Mechanism of Capsid Assembly for an Icosahedral Plant Virus

Abstract

Capsids of spherical viruses share a common architecture: an icosahedral arrangement of identical proteins. We suggest that there may be a limited number of common assembly mechanisms for such viruses. Previous assembly mechanisms were proposed on the basis of virion structure but were not rigorously tested. Here we apply a rigorous analysis of assembly to cowpea chlorotic mottle virus (CCMV), a typical, small, positive-strand RNA virus. The atomic resolution structure of CCMV revealed an interleaving of subunits around the quasi-sixfold vertices, which suggested that capsid assembly was initiated by a hexamer of dimers (Speir et al., 1995, Structure 3, 63-78). However, we find that the capsid protein readily forms pentamers of dimers in solution, based on polymerization kinetics observed by light scattering. Capsid assembly is nucleated by a pentamer, determined from analysis of the extent of assembly by size-exclusion chromatography. Subsequent assembly likely proceeds by the cooperative addition of dimers, leading to the T = 3 icosahedral capsid. At high protein concentrations, the concentration-dependent nucleation reaction causes an overabundance of five-dimer nuclei that can be identified by classical light scattering. In turn these associate to form incomplete capsids and pseudo-T = 2 capsids, assembled by oligomerization of 12 pentamers of dimers. The experimentally derived assembly mechanisms of T = 3 and pseudo-T = 2 CCMV capsids are directly relevant to interpreting the structure and assembly of other T = 3 viruses such as Norwalk virus and pseudo-T = 2 viruses such as the vp3 core of blue tongue virus.

Core-controlled polymorphism in virus-like particles

“…The protein concentration at which the assembly experiments have been carried (30 M) is well above the critical concentration for empty capsid assembly (Ϸ10 M) (44). Therefore, the existence of a threshold CP/Au ratio for VLP formation, even when the protein concentration is larger than the critical concentration for empty capsids, signifies that there is a rapid initial association step between protein subunits and the negatively charged cores that is independent of the structural requirements for capsid formation.…”

Controlling viral capsid assembly with templating

“…We show in appendix A that the average timescales of these phases for an individual capsid can be described by τ = τ nuc + τ elong , with and , where f is the subunit-subunit binding rate constant, C S is the concentration of free subunits, n nuc is the number of subunits in the nucleus. Because elongation requires N − n nuc assembly events, it introduces a minimum timescale for the overall assembly process, which is primarily responsible for the lag time in assembly kinetics reported in experiments [9,12,53], theory [21,48], and simulations [26,28,80], and results in a distribution of assembly times for an individual capsid that cannot be fit with a sum of pure exponential functions [81]. The observed assembly rate constant, f, can be considered an average quantity, since computational models [26,28] suggest that it varies for different intermediates and decreases due to excluded volume constraints as assembly nears completion.…”

“…For example, protein-protein interactions, and hence the critical subunit concentration (CSC), can be controlled in virus-like particle experiments by varying the salt concentration or pH [12,29,47]. Capsid proteins denature, however, if these parameters are changed too far from physiological conditions.…”

“…Thus, in addition to their biomedical and technological applications, studying viral capsids has revealed fundamental principles of assembly. Although specific assembly mechanisms are poorly understood for most viruses, a general mechanism has emerged for the spontaneous assembly of empty capsids [10][11][12][12][13][14][15]21,26,28,[47][48][49][50][51][52][53]. Assembly occurs through a sequential addition process in which individual subunits or larger intermediates [26,52] bind to a growing capsid.…”

Master equation approach to the assembly of viral capsids

“…A first model for the self-assembly of a small plant virus was pioneered by Zlotnick [25], exploring the assembly of a dodecagonal shape by a cascade of single order reactions. It has since been extended to more involved scenarios [6,26,27], including a study of the energy landscape underlying assembly [7], that is similar to approaches in protein folding [2] or the energy landscape description of association reactions [21,22,23]. These results have been used to investigate the possibility of inhibiting assembly via an anti-viral drug in the case of Herpes Virus [28].…”