NSF Award
0111025
The lifecycle of many spherical (icosahedral) viruses involves co-assembly of the protein<br/>capsid around the viral genome. However, the details of this process are largely<br/>unknown. Important questions remain about how capsid protein associates with the<br/>nucleic acid to initiate assembly and complete virion, and how the viral genome becomes<br/>organized within the capsid. We will examine assembly of Cowpea Chlorotic Mottle<br/>virus (CCMV), a plus strand RNA plant virus. This is an ideal model for studying in vitro<br/>assembly because CCMV capsid protein (CP) can assemble into empty capsids, virus-like<br/>particles with non-specific RNA, and infectious virions with viral RNA. We hypothesize<br/>that virion assembly proceeds stepwise by (i) non - specific binding of CP to RNA, (ii)<br/>specific binding to viral RNA, (iii) refolding of the RNA - protein complex, and (iv)<br/>finally, cooperative association of CP to the complex, leading to completion of the<br/>capsid. This hypothesis implies that the protein - protein interactions will affect the<br/>organization of the packaged RNA and that RNA structure will have a profound effect on<br/>capsid assembly.<br/><br/>Understanding the assembly of empty capsids is a preliminary to studying assembly of<br/>RNA-filled virions. Study of the protein-protein interaction is complicated because the<br/>CCMV capsid is an oligomer of 180 copies of CP. We have developed analyses of<br/>assembly that allow us to deduce elements of the mechanism and thermodynamic and<br/>kinetic parameters. Experimentally, assembly will be observed using light scattering,<br/>liquid chromatography, and electron microscopy.<br/>Assembly of RNA-filled capsids adds an additional layer of complexity. In vivo, only<br/>viral RNA is encapsidated; in vitro, encapsidation is promiscuous. The role of RNA in<br/>directing assembly remains to be determined. RNA binding will concentrate CP, but is<br/>also likely to affect CP - RNA quaternary structure. We have observed distinct CP - RNA<br/>complexes that are likely to play critical roles in assembly and specificity. We will<br/>physically and structurally characterize these complexes and their formation on viral and<br/>random RNA. Subsequent capsid assembly will be observed with electrophoresis, and<br/>solution assays using RNA with bound complex, bare viral RNA, and random RNA as<br/>substrates. We expect that protein - protein interaction energy, observed for formation of<br/>empty capsids, will probably contribute to the cooperativity of assembly. We anticipate<br/>that these studies will yield a detailed picture of a basic biological process, the assembly<br/>of an infectious virus. These studies will be a paradigm for understanding assembly of<br/>other viruses, identifying new targets for anti-viral intervention, and for manipulating<br/>virus assembly to yield novel nano-structures.
