The Self-assembly of Cowpea Chlorotic Mottle Virus
- Author(s): Garmann, Rees Frederik
- Advisor(s): Gelbart, William M
- Knobler, Charles M
- et al.
The first half of this dissertation (Chapters 1-3) deals with the in vitro self-assembly of Cowpea Chlorotic Mottle Virus (CCMV) from its single-stranded (ss) RNA genome and its capsid protein (CP). We have systematically investigated how the assembly depends on three previously unaddressed factors: (1) the length of the RNA; (2) the strength of attraction between the CP and the RNA; and (3) the electrostatic charge of the CP. Our results support a theoretically predicted but previously unverified self-assembly mechanism in which CP initially binds RNA in a disordered manner, and then reorganizes into the final icosahedral capsid structure. Additionally, we find that any length of RNA (from 140 to 12,000 nucleotides) can be efficiently packaged so long as two key requirements are met. First, the reaction mixture must contain a specific excess of CP to provide "charge-matching" between the relevant basic residues of the CP and the phosphate backbone of the RNA. Second, the assembly reaction must be carried out by a two-step, pH- and salt-dependent protocol.
In the second part (Chapters 4 and 5) we explore the potential use of self-assembled CCMV virus-like particles (VLPs), both as fluorescent nano-particles with controllable size (Chapter 4), and as gene delivery vectors (Chapter 5). We find that the fluorescent properties of dye-labeled polymers encapsidated by CCMV CP are robust against quenching by external quencher molecules. Additionally, we find that VLPs consisting of RNA genes packaged in CCMV capsids, once delivered to cytoplasm of mammalian cells, are released from their capsids and efficiently expressed, allowing them to be exploited for gene delivery.
In Chapter 6, we have examined the complicated branching networks of ss-RNA secondary structures by direct visualization with cryo-electron microscopy and confirmed a previously reported secondary structure of the entire genome of satellite tobacco mosaic virus. Finally, we discuss current preliminary data and future work in the Summary section.
Throughout this work we have repeatedly exploited the techniques of native agarose gel electrophoresis, velocity sedimentation, fluorescence spectroscopy (and microscopy), and electron microscopy.