Role of N-linked glycosylation sites in human ACE2 in SARS-CoV-2 and hCoV-NL63 infection

Angiotensin-converting enzyme 2 (ACE2) is a transmembrane protein known for its physiological role in the renin-angiotensin system that also serves as a receptor for entry of SARS-CoV-1, SARS-CoV-2, and the seasonal human coronavirus NL63 (hCoV-NL63). ACE2 contains seven N-linked glycosylation sites. Molecular simulation and binding analyses suggest that some of them are involved in the interaction with the Spike (S) proteins of hCoVs, but their relevance in S-mediated fusion and viral entry is poorly investigated. To address this, we determined the impact of all seven N-linked glycosylation sites in ACE2 on S-mediated SARS-CoV-2 and hCoV-NL63 infection as well as cell-to-cell fusion. We found that all mutant ACE2 proteins are expressed and localized at the cell surface, albeit ACE2 lacks all glycans at decreased levels. On average, changes in T92I, N322A, and N690A, as well as combined mutation of all N-linked glycosylation sites increased endocytic VSVpp infection mediated by early HU-1 as well as Omicron BA.2, BA.5, and XBB.1.5 SARS-CoV-2 S proteins. In comparison, only the lack of glycan at N322 in ACE2 enhanced syncytia formation and only in the case of HU-1 and XBB.1.5 S proteins. ... mehrChanges in N90A, T92I, and N322A increased infection by the early SARS-CoV-2 HU-1 strain about twofold to threefold but had lesser effects on infection by genuine Omicron variants. Despite reduced cell surface expression of ACE2, elimination of all N-linked glycosylation sites usually enhanced SARS-CoV-2 infection via the endocytic pathway while having little effect on entry at the cell surface in the presence of TMPRSS2. Our results provide insights into the role of N-linked glycans in the ability of human ACE2 (hACE2) to serve as receptors for coronavirus infection.
IMPORTANCE
Several human coronaviruses use angiotensin-converting enzyme 2 (ACE2) as a primary receptor for infection of human cells. ACE2 is glycosylated at seven distinct positions, and the role of glycans for the entry of SARS-CoV-2 and hCoV-NL63 into their target cells is incompletely understood. Here, we examined the impact of individual and combined mutations in hACE2 glycosylation sites on Spike-mediated VSV-pseudoparticle and genuine SARS-CoV-2 and hCoV-NL63 infection and cell-to-cell fusion. Our results provide new information on the role of glycans in hACE2 for infection by highly pathogenic and seasonal coronaviruses.