The Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus.
|Title||The Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus.|
|Publication Type||Journal Article|
|Year of Publication||2014|
|Authors||Garmann, RF, Comas-Garcia, M, Koay, MST, Cornelissen, JJLM, Knobler, CM, Gelbart, WM|
|Date Published||2014 Jun 25|
We have recently discovered (Cadena-Nava, et al. J. Virol. 2012: , 86, 3318) that the in vitro packaging of RNA by the capsid protein (CP) of Cowpea Chlorotic Mottle Virus is optimal when there is a significant excess of CP, specifically that complete packaging of all the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively-charged arginine-rich motifs (ARMS) of the CPs with the negatively-charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a "charge-matched" protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally-symmetric, nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid, through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario we prepare CP mutants with "extra" and "missing" (relative to wildtype) cationic residues and show that a correspondingly smaller and larger excess of CP is needed, respectively, for complete packaging of RNA.
IMPORTANCE: Cowpea Chlorotic Mottle Virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly - i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA - driven by electrostatic attraction - followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N-termini of the CP).
|Alternate Journal||J. Virol.|