Protein glycosylation is essential for SARS-CoV-2 infection

34 SARS-CoV-2 extensively N-glycosylates its surface spike (S) proteins. This post35 translational modification is essential to modulate protein conformation and host cell 36 invasion. Each S monomer can be modified with up to 22 N-glycans. To meet the high 37 demand of protein glycosylation during virus replication, SARS-CoV-2 upregulates the 38 expression of host N-glycosylation genes. Although a substantial amount of detail is 39 known about the structure of S protein N-glycans, the role of N-glycosylation in SARS40 CoV-2 infection remains largely undetermined. Here, we investigated the essentiality 41 of the host N-glycosylation pathway and viral N-glycans for SARS-CoV-2 infection. 42 When either monkey or human cells were preincubated with glycosylation inhibitors, 43 including FDA-approved iminosugars, virus infection was significantly reduced. This 44 infection phenotype was confirmed after RNAi knockdown of several glycosylation 45 genes. In addition, enzymatic deglycosylation of whole viral particles confirmed that 46 accessible oligosaccharides on the SARS-CoV-2 surface are essential for host cell 47 infection. Altogether, we show evidence that the normal functioning of the host N48 glycosylation machinery is essential not only for SARS-CoV-2 to infect, but also to 49 produce new functional virions. These findings open the door for developing new 50 approaches targeting N-glycosylation against COVID-19. 51 52