Production and Characterization of a Soluble Active Form of Tva the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

摘要

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of ∼25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.