In vitro virus-like particle assembly follows two distinct, competing pathways

1.

Virus-like particles (VLPs) formed from the murine polyomavirus major capsid protein VP1 are widely used as vaccine antigens and are being explored as nucleic acid and drug delivery vehicles. However, the factors controlling distinct VP1 capsid morphologies remain unclear. We investigated in vitro VP1 assembly with tRNA across NaCl concentrations of 0.15–1.0 M and tRNA mass ratios of 1:1–1:80 (w/w) using SEC-HPLC, transmission electron microscopy, and dynamic light scattering.

Two competing assembly pathways were identified. At low ionic strength (≤0.15 M NaCl), nucleic acid-templated assembly produced compact, tRNA-filled T=1 VLPs (∼28–30 nm). Assembly was maximal at tRNA ratios of 1:10–1:20, whereas excess or insufficient tRNA reduced yields. Increasing NaCl to 0.30 M lowered T=1 yields by 68–97%, and no T=1 particles were detected at ≥0.5 M NaCl.

Conversely, high ionic strength (≥0.5 M NaCl) promoted template-independent formation of hollow T=7 VLPs (∼55–60 nm). T=7 assembly was inhibited by tRNA and was highest without nucleic acid. At 1.0 M NaCl, reducing the tRNA ratio from 1:40 to 1:80 increased T=7 yield more than 11-fold, while removing tRNA produced the greatest assembly efficiency. Kinetic analyses further showed that VP1 concentration and ionic strength regulate nucleation and assembly rate in the template-driven pathway.

These findings show that electrostatic interactions govern pathway selection between tRNA-templated T=1 assembly and salt-driven T=7 self-assembly, providing practical guidance for controlling capsid morphology and cargo loading in VLP-based applications.