Characterization of the high Mr glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate

Abstract

The high Mr glycoprotein (gp300) of equine herpesvirus type 1 was found to have an Mr, estimated by SDS-PAGE, over 400000 and was confirmed as being a surface glycoprotein by 125I-labelling. In contrast to [3H]glucosamine, gp300 showed very low levels of [3H]mannose incorporation. The Mr of gp300 showed no detectable change upon treatment of purified virus with N-glycanase, and showed only a small change: in virus-infected cells treated with trunicamycin. In addition, gp300 failed to bind the lectin concanavalin A. Taken together, these results indicate a lack of N-linked carbohydrate on gp300. The major carbohydrate species were found to be composed primarily of O-linked chains, as indicated by the sensitivity of the protein to monensin, to exoglycanase enzymes specific for sugars present in O-linked chains and to mild alkaline borohydride treatment, which revealed three species of carbohydrate of Mr of > 10000, 2400 and 1100 respectively. Neuraminidase treatment and binding of Helix pomatia lectin indicated the presence of .alpha.-N-acetylglucosamine and sialic acid as terminal sugars. Immunological cross-reactivity of gp300 with a high Mr protein of equine herpesvirus type 4 was shown and it also exhibited a marked Mr variation in the vaccine strain Rhinomune.