Involvement of Integrin β3 O-Glycosylation in Human Cytomegalovirus Internalization Into Fibroblasts

Abstract

Glycosylation of viral receptors is essential during the initial steps of viral infection. While integrin β3 has been recognized as a cellular receptor facilitating human cytomegalovirus (hCMV) entry into fibroblasts, the specific contribution of its glycosylation remains poorly understood. In this study, recombinant integrin β3 was engineered, and O-glycopeptide profiling was performed using LC-MS/MS. hCMV infection, attachment, and internalization were investigated in MRC-5 cells expressing integrin β3 mutants with altered O-glycosylation sites, employing a range of virological assays. To further elucidate the functional relevance of glycosylation, its effect on receptor–ligand interactions, and downstream signaling events involved in viral entry was investigated. The glycomic analysis identified mucin-type O-GalNAc structures across all O-glycosylation sites, with serine 85 (S85) showing the highest glycosylation efficiency. Mutation at S85 reduced hCMV infection and impaired viral internalization, while attachment to the cell surface remained unaffected. Mechanistic studies revealed that the S85 mutation interfered with gH binding to integrin β3 and disrupted the activation of downstream signaling pathways required for viral entry. These results highlight the pivotal role of O-glycosylation at S85 in facilitating hCMV internalization into fibroblasts and underscore its potential as a therapeutic target in antiviral strategies aimed at blocking viral entry.