Virus-like particles (VLPs) leverage the intrinsic self-assembly properties of viral proteins, making them promising candidates for drug and nucleic acid delivery platforms. Mimicking native viruses without causing disease, VLPs can enable targeted cargo delivery by bypassing or exploiting cellular pathways for internalization. This study undertakes a comparative analysis between two VLP-forming systems: the envelope proteins VP19 and VP28 from the non-zoonotic White Spot Syndrome Virus (WSSV)—a pathogen with major impacts on global shrimp aquaculture—and the Capsid Protein (CP) of Bacteriophage MS2, a standard reference in self-assembly and structural virology. While VP19 and VP28 play key roles in WSSV infection, their assembly mechanisms are not well characterized; MS2 CP, on the other hand, is known for its precise, nucleic acid-directed assembly into 180-subunit capsids. Recombinant expression and in vitro reconstitution of VP19, VP28, and MS2 CP were performed to dissect the kinetics and thermodynamics of their particle formation. For MS2 CP, results reaffirm the central importance of nucleic acids in guiding assembly fidelity, stabilizing particle architecture, and regulating disassembly dynamics crucial for controlled cargo release. Extending these methods to the WSSV system, nucleic acids were also shown to significantly enhance the stability and integrity of VP19- and VP28-mediated assemblies. Direct comparisons revealed both conserved and distinct determinants of assembly, with variations in protein–nucleic acid interactions, optimal solution conditions, and particle robustness against environmental changes. These insights enabled the identification of parameters that maximize VLP yield, homogeneity, and functional stability across both systems.
By integrating structural, biophysical, and biochemical approaches, this work deepens mechanistic understanding of viral protein assembly and demonstrates how cross-system insights can guide the engineering of stable, functional VLPs. The findings establish foundational principles for rational VLP design with translational potential in nanomedicine and therapeutic delivery.