Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

Lennemann, Nicholas J.; Rhein, Bethany A.; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

N-linked glycans on GP1 are not required for entry into macrophages. Macrophages are major, early targets during filovirus infections, and others have suggested that CLECs may be important for entry into these cells (31) (32) (33) . Previous work has shown that murine MGL and SIGNR1, the murine homolog of DC-SIGN, are expressed on peritoneal macrophages (34, 35) . We used our most extensively deglycosylated mutants to determine the role of N-linked and O-linked glycans in entry into these cells. Since VSV is highly sensitive to the type I interferon response (36), resident peritoneal cells were isolated from IFNAR Ϫ/Ϫ mice for these studies. In addition, these mice lacked TIM-1, which eliminated the possibility for entry in epithelial cells that may have contaminated the preparations. Cells were treated for 3 days with murine macrophage colony-stimulating factor (M-CSF) and their phenotypes were determined prior to use. Eighty-five percent of adherent cells from the peritoneal cavity were CD11b ϩ /F480 ϩ , indicative of matured macrophages (see Fig. S7 in the supplemental material). These cells were highly permissive for VSV pseudotyped with GP (Fig. 5) . Entry mediated by 7Gm8G was en-hanced~3-fold over entry mediated by the WT, which is more consistent with our Vero cell data than with our CLEC utilization data. Furthermore, macrophages supported entry mediated by the 5G⌬muc mutation, although this was~15-fold decreased compared to the results for the WT. These findings demonstrated that neither O-or N-linked glycans on GP1 are absolutely required for entry into peritoneal macrophages.