Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection. Author summaryHeartland bandavirus (HRTV) and Dabie bandavirus (DBV) were recently identified as emerging tick-borne zoonotic viruses in the United States and Asia, respectively. As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. Here, we use genome-wide CRISPR/Cas9 knockout screens to determine the requirements of HRTV entry in mammalian cells. We found that the glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The infectivity of HRTV and DBV in glycosphingolipid biosynthesis-deficient cells was drastically reduced. We also found that glucosylceramide (GlcCer) plays a vital role in HRTV glycoproteins-mediated membrane fusion. The GlcCer targets the lipid-head-group binding pocket of HRTV glycoprotein in the host lysosomal membrane to form a stable lipid-protein complex, thereby facilitating viral fusion and entry. Our study reveals the detailed molecular mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provides a potential therapeutic target for tick-borne virus infection.

Automated summary

Due to climate change, emerging tick-borne zoonotic viruses such as HRTV and DBV have become a significant concern. However, the mechanisms of viral infection remain largely unknown. This study reveals that the glycosphingolipid biosynthesis pathway, especially GlcCer, is essential for HRTV and DBV infection. GlcCer targets the lipid-head-group binding pocket of Gn/Gc glycoproteins, facilitating viral fusion and entry. These findings provide valuable mechanistic insights and a potential therapeutic target for tick-borne virus infection.