Integrin β3 N125 glycosylation is essential for human cytomegalovirus entry into fibroblasts

Human cytomegalovirus (hCMV) infection is highly prevalent worldwide. N-glycosylation of viral receptors is a key factor in early viral infection. Integrin β3 functions as an entry receptor for hCMV infection in fibroblasts; however, the role of integrin β3 N-glycosylation in hCMV entry remains unclear. This study aims to investigate the involvement and mechanism of integrin β3 N-glycosylation in hCMV early infection. The N-glycopeptide profile of recombinant integrin β3 was examined using LC-MS/MS. To assess the effects of specific N-glycosite mutations, viral infection, attachment, and internalization in MRC-5 cells were evaluated through various virological techniques. Moreover, the role of integrin β3 N-glycosylation in receptor-ligand interactions and downstream viral entry signaling pathways was analyzed. Glycomics analysis revealed that integrin β3 N125 mainly contained complex-type glycans, with A2S1G1 as the major glycoform. The N125 mutation in integrin β3 led to a marked reduction in hCMV-induced cytopathic effects, viral DNA load, expression of immediate-early (IE) proteins, and the production of new hCMV particles. Further analysis revealed that this inhibitory effect occurred during the viral entry phase, as the N125 mutation significantly disrupted internalization without affecting viral attachment. Furthermore, the N125 mutation suppressed hCMV glycoprotein H (gH) binding to integrin β3 and inhibited activation of the integrin/Src and RhoA/cofilin signaling pathways. These findings demonstrate that integrin β3 N125 glycosylation is essential for hCMV entry into fibroblasts. More importantly, this study establishes a correlation between hCMV ligand-receptor glycosylation and viral entry signaling pathways, providing novel insights into glycobiological targets for hCMV internalization and potential strategies for antiviral drug and vaccine development.