A Single Glycosylation Site Within the Receptor-Binding Domain of the Avian Sarcoma/Leukosis Virus Glycoprotein Is Critical for Receptor Binding

Abstract

Retroviral envelope proteins are heavily glycosylated. In some cases, glycosylation has been shown to be important for folding, protein stability, immune evasion, or receptor usage. The receptor-binding subunit (SU or gp85) of the envelope protein (EnvA) of the avian sarcoma/leukosis virus, subtype A (ASLV-A), contains 11 potential N-linked glycosylation sites (NXS/T). To address the importance of N-linked glycosylation for the function of EnvA, we prepared a series of EnvA proteins lacking one or more of these carbohydrate addition sites. Using site-directed mutagenesis, we mutated the S or T in each NXS/T glycosylation sequon to A. We also prepared EnvAs bearing selected double and triple mutations. We examined each mutant EnvA for its ability to be expressed at the cell surface, proteolytically processed into gp85 and gp37, incorporated into MLV pseudotyped virions, and to support infection of cells expressing the ASLV-A receptor, Tva. Eight single mutations were well tolerated, and, in general, EnvA was able to tolerate double mutations of these glycosylation sites. Triple mutations were more variable in their effects. Of the three glycosylation sites important for EnvA function, two are important for folding (EnvA production and processing were severely impaired). For the third, although EnvA processing was impaired, significant amounts of processed EnvA were expressed at the cell surface and incorporated into virions. Nonetheless, this mutant EnvA, EnvAΔNg10, was unable to support infection. Further examination of EnvAΔNg10 revealed that it was unable to bind Tva and was severely impaired for binding to a monoclonal antibody which inhibits receptor binding. This work has therefore identified a single N-linked glycosylation site in the SU domain of EnvA that is critical for binding between EnvA and its receptor, Tva.