Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV

Abstract

The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. To study the maturation pathway of the S glycoprotein of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) within the host cell, a T7/vaccinia virus-based expression system coupled to immunoprecipitation with anti-S antibodies was used to test and analyze different forms of the S glycoprotein. The state of maturity of the S glycoprotein can be deduced from its sensitivity to hydrolysis by endoglycosidase H (EndoH) or N-glycosidase F (N-Gly F). A fully matured S glycoprotein will be modified with complex oligosaccharides which makes it resistant to cleavage by EndoH but not by N-Gly F. By exploiting this characteristic, it is then possible to determine which forms of the immunoprecipitated S protein are properly processed by the host cell. With this system, many different constructs of the S glycoprotein can be analyzed in parallel thus providing another method by which to study the functional domains of S involved in membrane fusion event that occurs during viral infection.

Fig. 1.

Restriction map and multiple cloning site of pKT-0. pKT-0 is a mammalian expression vector that allows a gene of interest to be highly expressed if it is inserted into the multiple cloning site, as shown on the map.

Fig. 2.

Time-course of S protein maturation. Cos7 cells transfected with pKT-S were radiolabeled and chased for 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, and 6 h respectively (lanes 1–7). Cos7 cells transfected with plasmid without insert are harvested at 6 h as negative control (lane 8). All the cell lysates were immunoprecipitated with rabbit α-SΔ10 antibodies and then separated on SDS-PAGE gels. In a separate experiment, the immunoprecipitated proteins (6 h posttransfection) were either treated (+) with EndoH (lane 10) or left untreated (−) as a control (lane 9). The S-specific bands and their molecular masses (in kDa) were indicated on the right. High-Range Rainbow Molecular Weight Markers (Amersham) were used to assess protein mass, as indicated on the left.

Fig. 3.

Treatment of the S-derived proteins in cell lysates with N-glycosidase F (N-Gly-F) and endoglycosidase H (EndoH). The full-length S was expressed in Cos-7 cells. Cells were resuspended in lysis buffer. The samples were either treated (+) with (A) N-Gly-F and (B) EndoH or mock-treated (−). Proteins were separated on SDS-PAGE gels. Western Blot was performed with rabbit-anti-S and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies. Lysates from mock-transfected cells were used as negative controls (lanes 3, 4, 7, and 8). Molecular masses of specific proteins are indicated on the right and masses of markers are indicated on the left in kilodalton.