Role for N-glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity

Functional and epidemiological data suggest that N-linked glycans on the SARS-CoV-2 Spike protein may contribute to viral infectivity. To investigate this, we created a panel of N-to-Q mutations at N-glycosylation sites proximal to the Spike S1-S2 (N61, N603, N657, and N616) and S2' (N603 and N801) proteolysis sites. Some of these mutations, particularly N61Q and N801Q, reduced Spike incorporation into Spike-pseudotyped lentivirus and authentic SARS-CoV-2 virus-like particles (VLPs). These mutations also reduced pseudovirus and VLP entry into ACE2-expressing cells by 80 to 90%. In contrast, glycan mutations had a relatively minor effect on cell surface expression of Spike, ACE2 binding, and syncytia formation. A similar dichotomy in function was observed when virus was produced in host cells lacking ER chaperones, calnexin and calreticulin. Here, while both chaperones regulated pseudovirus function, only VLPs produced in calnexin KOs were less infectious. Overall, Spike N-glycans are likely critical for SARS-CoV-2 function and could serve as drug targets for COVID-19.