Role of O-linked glycosylation modification on internalization and replication of avian leukosis virus subgroup J

Upon infection, viruses reprogram the host metabolic system to hijack metabolic resources for proliferation. Avian leukosis virus subgroup J (ALV-J), an avian oncogenic virus, poses significant challenges to the poultry industry. ALV-J infection upgrades monosaccharide N-acetylgalactosamine (GalNAc) and galactosyltransferase (core 1 β3-Gal-T) in DF-1 cells, both of which are crucial for O-linked glycosylation. Addition of GalNAc or overexpression of core 1 β3-Gal-T in DF-1 cells can promote ALV-J replication. ALV-J envelope protein (Env) undergoes complex post-translational modifications. Two O-linked glycosylation sites (T32 and T271) located in the head region of the ALV-J Env have been identified for the first time using liquid chromatography-mass spectrometry (LC-MS). The results of coimmunoprecipitation and flow cytometry indicate that mutations in T32 or T271 diminish ALV-J infection by affecting viral internalization, rather than attachment. The viral internalization efficiency was partially restored under a low pH environment. Incorporation of T32A and T271A into ALV-J led to significantly reduced replication capacity in vivo and viral shedding of the recombinant virus. These findings are valuable for our understanding of the roles of glycans in the ALV-J infection cycle, as well as for providing potential anti-ALV-J strategies.