Disassembly and reassembly of AP205 virus-like particles and the removal of bound RNA for cargo encapsulation

Acinetobacter phage 205 (AP205) is a single-stranded RNA virus. The AP205 capsid protein (CP) spontaneously self-assembles to form virus-like particles (VLPs). VLPs have been widely used in vaccine development due to their high immunogenicity and show great potential for drug encapsulation and delivery. However, recombinant AP205 VLPs contain significant quantities of host cell RNA, which hinders their implementation as therapeutics. Furthermore, the relationship between AP205 CP structure and its roles in VLP stability and RNA association remains poorly understood. Here, we developed a method - predominantly mediated by the chaotropic effect of urea - to disassemble and reassemble AP205 VLPs with high fidelity (>90 %). We also identified the AP205 RNA binding site at Lys14 and Ser30 and generated an AP205 mutant that successfully abrogated RNA binding while retained the ability to self-assemble into RNA-free VLPs. Building on these findings, we assessed the encapsulation of biomolecules such as RNA and proteins within AP205 VLPs and demonstrated that proteins as large as 38 kDa could be encapsulated within our RNA-free AP205 VLPs. Our findings present a homogenous, RNA-free VLP construct suitable for vaccine development and introduce a novel approach for the delivery of biomolecules and drugs through AP205 VLP cargo encapsulation.