Endoskeleton-Enhanced Virus-Like Particles (VLPs): Internal Cross-Linking of Physalis Mottle Virus and Stability Tests.

Virus-like particles (VLPs) often exhibit favorable chemical, thermal, and mechanical stability compared to other biologic materials. Nonetheless, enhancing their stability remains crucial for advancing VLP-based materials toward practical and commercial use. To address this, we introduced an "endoskeleton-armoring" approach aimed at reinforcing VLP stability. Using the VLP from physalis mottle virus (PhMV) as a model system, we created an internal polymer backbone by covalently linking the coat proteins within the capsid using a maleimide-PEG15-maleimide crosslinker. This contrasts with native VLPs, which rely solely on non-covalent interactions for structural integrity. The resulting armored VLPs demonstrated markedly improved thermal stability, enhanced resistance to various denaturants (including surfactants, extreme pH, chemical agents, and organic solvents), and superior mechanical robustness. In this chapter, we detail the methods to achieve the "endoskeleton-armoring" approach using PhMV as proof of concept, which can be applied to other VLPs and protein cages.