The lifecycle of many spherical (icosahedral) viruses involves co-assembly of the protein capsid around the viral genome. However, the details of this process are largely unknown. Important questions remain about how capsid protein associates with the nucleic acid to initiate assembly and complete virion, and how the viral genome becomes organized within the capsid. We will examine assembly of Cowpea Chlorotic Mottle virus (CCMV), a plus strand RNA plant virus. This is an ideal model for studying in vitro assembly because CCMV capsid protein (CP) can assemble into empty capsids, virus-like particles with non-specific RNA, and infectious virions with viral RNA. We hypothesize that virion assembly proceeds stepwise by (i) non - specific binding of CP to RNA, (ii) specific binding to viral RNA, (iii) refolding of the RNA - protein complex, and (iv) finally, cooperative association of CP to the complex, leading to completion of the capsid. This hypothesis implies that the protein - protein interactions will affect the organization of the packaged RNA and that RNA structure will have a profound effect on capsid assembly.
Understanding the assembly of empty capsids is a preliminary to studying assembly of RNA-filled virions. Study of the protein-protein interaction is complicated because the CCMV capsid is an oligomer of 180 copies of CP. We have developed analyses of assembly that allow us to deduce elements of the mechanism and thermodynamic and kinetic parameters. Experimentally, assembly will be observed using light scattering, liquid chromatography, and electron microscopy. Assembly of RNA-filled capsids adds an additional layer of complexity. In vivo, only viral RNA is encapsidated; in vitro, encapsidation is promiscuous. The role of RNA in directing assembly remains to be determined. RNA binding will concentrate CP, but is also likely to affect CP - RNA quaternary structure. We have observed distinct CP - RNA complexes that are likely to play critical roles in assembly and specificity. We will physically and structurally characterize these complexes and their formation on viral and random RNA. Subsequent capsid assembly will be observed with electrophoresis, and solution assays using RNA with bound complex, bare viral RNA, and random RNA as substrates. We expect that protein - protein interaction energy, observed for formation of empty capsids, will probably contribute to the cooperativity of assembly. We anticipate that these studies will yield a detailed picture of a basic biological process, the assembly of an infectious virus. These studies will be a paradigm for understanding assembly of other viruses, identifying new targets for anti-viral intervention, and for manipulating virus assembly to yield novel nano-structures.
