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 membrane proteoglycans carrying heparan sulfate chains (HSPGs). A secondary binding step requires gD and presumably another, hitherto unidentified cellular receptor. By use of a virus overlay protein binding assay (VOPBA), cosedimentation analyses, and affinity chromatography, we identified three species of cell membrane constituents that bind PrV. By treatment with EDTA, peripheral HSPGs of very high apparent molecular mass (>200 kDa) could be extracted from Madin-Darby bovine kidney cells. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzymatic digestion with heparinase or papain. Cosedimentation analyses indicated that binding between PrV and high-molecular-weight HSPG depended on the presence of gC in the virion. In addition, adsorption of radiolabeled PrV virions to cells could be inhibited by the addition of purified 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 presence of heparan sulfate, since reactivity was abolished after suppression of glycosaminoglycan biosynthesis with NaClO3 and after heparinase treatment. In addition to HSPG, in cellular membrane extracts obtained by treatment with mild detergent, a 85-kDa membrane protein was demonstrated to bind PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents that bind PrV: a peripheral HSPG of high molecular weight, an integral HSPG of approximately 140 kDa, and an integral membrane protein of 85 kDa. It is tempting to speculate that interaction between PrV and the two species of HSPG mediates primary attachment of PrV and that the 85-kDa protein is involved in a subsequent attachment step.
Karger
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Institute of Molecular and Cellular Virology, Friedrich Loeffler Institutes, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany.
Mettenleiter
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TC
D000818:Animals; D002417:Cattle; D002460:Cell Line; D011558:Herpesvirus 1, Suid / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D011991:Receptors, Virus / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012107:Research Design; D012995:Solubility; D013552:Swine; D014508:Urea; D014759:Viral Envelope Proteins / Q000378:metabolism; D014867:Water
D011509:Proteoglycans; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D014867:Water; D014508:Urea