From a structural perspective, the identified glycan attachment sites form two clusters (Figure ), one associated with the RBD domain of S (N122, N165, N331, N343); and one close to the furin cleavage site (N61, N616, N657). We cannot exclude the caveat that some of these glycan attachment site mutations could also affect trafficking of the virus including protein processing in cells. The reduced infectivity of certain glycan mutants could reflect more global changes in Spike structure and glycosylation. To further evaluate this subset of identified residues, the ability of CV-N to irreversibly inactivate pseudovirus mutants (N61D, N122D, N331D, N343D, N603D, N657D) was tested using the the same protocol as in previous experiments with WT pseudovirus. The data indicated that N61D, N657D, N122D, N331D, and N343D did not lose their infectivity upon CV-N treatment and washout compared to the WT control (Figure ). As before, western blot analysis confirmed the removal of un-bound CV-N from "CV-N treated" glycan single attachment-site SARS-CoV-2 pseudovirus mutants (Supplemental Figures S8 to S13).