Investigation of plant virus-like particle formation in bacterial and yeast expression systems

Plant virus-like particles (VLPs) are highly ordered protein nanostructures with potential for biointerface engineering, targeted delivery, and templated nanomaterial synthesis. We investigated the self-assembly behavior and nucleic acid (NA) encapsulation capacity of coat proteins (CPs) from three sobemoviruses, focusing on ryegrass mottle virus (RGMoV) CP (RGCP). Expression in Pichia pastoris using a chromosome-integrated system enabled successful VLP formation, in contrast to the aggregate-prone assembly in Escherichia coli and Saccharomyces cerevisiae , likely due to the strong single-stranded DNS (ssDNA)-binding affinity of RGCP. RNA-sequencing and RT-PCR confirmed selective encapsidation of CP mRNA, highlighting sequence-compactness-driven self-packaging. Gel shift assays and a BacterioMatch II system revealed the preferential binding to ssDNA, which affected the assembly outcomes. Reassembly experiments with a CpG oligonucleotide demonstrated dose-dependent NA encapsulation and polymorphic capsid formation, with T = 1 and T = 3 symmetry particles forming at 1.5 µg/µl and T = 3 particles at 3 µg/µl. The observed structural transitions reflected the influence of NA size, secondary structure, pH, and potential CP truncation on capsid morphology. These findings suggested that RGMoV-derived VLPs are versatile and thermally stable bio-nanocontainers with tunable interior loading, offering novel strategies for constructing functional biointerfaces and nucleoprotein-based nanomaterials.