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.
Highlights
The in vivo assembly of virus-like particles (VLP), multi-protein structures, in unnatural host systems results many times in predominantly malformed structures because the host biology provides neither the adequate spatial and temporal protein expression conditions nor the ideal thermodynamic environment for their assembly into VLP
Mathematical models built on stability, kinetic and thermodynamic principles of protein macrostructures and respective assembly pathways are key elements to better understand and design VLP assembly control strategies
Virus-like particles (VLP) are multi-protein structures with potential to be used in biomedicine as therapeutic or prophylactic vaccines against many worldwide diseases
Summary
The in vivo assembly of virus-like particles (VLP), multi-protein structures, in unnatural host systems results many times in predominantly malformed structures because the host biology provides neither the adequate spatial and temporal protein expression conditions nor the ideal thermodynamic environment for their assembly into VLP. The problem of capsid assembly was initially addressed as a cascade of low-order reactions with driving force the thermodynamic-equilibrium relationship between monomers and complete capsids [2] This formalism uses two basic parameters to control virus capsid assembly, namely the concentration of structural subunits and the Gibbs free energy of subunit association (DG0n). If the association energy in the initial assembly steps are much higher than that of subsequent steps, fast depletion of free subunits occurs which leads to the accumulation of stable intermediates and subsequently to kinetically trapped products. This phenomenon can be observed at very high protein concentrations [4].
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