Pteropine orthoreoviruses use cell surface heparan sulfate as an attachment receptor

Pteropine orthoreoviruses (PRV) are an emerging group of fusogenic, bat-borne viruses from the orthoreovirus genus. Since the isolation of PRV from a patient with acute respiratory tract infections in 2006, the zoonotic potential of PRV has been further highlighted following subsequent isolation of PRV species from patients in Malaysia, Hong Kong and Indonesia. However, the entry mechanism of PRV is currently unknown. In this study, we investigated the role of previously identified mammalian orthoreovirus (MRV) receptors, sialic acid and junctional adhesion molecule-1, for PRV infection. However, none of these receptors played a significant role in PRV infection, suggesting PRV uses a distinct entry receptor from MRV. Given its broad-tissue tropism, we hypothesized that PRV may use a receptor that is widely expressed in all cell types, heparan sulfate (HS). Enzymatic removal of cell surface heparan sulfate by heparinase treatment, and genetic ablation of HS biosynthesis genes, SLC35B2, EXT-1, NDST-1 and B3GAT3 significantly reduced infection with multiple genetically distinct PRV species. Replication kinetic of PRV3M in HS-knockout cells revealed that HS plays a crucial role in the early phase of PRV infection. Mechanistic studies demonstrated that HS was an essential host-factor for PRV attachment and entry into cells. To our knowledge, this is the first report on the use of HS as an entry receptor by a non-enveloped virus.