Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus

Abstract

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318 -3326, 2012, doi:10.1128/JVI.06566-11) 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 of 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 the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. IMPORTANCECowpea 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).

A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

“…While colocalization of NS2A with double-stranded RNA has been described (10), interaction with the ssRNA packaged into virions has not been described. Besides charge matching between the positively charged motifs in the capsid protein and the negatively charged phosphate backbone of the RNA, the presence of multiple sites within the ssRNA genomes that contact the capsid proteins during assembly, so-called packaging signals, have been described to be important for efficient and specific packaging of ssRNA genomes (30)(31)(32). Whether NS2A has a role in virion assembly at the level of RNA packaging by for example assisting in condensing the RNA remains to be investigated.…”

Effects of RNA branching on the electrostatic stabilization of viruses

“…Non-specific electrostatic interactions have emerged as the driving force for virus assembly through both the experimental as well as the theoretical studies [9,14,24,27,28]. In our two simple models we generalized the implementation of electrostatic interactions by coupling it to RNA topology.…”

“…In the prevailing paradigm this assembly is predominantly driven by generic, nucleotide sequence independent, electrostatic interactions [2] between the negative charges on the RNA phosphate backbone and the positive charges on the virus capsid proteins (CP) [3][4][5][6][7][8]. Recent experiments have indeed abundantly verified the importance of the "chargematching hypothesis", based on the preponderance of electrostatic interactions between the capsid proteins and the RNA for proper genome packaging [9].…”

Revealing the density of encoded functions in a viral RNA

“…The PS fragments and the variant sites, with the exception of the insert variants, are of the same length, have the same net charge and are predicted to share common secondary structures. Under ensemble assembly conditions (CP ≥ 10 μM) B34U triggers T = 1 capsid formation, although much less efficiently than B3 (15), i.e., the sequencespecific RNA discrimination is dramatically reduced at higher concentration, and reassembly can be driven almost exclusively by electrostatic interactions (26). In contrast, the outcomes of single-molecule in vitro reassembly, carried out at nanomolar concentrations, are more sensitive to sequence-specific effects and may reflect more accurately conditions in vivo.…”