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

## TL;DR

This study shows how O-linked glycosylation affects the internalization and replication of ALV-J, an avian virus, and identifies key sites that could help develop anti-viral strategies.

## Contribution

The first identification of O-linked glycosylation sites (T32 and T271) in ALV-J Env and their role in viral internalization and replication.

## Key findings

- ALV-J replication is enhanced by GalNAc and core 1 β3-Gal-T in DF-1 cells.
- Mutations in T32 or T271 reduce ALV-J infection by impairing internalization.
- T32A and T271A mutations significantly lower ALV-J replication and viral shedding in vivo.

## Abstract

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.

The online version contains supplementary material available at 10.1186/s13567-025-01709-3.