Co-release of tick-borne encephalitis virus RNA and structural proteins via virus-like particles and extracellular vesicles.

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that causes severe neurological diseases in humans. The assembly of flaviviruses is initiated by the interaction between capsid (C) proteins and viral genomic RNA, yet the molecular determinants that govern RNA encapsidation remain unclear. In this study, we established a TBEV virus-like particle (VLP) system to analyse viral factors that influence viral RNA incorporation independently of productive infection. Using a reporter-containing TBEV minigenome, we investigated the contribution of UTRs to the incorporation of RNA into extracellular particles. Truncation of the 5' UTR, 3' UTR or both did not significantly affect the levels of minigenome RNA detected in pelleted extracellular fractions, indicating that RNA incorporation occurs largely in a non-specific manner. We further examined the role of C protein dimerization by introducing alanine substitutions into residues that form the α2-α2' and α4-α4' dimer interfaces. Paradoxically, these substitutions increased the levels of minigenome RNA detected in pelleted extracellular particles without altering intracellular RNA expression, indicating a complex relationship between the integrity of the C protein dimer interface and levels of extracellular viral RNA. Finally, we showed that TBEV proteins and minigenome RNA can be detected in extracellular vesicle (EV)-associated fractions under VLP-producing conditions. Using immunoaffinity purification, we demonstrate the presence of viral components in EVs, underscoring EV-associated release as a factor that complicates the analysis of flaviviral particle assembly.