Envelope plasmids for all the 22 single-site glycan SARS-CoV-2 pseudoviruses mutants were produced in the Bieniasz Lab by expressing C-terminally truncated, human-codon-optimized SARS-CoV-2 spike protein (pSARS-CoV-2Δ19) as previously described . Using this plasmid as the template, asparagine at N-linked glycosylation sites were replaced by aspartate in an overlap-extension PCR amplification with primers that incorporated the corresponding nucleotide substitutions. Mutations in the O-linked glycosylation sites in the RBD region (S323, T325) were conducted by a similar process by replacement with alanine. The purified PCR products were then inserted into the pCR3.1 expression vector with NEBuilder® HiFi DNA Assembly. Some mutations within or adjacent to the RBD region, including N282D, N331D, N343D, and double mutation S323A/T325A, were also introduced in spike bearing the R683G substitution, the latter of which alters the furin cleavage site . The plasmids were amplified and validated via gene sequencing for confirmation, prior to being used for SARS-CoV-2 pseudovirus production.