L-A virus is a dsRNA virus of the Totiviridae family found in Saccharomyces cerevisiae yeast [1]. With evidence that the native L-A host, S. cerevisiae, is abundant in the natural human environment [2], it is expected that the capsids of this virus could be adapted for immune response-neutral nanodelivery of biomolecules into mammalian cells. To this end, our previous studies have developed a system for the synthesis of L-A virus capsids in S. cerevisiae yeast. Such particles, consisting of recombinant viral capsid proteins and resembling native virions in structure, yet lacking of genetic material, are more commonly known as virus-like particles (VLPs). VLPs are becoming an increasingly popular object for drug nanodelivery studies due to their biocompatibility, safety, monodispersity, natural tissue tropism, and the possibility of easy chemical and genetic modification of the inner and outer surface [3].
One of the key steps in the application of VLPs to nanocarrier applications is disassembly-reassembly studies. This method helps to get rid of cellular proteins and other molecules that are encapsulated during self-assembly in the cell, often increases the stability of the reassembled particles, and is a popular method for encapsulating larger cargoes such as proteins or nucleic acids [3]. Therefore, in order to apply the L-A VLPs synthesized by Saccharomyces cerevisiae to nanocarrier applications, the main objective of this study was to find the conditions for both their disassembly and reassembly.
Particle disassembly was examined under different conditions: high ionic strength, basic and acidic pH, and reducing and denaturing reagents (β-mercaptoethanol, dithiothreitol, urea). Following the overnight treatment, dynamic light scattering (DLS) method was used to assess the particle size. Small structures of 14–18 nm were only detected in samples containing urea, suggesting that the L-A VLPs had disassembled. Consequently, further reassembly experiments were conducted using particles that had been disintegrated using 2 M urea. The disassembled particles were dialyzed overnight in Tris-HCl-based buffers with different ionic strengths and Tween 80, and then reassembled particles were precipitated by ultracentrifugation through sucrose cushion. As evidenced by the initial experiments, L-A VLPs did reassemble during dialysis, regardless of the buffer solution. Future research will be conducted to determine the efficiency of the reassembly, as well as the prospects of cargo encapsulation.