Using pseudoviruses containing single glycan attachment-site mutations in the spike protein, initial infection by the mutant pseudoviruses, lectin inhibition of pseudovirus infection, and the irreversible inactivation by CV-N were found to be altered. The identified mutations were a subset of glycan attachment sites present in the S1 subunit of the Spike protein. The locations of the critical glycan attachment site mutations could be grouped into two clusters, one located near the receptor binding domain (N122, N331, N343) and the second proximal to the furin cleavage site between the S1 and S2 subunits (N61, N657). Some of the glycan attachment site mutant SARS-CoV-2 pseudoviruses, especially mutations within the S1 subunit, exhibited significantly reduced infectivity compared to WT (Supplemental Figure ). The changes in overall infectivity could indicate that changes in the overall structure of Spike or pattern of glycosylation could occur, though the changes in infectivity still allowed measurement of the effects of CV-N inhibition on pseudovirus mutant infection (for mutant infectivity levels, see Supplemental Figure , and for CV-N dose responses, see Supplemental Figure ).