Innate immune-regulated sulfated glycosaminoglycans are associated with progeny respiratory syncytial virus retention at the cell surface in immortalized cell lines.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), members of the Pneumoviridae family, are major causes of acute respiratory tract infections globally. Although glycosaminoglycans (GAGs), including heparan sulfate (HS), are implicated in viral attachment, their roles beyond entry remain incompletely defined. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in interferon receptor-deficient cells to identify host factors commonly required for RSV and hMPV infection. The screen identified multiple genes involved in GAG biosynthesis and sulfation, including solute carrier family 35 member B2 (SLC35B2) and β-1,3-glucuronyltransferase 3 (B3GAT3). GAG sulfation promoted viral attachment and internalization of both viruses. Beyond entry, GAG sulfation influenced post-entry RSV dynamics. Genetic disruption of GAG biosynthesis or sulfation, as well as pharmacological inhibition, reduced cell-associated RSV while increasing infectious particles released into the supernatant, indicating that sulfated GAGs are associated with RSV particle retention at the cell surface in immortalized cell-line systems. This phenotype was not observed for hMPV, supporting a virus-specific requirement for GAG sulfation during RSV infection. Furthermore, RSV infection increased cell-surface GAG-reactive signal in a mitochondrial antiviral signaling protein (MAVS)-dependent manner. Loss of MAVS abolished this increase and was associated with increased released RSV titers, suggesting that innate immune signaling may influence viral release either directly through antiviral effects or indirectly through changes in GAG-related cell-surface properties. Collectively, our findings support a dual role for sulfated GAGs in Pneumoviridae infection in immortalized cell-line systems, facilitating viral entry for both RSV and hMPV while selectively restricting RSV dissemination through modulation of viral particle retention at the cell surface.