On the Effect of Thermodynamic Equilibrium on the Assembly Efficiency of Complex Multi-Layered Virus-Like Particles (VLP): the Case of Rotavirus VLP

Abstract

Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins.

Double-layered rotavirus-like particles are efficient carriers to elicit strong CTL responses to delivered heterologous antigens

“…The proteins forming the chimeric VLPs showed stoichiometries of about 2.7 in densitometry assays (data not shown). These values are similar to those previously reported for VP2/VP6 VLPs [24]. Finally, electron microscopy revealed DLPs morphologically indistinguishable from wild type VLPs (Fig.…”

Formation of self-assembled triple-layered rotavirus-like particles (tlRLPs) by constitutive co-expression of VP2, VP6, and VP7 in stably transfected high-five insect cell lines

“…Investigating the production of RLPs in baculovirus expression system, it has been shown that RV VLP2/6/7 and DLP2/6 formation account for 12% and 88% of the total mass of proteins expressed, respectively [Vieira et al, ; Roldao et al, ]. Wasteful accumulation of unassembled proteins and formation of incomplete RLPs were also reported [Palomares et al, ; Vieira et al, ; Roldao et al, ]. In addition, the formation and stability of RLPs are shown to be highly dependent on the pH, ionic strength, and temperature [Mellado et al, ].…”

Exploiting virus-like particles as innovative vaccines against emerging viral infections

“…Computational structure modeling and bioinformatics techniques have been increasingly applied on design of VLPbased vaccines (Ding et al, 2010;Pankavich and Ortoleva, 2012;Roldão et al, 2012). Currently, there are 540 completed icosahedral virus capsid structures available from VIPERdb database (http://viperdb.scripps.edu/), from which the designing principle of multi-molecular assembly could be deduced.…”

“…It is practically very difficult to sufficiently control all of these factors during VLP assembly in vivo. As an alternative, therefore, disassembly and reassembly in vitro were developed and optimized for several VLPs (McCarthy et al, 1998;Ren et al, 2006;Xing et al, 2010;Link et al, 2012;Roldão et al, 2012). The disassembly and reassembly in vitro allows easy control over encapsulated host cell impurities and lead to increase the homogeneity, stability, and reactivity of VLPs (Zhao et al, 2012a(Zhao et al, , 2012b.…”