FIG 2.

SERINC5 is N-glycosylated: the higher-molecular-weight species in virions contains complex glycans, while the lower-molecular-weight species that predominates in transfected cells contains immature, high-mannose glycans. (A) HEK293 Tet-On TRE-HIV1-ΔNef cells were transfected with either pBJ5-S5-iHA or an empty vector. Twenty-four hours later, the cells were treated with 0.5 μg/ml of doxycycline for 8 h to induce virus production. The virion pellets obtained by centrifugation through 20% sucrose cushions were suspended in 1× TCEP reducing buffer and then treated with the indicated glycosidases at 37°C for 1 h. The reaction products were first immunoblotted for SERINC5 (HA; top panel), and then the membrane was stripped and reprobed with anti-gp120 (bottom panel). The star indicates a nonspecific band. (B) HEK293 cells were transfected with 100 ng of pBJ5-S5-iHA and 1.5 μg of pNL43ΔNef; 24 h later, the cells and virions were isolated as described above. The virions were treated with PNGase F (or not) for 1 h at 37°C and immunoblotted for SERINC (HA). (C) SERINC3/5 double KO cells were transfected with pNL43ΔNef and 100 ng of pBJ5-S5-iHA. Virions were isolated as described above. The virions were treated with PNGase F as described for panel A and immunoblotted for SERINC5 (HA). (D) The cells from the experiment shown in panel B were suspended in 1× TCEP reducing buffer, and whole-cell extracts were prepared as described in Materials and Methods. An aliquot of the whole-cell extracts was treated with endoglycosidase H (Endo-H) or PNGase F for 1 h at 37°C. The “input” is untreated lysate left at 4°C. The reaction products were blotted for SERINC5 (HA).