Interactions between Tick-Borne Encephalitis Virus Nonstructural Protein 1 and Blood–Brain Barrier Tight Junction Proteins: Potential Clues to Strain-Specific Neuropathogenicity

Abstract Tick-borne encephalitis virus (TBEV) invades the central nervous system (CNS) through strain-specific mechanisms that remain poorly understood. For mosquito-borne orthoflaviviruses such as dengue and yellow fever viruses, the nonstructural protein 1 (NS1) has been shown to disrupt endothelial barrier integrity by targeting tight junction proteins (TJPs), facilitating viral neuroinvasion. However, comparable mechanisms in TBEVs remain largely unexplored. Here, we investigated the potential interaction of NS1 from high (Hypr)- and low (Vs)-pathogenic TBEV strains with different blood–brain barrier (BBB) TJPs, using AlphaFold3 (AF3) multimer modeling and in vitro binding assays. AF3 modeling suggested that NS1 from the highly pathogenic strain may interact with multiple TJPs, including junctional adhesion molecule A (JAM-A), a key component of the paracellular barrier. In contrast, the low-pathogenic Vs showed a more restricted interaction profile. Experimental validation using recombinant NS1 proteins revealed strain-specific binding profiles: Hypr NS1 displayed high-affinity, saturable direct binding to immobilized JAM-A (KD,app = 0.81 nM), whereas Vs NS1 showed no binding. Immunofluorescence assays on human lung epithelial (A549) and BBB (hCMEC/D3) cells further demonstrated colocalization between Hypr NS1 and JAM-A, supporting a potential interaction. No direct binding to ZO-1, a barrier scaffold lacking an extracellular domain, was observed for either strain. This differential interaction profile may be modulated by 22 amino acids localized to the Wing and β-Ladder domains within NS1, which distinguish Hypr from Vs. Notably, despite its limited interactions with the investigated TJPs, the Vs strain is associated with slow-progressive infections that can culminate in chronic neurological disease. This highlights the need for further studies into noncanonical pathways of neuroinvasion.