Cowpea Chlorotic Mottle Virus

Stan Maassen 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.

Viruses provide a whole new set of building blocks for the development of new materials and as such have found application in, for example, materials science, nanotechnology, and medicine. Their monodispersity and high degree of symmetry surpass any synthetic nanoparticle currently available, and due to the huge amount of different viruses found on Earth there are many shapes and sizes to choose from. In particular the capsid of viruses, that surrounds their genome, is commonly studied. This protein shell transports and protects the genome, and releases the viral genetic material once it has entered a host cell to allow reproduction of the viral components. After reproduction, these reassemble into full viruses which are released from the host. This assembly and disassembly behavior requires specific inter-subunit interactions to provide stability of the virus during transportation while allowing disassembly to infect a host. Both for the purpose of understanding viral reproduction and treating viral infections as well as for using viral components for various applications and being able to predict the structures that will be formed from them, gaining insight in these interactions is crucial.

The work described in this thesis extends our knowledge on viral assembly, by studying the assembly and confinement conditions of the cowpea chlorotic mottle virus (CCMV). By introducing microscale thermophoresis (MST) as a new way to study (self-)assembly, we were able to study and compare the assembly behavior of native CCMV capsid protein (CP) with that of two genetically modified versions of this protein over a wide range of conditions (Chapter 3). MST was also used in combination with isothermal titration calorimetry (ITC) to study CCMV CP assembly into virus-like particles (VLPs) templated by polyanionic species at neutral pH (Chapter 4). Continuing the study of polyanion-templated assembly, we determined the minimum length of single-stranded (ss)DNA, correlating to a minimal electrostatic interaction, required to induce viral assembly at neutral pH (Chapter 5).

To gain insight into the physio-chemical conditions inside a protein cage, we studied the pH conditions inside CCMV-based capsids, using a negatively charged, pH-responsive fluorescent probe (Chapter 6). These results, combined with a theoretical model, show that the pH inside a protein cage is not necessarily the same as in the bulk solution, which may be of interest for catalytic purposes or the development of new virus-based materials. To aid in the development of new CCMV-based materials and applications, we developed new methods for VLP assembly and functionalization, and studied polymerization reactions inside CCMV-based capsids (Chapters 7 & 8).  Overall, we see great potential for the application of viruses in various fields, however, to meet this potential, further research towards the assembly and confinement properties of virus(‑like) particles is required. The studies presented in this thesis extend our understanding of these structures, however building on the research shown here new questions arise. Instead of studying the assembly of virus particles and the energies involved by looking at an average behavior of many particles in a solution, it might be interesting to follow separate components, e.g. a single CP or a single polyanion chain. This potentially provides new information on viral assembly on a smaller scale, rather than giving an average of millions of particles, and may allow for the development of a more accurate understanding of assembly pathways. Furthermore, more knowledge on the interactions between various components is required to be able to accurately design specific structures. Also, knowing the pH inside protein cages can deviate from it surrounding, what other differences in physiochemical properties occur inside such structures? And how is the cargo affected by this? A difference in pH causes changes in the protonation state of the cargo, while molecular crowding may affect the mobility or even the mechanical properties of the cargo. These are issues to consider when designing new materials based on viruses. Lastly, the data obtained for CCMV as a model virus and the techniques used to obtain them are potentially applicable over a wider range of viruses. Future research using other viruses is required to determine if these data are a general trend for virus assembly and confinement properties in general.