Multiscale Simulations and Cryo-Electron Microscopy Reveal the Transition Pathway of Dengue Virus-like Particle Nanoassembly

Dengue virus (DENV) continues to impose a global health burden, and virus-like particles (VLPs) are promising vaccine candidates, owing to their ability to elicit broadly neutralizing antibodies. However, the lack of nanoscale structural insights into the VLP maturation process has limited rational engineering. Here, we report the cryo-electron microscopy (cryo-EM) structure of immature DENV serotype 2 VLPs, revealing prM-E spikes arrayed on a <i>T</i> = 1 shell, consistent with mature DENV-2 VLPs and distinct from the virion, which exhibits a <i>T</i> = 3 icosahedral lattice. To connect the experimentally determined immature and mature endpoint structures, we employed a multiscale molecular dynamics (MD) framework to assess sterically feasible transition pathways. The simulations support the steric feasibility of a sliding-rotating rearrangement in which trimeric prM-E spikes reorganize into flat E dimers (E-E) without clashes. In virions, trimers reorganize into extended rafts of three parallel E dimers, whereas in VLPs, which lack the long-range symmetry and geometric constraints of the <i>T</i> = 3 lattice, maturation proceeds via a less extensive rearrangement in which neighboring monomers form small triangular clusters of three dimers. In addition, the simulations reveal pronounced lipid core mobility during maturation, including transient and spatially localized lipid protrusion events that preferentially occur near regions undergoing protein rearrangement, consistent with a potential role for dynamic membrane remodeling in accommodating maturation-associated structural changes. We also established a stable Chinese hamster ovary (CHO-K1) producer cell line, enabling efficient production of immature DENV serotype 2 VLPs. Together, this work defines a structure-dynamics framework that links steric feasibility, membrane composition, and particle stability and outlines process-relevant, testable hypotheses to inform future engineering of dengue VLPs that may ultimately guide vaccine design.