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

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

Viruses and virus-like particles in biotechnology: Fundamentals and applications

Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this chapter, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitoring, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly

SLP assembly efficiency as function of the initial vp2 concentration, [vp2]₀, for different Gibbs free energy of vp2 subunit association values, ΔG⁰_vp2,3-mer.

<p>The full line represents <b>ΔG⁰_vp2,3-mer</b> = −4.08 kcal.mol⁻¹ as estimated by Zlotnick 1994, the dash lines represent <b>ΔG⁰_vp2,3-mer</b>>−4.08 kcal.mol⁻¹ and the dot lines represent <b>ΔG⁰_vp2,3-mer</b><−4.08 kcal.mol⁻¹. <b>K_d,app</b> represents the pseudo-critical concentration that satisfies the constraint <b>[1]</b> (concentration of unassociated vp2 subunits at equilibrium - <b>[1]</b> = <b>[svp2]_e</b>) = <b>[20]</b> (complete SLP) = <b>K_d,app</b> at equilibrium for a specific <b>ΔG⁰_vp2,3-mer</b>.</p

Kinetics of <i>in vitro</i> RLP assembly and disassembly by 90° light scattering.

<p>Transition of DLP to RLP under standard condition (TNC buffer at pH 5.5, 25°C, NaCl 0.1 M and Ca²⁺ 1 mM) (graph A) and upon increase in pH (8 – graph B), Ca²⁺ (5 mM – graph C), temperature (35°C – graph D) and NaCl (0.5 M – graph E). Dissociation of RLP to DLP at 25°C in TNC with 1 mM of EDTA (grey triangle) or in D-PBS with 1 mM of EGTA (black diamond) (graph F). The arrows indicate the times at which vp7 was added to DLP (graphs A to E) or the addition of chelating agents (graph F).</p

<i>In vitro</i> assembly and disassembly of RLPs.

<p><i>In vitro</i> assembly and disassembly of RLPs.</p

The concentration of assembly intermediates for three initial protein concentration scenarios:

<p>1) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (stoichiometric ratios – white bars); 2) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 0.78 M (limitation of vp7 – black bars); 3) <b>[vp2]₀</b> = 12 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (excess of vp2 – grey bars). The Gibbs free energy of vp2, vp6 and vp7 subunit association was assumed to be equal (<b>ΔG⁰_vp2,3-mer</b> = <b>ΔG⁰_vp6,13-mer</b> = <b>ΔG⁰_vp7,13-mer</b> = −4.08 kcal.mol⁻¹).</p

The effect of ΔG⁰_vp7,13-mer on RLP assembly efficiency when the initial protein concentration of vp2, vp6 and vp7 is 1.2 M, 7.8 M and 7.8 M, respectively, and the Gibbs free energy of vp2 and vp6 subunit association (ΔG⁰_vp2,3-mer and ΔG⁰_vp6,13-mer, respectively) is equal to −4.08 kcal.mol⁻¹.

<p><b>ΔG⁰_vp7,13-mer</b> varies between −4.08×[0.05 to 1.4] kcal.mol⁻¹.</p