Self-Assembly of an Alphavirus Core-like Particle Is Distinguished by Strong Intersubunit Association Energy and Structural Defects

Abstract

Weak association energy can lead to uniform nanostructures: defects can anneal due to subunit lability. What happens when strong association energy leads to particles where defects are trapped? Alphaviruses are enveloped viruses whose icosahedral nucleocapsid core can assemble independently. We used a simplest case system to study Ross River virus (RRV) core-like particle (CLP) self-assembly using purified capsid protein and a short DNA oligomer. We find that capsid protein binds the oligomer with high affinity to form an assembly-competent unit (U). Subsequently, U assembles with concentration dependence into CLPs. We determined that U-U pairwise interactions are very strong (ca. −6 kcal/mol) compared to other virus assembly systems. Nonetheless, assembled RRV CLPs appeared morphologically uniform and cryo-EM image reconstruction with imposed icosahedral symmetry yielded a T=4 structure. However, 2D class averages of the CLPs show that virtually every class had disordered regions. These results suggested that irregular cores may be present in RRV virions. To test this hypothesis, we determined 2D class averages of RRV virions using authentic virions or only the core from intact virions isolated by computational masking. Virion-based class averages were symmetrical, geometric, and corresponded well to projections of image reconstructions. In core-based class averages, cores and envelope proteins in many classes were disordered. These results suggest that partly-disordered components are common even in ostensibly well-ordered viruses, a biological realization of a patchy particle. Biological advantages of partly-disordered complexes may arise from their ease of dissociation and asymmetry.

Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

“…However, collectively the data presented in this report indicate that the interactions, or at least the strength thereof, between the SINV capsid protein and the genomic RNA may be context dependent. The disseminated pattern of binding of the viral genomic RNA to capsid observed in purified viral particles supports a model where the C:R interactions are largely nonspecific, and perhaps based on charge-charge interactions [25,27,28]. However, from this data set it is impossible to identify if the interactions between the capsid protein and cargo RNA are mutually saturating (where each capsid monomer interacts with~50 nt of genomic RNA), or unsaturated (where not every nt is associated with capsid monomer) but nonspecifically distributed along the length of the genome.…”

“…Several studies have indicated that the association of the alphaviral capsid protein with viral RNA is nonspecific in regards to primary nucleotide sequence and secondary structure, and is likely driven by electrostatic interactions between the N-terminus of Capsid and the phosphodiester RNA backbone [24][25][26][27][28]. While these studies primarily focused on the interactions of nucleic acids and the viral capsid proteins in vitro, it is highly likely that these observations are valid during bona fide infections as evidenced by the distributive nature of the Capsid:RNA interactions observed in particles.…”

“…The interactions between the alphaviral capsid protein and the viral genomic RNA is an under characterized, yet vitally important, RNA:protein interaction. Alphaviral in vitro assembly systems have largely indicated that the assembly of Capsid:nucleic acid complexes is a nonspecific process [24][25][26][27][28]. However it should be noted that the direct interaction between the viral capsid protein and viral RNA has not been exhaustively characterized in the cytoplasm of infected cells, or in mature viral particles leaving our understanding of the molecular interactions between these essential components of the virus incomplete.…”

Why Enveloped Viruses Need Cores—The Contribution of a Nucleocapsid Core to Viral Budding

“…Motivated by the recent observation that alphavirus nucleocapsids do not require icosahedral symmetry (53) to be infectious, we model the nucleocapsid as an isotropic sphere of radius similar to that estimated from cryoelectron microscopy (CryoEM) experiments, r NC z 18.0 nm. Because complete NCs have significantly higher rigidity than lipid membranes or GP-coated vesicles (54,55), we model the NC as infinitely rigid.…”

Identification of Chikungunya virus nucleocapsid core assembly modulators

“…CLPs can be encapsidated by viral glycoproteins, and the newly formed virus-like particles are able to enter and disassemble within a new cell (50,51). A 27mer DNA oligo interacts in a 1:1 molar ratio to neutralize the basic N-terminus of the capsid protein (52,53) to initiate CLP assembly. CLPs are 40 nm in diameter and structurally similar to cores in virions (52)(53)(54)(55).…”