Abstract
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 Asn(150); yet, the functional consequences of this modification have remained unclear. Here, we characterize the Asn(150)-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 Asn(150) 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 Asn(150) motif across BTV serotypes underscores its functional importance and highlights NS3/NS3A glycosylation as a potential target for broad-spectrum antivirals or vaccine strategies.IMPORTANCEBluetongue virus (BTV) is an economically significant arbovirus that causes hemorrhagic disease in ruminants and restricts global livestock trade. Although the N-linked glycosylation site at Asn(150) in the nonstructural protein NS3/NS3A is strictly conserved across BTV serotypes, its functional role has remained unclear. Here, we demonstrate that glycosylation at Asn(150) acts as a targeting signal that promotes efficient trafficking of NS3/NS3A to the plasma membrane and its enrichment within lipid raft-associated microdomains that support virus egress. These findings reveal that this viral glycan plays a critical role in directing NS3/NS3A to specific membrane microenvironments, providing new insight into how BTV exploits host membrane organization to facilitate its replication cycle. The strict conservation of this glycosylation site among typical BTV serotypes highlights its potential as a broad-spectrum antiviral target and warrants further investigation into the role of NS3/NS3A glycosylation in vaccine development and interspecies transmission.