Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

Bluetongue virus (BTV) is an economically important arbovirus of ruminants worldwide. The nonstructural glycoprotein NS3/NS3A contains a uniquely conserved N-linked glycosylation site at Asn150; yet, the functional consequences of this modification have remained unclear. Here, we characterize the Asn150-linked glycan and show that it predominantly comprises high-mannose structures that undergo maturation specifically in mammalian cells. Using a reverse-genetics system, we demonstrate that N-linked glycosylation is not required to maintain NS3/NS3A stability or its interaction with outer capsid proteins, but is essential for efficient virion egress. Ablation of this site (N150Q) markedly impaired plasma-membrane localization and reduced partitioning into detergent-resistant lipid raft microdomains. These defects resulted in reduced extracellular titers and attenuated multicycle spread. Together, these findings support a model in which the Asn150 glycan functions as a glycan-dependent targeting signal that biases NS3/NS3A toward plasma-membrane microdomains enriched in lipid rafts, thereby promoting productive virus release. In AG129 mice, the N150Q mutant exhibited attenuated virulence, characterized by reduced viremia, restricted tissue dissemination, and minimal pathology, in contrast to the severe systemic disease caused by the wild-type virus. The strict conservation of the Asn150 motif across BTV serotypes underscores its functional importance and highlights NS3/NS3A glycosylation as a potential target for broad-spectrum antivirals or vaccine strategies.