IDENTIFICATION OF CELL-SURFACE MOLECULES THAT INTERACT WITH PSEUDORABIES VIRUS

The alphaherpesvirus pseudorabies virus (PrV) has been shown to attach to cells by interaction between the viral glycoprotein gC and cell me mbrane proteoglycans carrying heparan sulfate chains (HSPGs). A second ary binding step requires gD and presumably another, hitherto unidenti fied cellular receptor. By use of a virus overlay protein binding assa y (VOPBA), cosedimentation analyses, and affinity chromatography, we i dentified three species of cell membrane constituents that bind PrV. B y treatment with EDTA, peripheral HSPGs of very high apparent molecula r mass (>200 kDa) could be extracted from Madin-Darby bovine kidney ce lls. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzym atic digestion with heparinase or papain. Cosedimentation analyses ind icated that binding between PrV and high-molecular-weight HSPG depende d on the presence of gC in the virion. In addition, adsorption of radi olabeled PrV virions to cells could be inhibited by the addition of pu rified high-molecular-weight HSPG. By using urea extraction buffer, a second species of HSPG of approximately 140 kDa could be solubilized. Binding of PrV to this HSPG in the VOPBA was also dependent on the pre sence of heparan sulfate, since reactivity was abolished after suppres sion of glycosaminoglycan biosynthesis with NaClO3 and after heparinas e treatment. In addition to HSPG, in cellular membrane extracts obtain ed by treatment with mild detergent, a 85-kDa membrane protein was dem onstrated to bind to PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents tha t bind PrV: a peripheral HSPG of high molecular weight, an integral HS PG of approximately 140 kDa, and an integral membrane protein of 85 kD a. It is tempting to speculate that interaction between PrV and the tw o species of HSPG mediates primary attachment of PrV and that the 85-k Da protein is involved in a subsequent attachment step.